Preface

Chapter 7 **HMGB Proteins from Yeast to Human. Gene Regulation, DNA**

Chapter 8 **Endophytic Yeast and Hosts: A Mutualistic Association Friendly**

Vizoso-Vázquez Ángel, Barreiro-Alonso Aida, Rico-Díaz Agustín, Lamas-Maceiras Mónica, Rodríguez-Belmonte Esther, Becerra Manuel, González-Siso María Isabel and Cerdán María Esperanza

**Repair and Beyond 139**

**VI** Contents

**to the Environment 169** Esperanza del Pilar Infante Luna

**Section 3 Yeast Versus Plant - Unknown Territory 167**

Yeast-based biotechnology traditionally regards the empirical production of fermented drinks and leav‐ ened bread. Although these processes are known to man for millennia, they keep posing challenges and fuelling considerable amount of research. Still, feasible and economically viable applications of yeastdriven technology are presently very diverse, critically contributing to the economical welfare of many differently developed countries and representing at world scale a business volume worth billions.

Yeast biotechnology follows essentially two main strategies. One uses the organism as a cell factory for the efficient production of fine chemicals and molecules to apply on pharma or research or bulk chemi‐ cals as in the case of biofuels. Another strategy uses the organism as bio-tool applied *in loco* to execute specific tasks. Examples can be food preservation implicating on shelf-life span, the concentrative re‐ moval of metals from industrial or mining residues or bioremediation of environmental disasters like oil spillage in the sea.

Yeasts are a very large group of microorganisms, harbouring a large number of species, and are genetical‐ ly very diverse. Like bacteria, they are present in all types of ecosystems, including human microbiome. Still, derived from the traditional applications abovementioned, most of the yeast biotechnologies of to‐ day are still based on the utilization of *Saccharomyces cerevisiae* strains, either wild-type isolates from spe‐ cific environmental niches or engineered, be it genetically, metabolically or evolutionarily. The possibilities associated with yeast biodiversity have therefore been largely disregarded.

The big disproportion between the numbers of applications that use *S. cerevisiae* in relation to other spe‐ cies has other reasons besides tradition. Successful biotechnology depends on detailed knowledge, not only of the process but also mainly of the organism that governs it. *S. cerevisiae* is by far the most wellstudied yeast species and one of the best well-known organisms, maybe just overcome by *Escherichia coli*. It is easier and cheaper to research comparing to higher eukaryote models and does not pose ma‐ nipulation ethical problems or health concerns. Importantly, yeasts are recognized as excellent models of cell and molecular biology for higher eukaryotes, including humans. Not only do yeasts contribute with key discoveries to understand cellular processes, but they also contribute with crucial knowledge about complex diseases, some of which can only be studied in yeast, like mitochondrial metabolic dis‐ eases or neurodegenerative disorders like Batten or Huntington diseases.

Yeast diversity of species and strains constitutes per se a huge potential for biotechnology. It though re‐ quires considerable reliability, which is only obtained with more research and knowledge. Many species have been poorly addressed by research and are consequently less applied, most of them not at all. The general designation of the non-*Saccharomyces* yeasts as *non-conventional* well translates this disproportion. Two exceptions can be pinpointed. *Schizosaccharomyces pombe*, the fission yeast, has been largely used as a model to understand cell cycle control. *Candida* species have been largely used to address host defences against pathogens and yeast-promoted tissue invasion and infection. This last case has gained particular importance in view of the increasing severity and prevalence of human yeast infections.

In spite of the huge potential that yeasts present for biotechnology and health, research with yeasts has presently entered a stalling period, yeasts being delegated to non-mainstream scientific interests difficult to fund. Of course, much has been achieved in the last 50 years, but yeasts, including *S. cerevisiae*, still present many challenges and still can surprise by unveiling unprecedented scientific knowledge, opening

new strands for application and innovation. Moreover, it is not only the organism itself that appears to have become *old fashion*. Research in the last 20 years has focused essentially on molecular aspects. This was fuelled by the growing amenability of yeast genetic manipulation and availability of molecular tools. This enabled the building up of a robust pile of knowledge on signalling, transcription and post-transla‐ tional modification and regulation, trafficking, chromosomal structure and behaviour and so on and so forth. But the actual function of the yeast cell transcends the mechanistic vision of intertwining enzymatic production lines that we can manipulate at our will to yield interesting metabolites for our profit. There are knurls, kinks and nuts. Critically, new and old unanswered questions cloud a full-picture understand‐ ing of yeast biology and preclude biotechnology application success. It is necessary to not lose the ability to assess biochemical and biophysical aspects in order to really understand function.

This book provides some insights into aspects of yeast science and yeast-based biotechnology less fre‐ quently addressed in the literature but nonetheless decisive to improve knowledge and, accordingly, boost up yeast-based innovation. These generally regard how the yeast cells dispose of ions, implicating in how they control the electric plasma membrane potential (Chapter 1), in how they act as biosorbents of heavy metal ions without suffering from their toxicity (Chapter 2) and their ability to transfer elec‐ trons as a whole cell (Chapter 3). All of these have clear technological implications in the use of yeasts in bioremediation, biomining or the development of yeast-based fuel cells. All these processes are ad‐ dressed as expected in *S. cerevisiae*, in which metabolic engineering for the specific production of certain dine and bulk chemicals is reviewed (Chapter 4). But also the biotechnological potential of several socalled non-conventional yeast species is briefly evaluated (Chapter 5), for the creation of yeast-based microbial fuel cells (Chapter 3) as well as for fermentative processes, either alone or in mixed fermenta‐ tions (Chapter 5). Moreover, the mechanisms and molecules involved in cellular relief from hydroper‐ oxides, which participate in tissue injury and in the onset and progression of degenerative diseases in humans (Chapter 6). These mechanisms are crucial to understand ageing, as are the mechanisms in‐ volved in DNA protection (Chapter 7), needless to stress how much these studies are crucial for phar‐ macological and clinical development. Finally, the last but an important chapter (Chapter 8) presents a whole new view on how yeasts can also colonize plants, living as endophytic microorganisms. This could parallel the commensality of yeasts in the human tissues. Yet, too few information is still available to know whether in plants, like in mammals, yeasts can change drastically their biology, shifting com‐ mensalism into parasitism and developing an infection. This is a quite promising line of work, bearing in mind the growing worldwide agriculture problem posed by plagues that either resist chemical treat‐ ment or cannot be managed that way because of health-risk concerns. New and revolutionary strategies are needed to control yeast and fungi pathogens in plants as in animals, which can only be achieved through continuous research effort.

> **Cândida Lucas** Full Professor Institute of Science and Innovation for Bio-Sustainability (IB-S) Centre of Molecular and Environmental Biology Research (CBMA) University of Minho, Portugal

> **Célia Pais** Associated Professor Centre of Molecular and Environmental Biology Research (CBMA) Biology Department, University of Minho, Portugal

**Yeasts - Indispensible Biotech Playwers**

new strands for application and innovation. Moreover, it is not only the organism itself that appears to have become *old fashion*. Research in the last 20 years has focused essentially on molecular aspects. This was fuelled by the growing amenability of yeast genetic manipulation and availability of molecular tools. This enabled the building up of a robust pile of knowledge on signalling, transcription and post-transla‐ tional modification and regulation, trafficking, chromosomal structure and behaviour and so on and so forth. But the actual function of the yeast cell transcends the mechanistic vision of intertwining enzymatic production lines that we can manipulate at our will to yield interesting metabolites for our profit. There are knurls, kinks and nuts. Critically, new and old unanswered questions cloud a full-picture understand‐ ing of yeast biology and preclude biotechnology application success. It is necessary to not lose the ability

This book provides some insights into aspects of yeast science and yeast-based biotechnology less fre‐ quently addressed in the literature but nonetheless decisive to improve knowledge and, accordingly, boost up yeast-based innovation. These generally regard how the yeast cells dispose of ions, implicating in how they control the electric plasma membrane potential (Chapter 1), in how they act as biosorbents of heavy metal ions without suffering from their toxicity (Chapter 2) and their ability to transfer elec‐ trons as a whole cell (Chapter 3). All of these have clear technological implications in the use of yeasts in bioremediation, biomining or the development of yeast-based fuel cells. All these processes are ad‐ dressed as expected in *S. cerevisiae*, in which metabolic engineering for the specific production of certain dine and bulk chemicals is reviewed (Chapter 4). But also the biotechnological potential of several socalled non-conventional yeast species is briefly evaluated (Chapter 5), for the creation of yeast-based microbial fuel cells (Chapter 3) as well as for fermentative processes, either alone or in mixed fermenta‐ tions (Chapter 5). Moreover, the mechanisms and molecules involved in cellular relief from hydroper‐ oxides, which participate in tissue injury and in the onset and progression of degenerative diseases in humans (Chapter 6). These mechanisms are crucial to understand ageing, as are the mechanisms in‐ volved in DNA protection (Chapter 7), needless to stress how much these studies are crucial for phar‐ macological and clinical development. Finally, the last but an important chapter (Chapter 8) presents a whole new view on how yeasts can also colonize plants, living as endophytic microorganisms. This could parallel the commensality of yeasts in the human tissues. Yet, too few information is still available to know whether in plants, like in mammals, yeasts can change drastically their biology, shifting com‐ mensalism into parasitism and developing an infection. This is a quite promising line of work, bearing in mind the growing worldwide agriculture problem posed by plagues that either resist chemical treat‐ ment or cannot be managed that way because of health-risk concerns. New and revolutionary strategies are needed to control yeast and fungi pathogens in plants as in animals, which can only be achieved

> **Cândida Lucas** Full Professor

> > **Célia Pais**

Associated Professor

University of Minho, Portugal

Institute of Science and Innovation for Bio-Sustainability (IB-S) Centre of Molecular and Environmental Biology Research (CBMA)

Centre of Molecular and Environmental Biology Research (CBMA)

Biology Department, University of Minho, Portugal

to assess biochemical and biophysical aspects in order to really understand function.

through continuous research effort.

VIII Preface

**Provisional chapter**

#### **The Plasma Membrane Electric Potential in Yeast: Probes, Results, Problems, and Solutions: A New Application of an Old Dye? Probes, Results, Problems, and Solutions: A New Application of an Old Dye?**

**The Plasma Membrane Electric Potential in Yeast:** 

DOI: 10.5772/intechopen.70403

Antonio Peña, Norma Silvia Sánchez and Martha Calahorra Martha Calahorra Additional information is available at the end of the chapter

Antonio Peña, Norma Silvia Sánchez and

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.70403

#### **Abstract**

For a long time, estimations and actual measurements of the electric plasma membrane potential (PMP) in whole yeast cells have been the subject of studies by several groups without reliable results. The conditions in the measurements, as well as precautions required to perform them, are described here. Essentially, two approaches using different dyes are reviewed: (a) qualitative estimations by following fluorescence changes under different energization conditions and (b) measurements of the PMP by the accumulation of dyes. An analysis is presented regarding the conditions recommended to obtain more consistent results when following the fluorescence changes. Also, measurements of accumulation of different dyes, and the necessary conditions to perform them, are analyzed. In particular, using acridine yellow appears to be a trustworthy method, with few reserves, both to follow in real time the qualitative changes of the PMP by fluorescence changes and to assess actual PMP values by measuring the accumulation of the dye.

**Keywords:** plasma membrane potential, yeast, acridine yellow, fluorescent monitors

#### **1. Introduction**

#### **1.1. Qualitative estimation of the electric plasma membrane potential (PMP) in yeast**

After the original proposal for the mechanism of K+ transport in yeast [1], it was shown [2, 3] that this ion is transported because a H+ -ATPase exists in the plasma membrane, pumping protons outside, therein generating an electric membrane potential difference (PMP), negative

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons

inside. This is then responsible mainly for the uptake of cations and other molecules required by the cells through different active uptake systems of which the most prominent is Trk1p [4]. This mechanism was also described for *Neurospora crassa* [5] and many other fungi and also for plants. These findings gave rise to a series of attempts to follow changes in the plasma membrane potential (PMP) in whole yeast cells, initially by measuring the accumulation of cationic compounds [6, 7]. Results were though far from satisfactory, mainly because of the slow rate at which these molecules entered the cells.

The use of fluorescent indicators originally to estimate the membrane potential in mitochondria started a long time ago [8, 9] by observing ethidium bromide fluorescence quenching under different energy states. Also, measurements of the PMP in animal cells were performed with reasonable results [10–12]. Following these studies and using whole yeast cells, fluorescence

**Figure 1.** Graphic interpretation of the fluorescence changes of ethidium bromide in yeast under different conditions [13]. (A) When glucose alone is added, fluorescence of the dye is moderately increased, but a large part of it is accumulated by the mitochondria, where its fluorescence is quenched. (B) When oxygen is exhausted, mitochondria are partially de-energized (they still can maintain most of its membrane potential by using ATP generated in glycolysis). (C) Upon the addition of H2 O2 , mitochondrial membrane potential is fully recovered, resulting in fluorescence quenching. (D) The addition of an uncoupler (FCCP) produces the full collapse of the mitochondrial membrane potential, the uniform distribution of the dye in the cytoplasm, and a large increase of fluorescence. The small oval shape represents a mitochondrion.

changes of ethidium bromide under different energization conditions were studied [13]. It was found that in order to observe the fluorescence increase derived from the accumulation of the dye, it was more convenient to use starved yeast cells, which required the addition of glucose as a substrate. **Figure 1** shows a schematic representation of the results observed and their interpretation. It was found that in the presence of glucose, fluorescence showed a rather small increase, and after a few seconds was followed by another one, coincident with the oxygen exhaustion in the medium that as expected could be reversed by the addition of H2 O2 . This led us to suspect that the initial fluorescence increase was actually composed of not only an increase due to the accumulation of the dye in the cytoplasm but also a decrease due to its accumulation in mitochondria, according to what had been described before to what was happening in the isolated mitochondria [9]. Moreover, it was found that, also as expected, the addition of an uncoupler resulted in a much larger increase of fluorescence, consistent with the idea that collapsing the membrane potential of the mitochondria resulted in an efflux of the dye and its uniform distribution in the cytoplasm, where the dye was highly fluorescent [14]. The actual fluorescence changes observed in the spectrofluorometer were similar to those shown in **Figure 2** with acridine yellow.

inside. This is then responsible mainly for the uptake of cations and other molecules required by the cells through different active uptake systems of which the most prominent is Trk1p [4]. This mechanism was also described for *Neurospora crassa* [5] and many other fungi and also for plants. These findings gave rise to a series of attempts to follow changes in the plasma membrane potential (PMP) in whole yeast cells, initially by measuring the accumulation of cationic compounds [6, 7]. Results were though far from satisfactory, mainly because of the slow rate at

The use of fluorescent indicators originally to estimate the membrane potential in mitochondria started a long time ago [8, 9] by observing ethidium bromide fluorescence quenching under different energy states. Also, measurements of the PMP in animal cells were performed with reasonable results [10–12]. Following these studies and using whole yeast cells, fluorescence

**Figure 1.** Graphic interpretation of the fluorescence changes of ethidium bromide in yeast under different conditions [13]. (A) When glucose alone is added, fluorescence of the dye is moderately increased, but a large part of it is accumulated by the mitochondria, where its fluorescence is quenched. (B) When oxygen is exhausted, mitochondria are partially de-energized (they still can maintain most of its membrane potential by using ATP generated in glycolysis). (C)

(D) The addition of an uncoupler (FCCP) produces the full collapse of the mitochondrial membrane potential, the uniform distribution of the dye in the cytoplasm, and a large increase of fluorescence. The small oval shape represents

, mitochondrial membrane potential is fully recovered, resulting in fluorescence quenching.

which these molecules entered the cells.

4 Old Yeasts - New Questions

Upon the addition of H2

a mitochondrion.

O2

Several years later, a new procedure, under the same principles was proposed by following the fluorescence changes of the cyanine DiSC<sup>3</sup> (3) [14]. Since then, perhaps the most used monitor to follow changes of the PMP in yeast is DiSC<sup>3</sup> (3), a high affinity probe that in the presence of a substrate (glucose) is concentrated in the cytoplasm, and similarly to ethidium bromide, also in the mitochondria [16]. Although the latter can be avoided by using low concentrations

**Figure 2.** Fluorescence changes of 25 μM acridine yellow at 470–500 nm when added to *S. cerevisiae* cells. Incubation was carried out in 10 mM MES-TEA buffer, pH 6.0; 10 μM BaCl<sup>2</sup> ; 20 mM glucose in a final volume of 2.0 mL. Where indicated, 8 μL of 3% H<sup>2</sup> O2 , 10 μM CCCP or 5 mM KCl were added. Representative figure of one experiment similar to that already reported [15]. Fluorescence was followed in an SLM Aminco spectrofluorometer with continuous stirring and at a temperature of 30°C. Excitation and emission fluorescence were 470 and 500 nm, respectively. Phototube voltage was 600 V, and slits were set at 8 nm. AU, arbitrary units.

of an uncoupler, still the high affinity and binding of the dyes to the cell components turns it difficult to calculate their free concentration inside, in order to accurately measure the PMP through its accumulation. Other authors [17] also used DiOC<sup>6</sup> (3) with this same purpose with rather uncertain values. Thus, results using cationic molecules, mostly dyes, have been under discussion for many years without arriving to a general consensus about the methods, and even less regarding the results obtained, especially when it comes to the real values of the PMP in yeast.

#### **2. The quantitative measurement of the PMP**

DiSC<sup>3</sup> (3) has been one of the most used monitors to estimate changes of the PMP in yeast [14, 16, 18–22] by following its fluorescence changes under different energy conditions of yeast cells. Attempts have also been made to assess the actual PMP values from the internal and external concentration ratios between the cells and the media. This implies (a) measuring the concentration and the amount of the dye or any cationic agent remaining after incubation with the cells; (b) knowing the amount originally added that taken up by the cells can be obtained; (c) the internal concentration of the dye can be obtained by previously measuring the internal water volume of the cells to acquire the internal concentration. Finally, by using the Nernst equation, the value of the PMP, in millivolts can be obtained from:

$$\mathbf{E} = -(\text{RT/ZF}) \ln \mathbf{C}\_{\text{in}} / \mathbf{C}\_{\text{out}} \tag{1}$$

where R is the gas constant, Z the charge of the ion, F is the Faraday constant, Cin and Cout the internal and external dye concentrations. Then, PMP is approximately equal to:

$$\text{PMP} = -60(\log \left[ \mathbf{C}\_{\text{in}} \right] / \left[ \mathbf{C}\_{\text{out}} \right]). \tag{2}$$

In this way, approximate values of the PMP have been obtained [14, 15, 17]. However, those values are subject to many errors and uncertainties that will be discussed below.

Studies with *Saccharomyces cerevisiae* [18] and *Rhodotorula glutinis* [19] proposed another approach to measure the actual PMP by following the changes of the *λ*max of the fluorescence spectrum of this monitor as an indicator, which has been also used by other authors [20, 23]. Within the claims to actually measure the plasma membrane potential in yeast, some values reported are too low to explain, among others, the large accumulation of K+ , which can reach internal concentrations of around 300 mM against micromolar external concentrations of the cation. Results are hard to rationalize, even considering that one part of the cation redistributes into the vacuole [24] and another is neutralized by the accumulation of bicarbonate when glucose is the substrate [25]. The use of fluorescent probes not only for the estimation of plasma membrane potential but also for many other purposes was partially reviewed by Slavik [26], and more recently, for yeast cells [16, 19]. However, this topic continues to be unsolved, and it is our belief that several aspects should be considered. The problems to estimate and measure the PMP in yeast imply a large series of factors that may affect results, such as the following: (a) the influence of the binding of the cationic monitor to the surface of the cell; (b) the use of an adequate buffer, avoiding organic molecules and other cations that may be taken up by the cells; (c) the concentration of the dye, critical and different for each one and probably for different yeast strains or species to observe the fluorescence changes and accumulation; (d) the accumulation of the dye by the mitochondria; (e) the binding of the dye inside the cells, and (f) the use of starved cells that allows observing changes due to energization of the cells by adding a substrate.

#### **3. Interaction, uptake, distribution, and binding inside the cells**

#### **3.1. Binding to the surface**

The first interaction of the dyes is, of course, with the negatively charged cell surface. Since the first studies performed [14], an immediate increase of fluorescence of DiSC<sup>3</sup> (3) was observed upon its interaction with the cells that could be diminished by the addition of low concentrations of a divalent cation eventually recommending the use of BaCl<sup>2</sup> to avoid binding [16].

#### **3.2. Uptake**

of an uncoupler, still the high affinity and binding of the dyes to the cell components turns it difficult to calculate their free concentration inside, in order to accurately measure the PMP

rather uncertain values. Thus, results using cationic molecules, mostly dyes, have been under discussion for many years without arriving to a general consensus about the methods, and even less regarding the results obtained, especially when it comes to the real values of the

(3) has been one of the most used monitors to estimate changes of the PMP in yeast [14, 16, 18–22] by following its fluorescence changes under different energy conditions of yeast cells. Attempts have also been made to assess the actual PMP values from the internal and external concentration ratios between the cells and the media. This implies (a) measuring the concentration and the amount of the dye or any cationic agent remaining after incubation with the cells; (b) knowing the amount originally added that taken up by the cells can be obtained; (c) the internal concentration of the dye can be obtained by previously measuring the internal water volume of the cells to acquire the internal concentration. Finally, by using

the Nernst equation, the value of the PMP, in millivolts can be obtained from:

internal and external dye concentrations. Then, PMP is approximately equal to:

values are subject to many errors and uncertainties that will be discussed below.

reported are too low to explain, among others, the large accumulation of K+

<sup>E</sup> <sup>=</sup> − (RT /ZF) lnCin / <sup>C</sup>out (1)

where R is the gas constant, Z the charge of the ion, F is the Faraday constant, Cin and Cout the

PMP <sup>=</sup> − 60 (log[Cin]/[Cout]). (2)

In this way, approximate values of the PMP have been obtained [14, 15, 17]. However, those

Studies with *Saccharomyces cerevisiae* [18] and *Rhodotorula glutinis* [19] proposed another approach to measure the actual PMP by following the changes of the *λ*max of the fluorescence spectrum of this monitor as an indicator, which has been also used by other authors [20, 23]. Within the claims to actually measure the plasma membrane potential in yeast, some values

internal concentrations of around 300 mM against micromolar external concentrations of the cation. Results are hard to rationalize, even considering that one part of the cation redistributes into the vacuole [24] and another is neutralized by the accumulation of bicarbonate when glucose is the substrate [25]. The use of fluorescent probes not only for the estimation of plasma membrane potential but also for many other purposes was partially reviewed by Slavik [26], and more recently, for yeast cells [16, 19]. However, this topic continues to be unsolved, and it is our belief that several aspects should be considered. The problems to estimate and measure the PMP in yeast imply a large series of factors that may affect results, such

(3) with this same purpose with

, which can reach

through its accumulation. Other authors [17] also used DiOC<sup>6</sup>

**2. The quantitative measurement of the PMP**

PMP in yeast.

6 Old Yeasts - New Questions

DiSC<sup>3</sup>

Using starved cells, the addition of a substrate, usually glucose, is required in order to generate the PMP [14]. Most probes appear to enter the cells by free diffusion. However, ethidium bromide, at least at certain concentrations, seems to be transported into the cell through the K+ transport system [27]. Monitors are generally cationic, with a delocalized electron structure that nonetheless does not eliminate their positive charge. In general, anionic molecules do not seem to enter the cells [28]. The main relevant characteristic to the topic of this review is that cationic molecules employed seem to be driven inside by the plasma membrane electric potential difference, and because of this, they can be used to follow changes of the membrane potential under varied conditions [16, 18, 19, 21, 22].

#### **3.3. The internal distribution**

Once the dyes enter the cells, they are not uniformly distributed inside due to the negative inside membrane electric potential difference of the mitochondria. Experiments show [16] that in fact the changes observed in the dyes' fluorescence within yeast cells mainly when using ethidium bromide, DiSC<sup>3</sup> (3), and acridine yellow [15] depend on the addition of a substrate, generally glucose. When this is added (a) a slow increase of fluorescence is seen, then after a few seconds that is followed by another small increase, coincident with the exhaustion of oxygen that can be reversed by the addition of a small concentration of H2 O2 , (b) if then a low concentration of an uncoupler around 5–15 μM is added, such as CCCP or FCCP, a much larger increase of fluorescence is observed. Finally, (c) when a concentration of KCl enough to be transported inside is added, a decrease of the fluorescence is seen. One additional observation with at least two dyes, DiSC<sup>3</sup> (3) [15, 16] and acridine yellow is that they under none of these conditions enter the vacuole. **Figure 2** shows the results of one experiment in which these fluorescence changes were observed with acridine yellow but are similar to those observed with DISC<sup>3</sup> (3) or ethidium bromide.

#### **3.4. Interpretation of results**

Results shown in **Figure 2** are interpreted as follows: in the presence of a substrate necessary to energize the cells, the dye is transported inside, driven by the PMP. Once inside, it is also taken by the mitochondria, where a large accumulation takes place, resulting in quenching of most of the fluorescence. This observation is supported by the small fluorescence increase when oxygen in the medium is exhausted by respiration that can be reversed by the addition of H2 O2 . When oxygen is exhausted, a partial deenergization of mitochondria occurs. Both these changes are small because even in the absence of oxygen, mitochondria are energized by the ATP produced in glycolysis. Adding then a low concentration (5–15 μM) of CCCP or FCCP depolarizes mitochondria, producing the efflux of the dye from the organelles and its uniform distribution in the cytoplasm, resulting in a large fluorescence increase (dequenching). After this, K+ addition, since the ion must be taken up through a PMP-driven transport partially neutralizing it, produces a large decrease of the fluorescence. Another important result in this respect is that the addition of a similar concentration of NaCl does not produce the fluorescence decrease resulting from the addition of KCl because Na<sup>+</sup> affinity for the transporter is much lower than that of K+ (not shown).

#### **3.5. Binding to the internal cell components**

One important characteristic of the monitors employed is the low fluorescence they show only in the buffered medium, which is largely increased by their interaction with the internal components of the cell [14, 15]. This characteristic is actually the basis for the fluorescencebased studies of the PMP and its changes under the different conditions. However, knowing the amount bound to the internal components of the cells becomes one of the main problems when trying to measure the actual values of the PMP by their accumulation.

#### **4. Evidence from microscope images**

Additional evidence is provided by looking at the cells under the microscope. **Figure 3** shows the images obtained with acridine yellow in a similar experiment to one already reported [15]. It can be clearly seen that in the presence of glucose alone the dye is concentrated in the mitochondria, but fluorescence is rather faint, which confirms that in fact, most of the dye is accumulated by these organelles; its fluorescence is quenched because of its large accumulation (**Figure 3A**). Then, particularly with acridine yellow, upon the addition of a low concentration of an uncoupler (10 μM CCCP), fluorescence of the dye increases remarkably and gets uniformly distributed in the cytoplasm (**Figure 3B**). The subsequent addition of KCl results in a general decrease of the fluorescence (**Figure 3C**). It is important to point out that the dye does not enter the vacuole.

The Plasma Membrane Electric Potential in Yeast: Probes, Results, Problems, and Solutions:... http://dx.doi.org/10.5772/intechopen.70403 9

**Figure 3.** Microscopic images following fluorescence changes of acridine yellow in yeast as a result of differences in PMP. In (A), the cells were incubated in 10 mM MES-TEA, pH 6.0, 20 mM glucose and 50 μM acridine yellow and observed 5 min later. Then, 10 μM CCCP was added, and 5 min later, image B was obtained. Image C was obtained 5 min after adding 10 mM KCl to the same preparation of image B. Similar figure to that of [15].

#### **5. The problems and solutions tested**

which these fluorescence changes were observed with acridine yellow but are similar to those

Results shown in **Figure 2** are interpreted as follows: in the presence of a substrate necessary to energize the cells, the dye is transported inside, driven by the PMP. Once inside, it is also taken by the mitochondria, where a large accumulation takes place, resulting in quenching of most of the fluorescence. This observation is supported by the small fluorescence increase when oxygen in the medium is exhausted by respiration that can be reversed by the addition

these changes are small because even in the absence of oxygen, mitochondria are energized by the ATP produced in glycolysis. Adding then a low concentration (5–15 μM) of CCCP or FCCP depolarizes mitochondria, producing the efflux of the dye from the organelles and its uniform distribution in the cytoplasm, resulting in a large fluorescence increase (dequench-

partially neutralizing it, produces a large decrease of the fluorescence. Another important result in this respect is that the addition of a similar concentration of NaCl does not produce

One important characteristic of the monitors employed is the low fluorescence they show only in the buffered medium, which is largely increased by their interaction with the internal components of the cell [14, 15]. This characteristic is actually the basis for the fluorescencebased studies of the PMP and its changes under the different conditions. However, knowing the amount bound to the internal components of the cells becomes one of the main problems

Additional evidence is provided by looking at the cells under the microscope. **Figure 3** shows the images obtained with acridine yellow in a similar experiment to one already reported [15]. It can be clearly seen that in the presence of glucose alone the dye is concentrated in the mitochondria, but fluorescence is rather faint, which confirms that in fact, most of the dye is accumulated by these organelles; its fluorescence is quenched because of its large accumulation (**Figure 3A**). Then, particularly with acridine yellow, upon the addition of a low concentration of an uncoupler (10 μM CCCP), fluorescence of the dye increases remarkably and gets uniformly distributed in the cytoplasm (**Figure 3B**). The subsequent addition of KCl results in a general decrease of the fluorescence (**Figure 3C**). It is important to point out that the dye

(not shown).

the fluorescence decrease resulting from the addition of KCl because Na<sup>+</sup>

when trying to measure the actual values of the PMP by their accumulation.

. When oxygen is exhausted, a partial deenergization of mitochondria occurs. Both

addition, since the ion must be taken up through a PMP-driven transport

affinity for the trans-

(3) or ethidium bromide.

observed with DISC<sup>3</sup>

8 Old Yeasts - New Questions

of H2 O2

ing). After this, K+

porter is much lower than that of K+

**3.5. Binding to the internal cell components**

**4. Evidence from microscope images**

does not enter the vacuole.

**3.4. Interpretation of results**

The general procedure to follow the fluorescence changes is as follows. The cells are added to an adequate buffer, and as a substrate, 20 mM glucose is used. A 2.0 mL final volume of the medium is important to ensure the effective continuous mixing of the incubation mixture. Adequate settings of the instrument regarding high voltage applied to the photomultiplier and the slit width should be chosen to get an adequate level of the fluorescence signal and its changes. We use an SLM Aminco spectrofluorometer with a cell holder provided with continuous magnetic stirrer and a constant temperature system. Different factors affecting the values obtained are the following:

*Buffer*. Salts that may be taken up by the yeast cells such as phosphate, sodium, or potassium salts should not be used to avoid their interference with the functioning of the cells. We prefer 10 mM MES (morpholinoethanesulfonic acid) taken to pH 6.0 with triehanolamine.

*Amounts of cells and dye*. With different monitors, different amounts of cells and dye concentrations should be assayed to find those in which the best tracings are obtained.

*External binding*. When fluorescence is followed incubating the cells in the presence of an adequate buffer, even in the absence of a substrate, an increase of fluorescence is observed, which appears to be due to the simple binding of the dyes to the negative external charges of the membrane. To avoid this, we started using a low concentration of CaCl<sup>2</sup> [14], but later realized that it was better to use a low concentration of 10–20 μM BaCl<sup>2</sup> [16].

*Accumulation in the mitochondria*. Uncouplers in yeast, at least *S. cerevisiae*, show an interesting behavior. They produce a very clear stimulation of respiration at concentrations around 2–5 μM, while to inhibit K+ transport, a process dependent on PMP, several times higher concentrations are required [16, 27]. This indicates that low concentrations can uncouple the mitochondria, without affecting the PMP. The solution then is to use low concentrations (5–15 μM) of either CCCP or FCCP to eliminate the accumulation of the dyes by the mitochondria.

*Binding to the internal components*. This factor is impossible to eliminate, and it is the actual basis of the analysis of the PMP by fluorescence, since as already mentioned, the monitors used in these studies show very low fluorescence values in water and require their interaction with the internal components of the cells. However, as it will be seen ahead, binding values can be measured.

#### **6. Conclusions from the fluorescence changes**

Changes of fluorescence of different monitors are an excellent way to follow not only the qualitative variations of the PMP in yeast, depending on the dye and conditions used. This has been shown with ethidium bromide [13, 27]. Regarding the claims that follow the displacement of the maximal fluorescence, peaks can provide the way to determine the actual value of the PMP and should be taken with caution [19]. Two reasons allow to affirm this (a) as mentioned before, the values obtained are too low to explain the large accumulation of K+ of around 300 mM inside the cells in the presence of micromolar concentrations outside, even considering that part of it is within the vacuole [24], or partially neutralized by the simultaneous accumulation of bicarbonate [16], and (b) although the authors state that the PMP can be fully collapsed by the addition of 10–20 μM of the usual uncouplers, CCCP or FCCP, results show that the concentrations required to stimulate respiration are 5–10 μM and those to inhibit K+ uptake are more than 20 μM [16]. Moreover, this anomalous incapacity of uncouplers to freely transport H+ through the yeast plasma membrane has been used to measure the internal pH of yeast cells by following the distribution of 2,4-dinitrophenol [2, 29]. When using acridine yellow [15], the fluorescence changes observed when deenergizing the mitochondria are particularly large. This means that this dye may be particularly useful to monitor the changes of the electric potential difference of mitochondria.

#### **7. Actual measurement of the PMP by the probe accumulation**

Initial attempts to measure the PMP based on the accumulation of cationic agents [6, 7] were unreliable because of the slow entrance of the cationic agents used and the apparently incomplete equilibrium reached between the inside and outside of the cells. Previous calculations with the simple accumulation of different dyes, either ethidium bromide [13], or DiSC<sup>3</sup> (3) [14, 16], gave results that appeared too high, and the variations under the different conditions too small. As already pointed out, more recently [15], acridine yellow was found to require higher concentrations than DiSC<sup>3</sup> (3) to observe the fluorescence changes usually described (**Figure 2**). Its behavior was also similar to that reported for other indicators when observed under the microscope (**Figure 3**). In summary, its activity was typical of that observed with other dyes, such as ethidium bromide or DiSC<sup>3</sup> (3). One difference is that while DiSC<sup>3</sup> (3) had to be used at nM concentrations, this dye required a concentration of 50 or even 150 μM to clearly observe changes in its accumulation.

#### **7.1. The PMP measured by the accumulation of acridine yellow**

concentrations are required [16, 27]. This indicates that low concentrations can uncouple the mitochondria, without affecting the PMP. The solution then is to use low concentrations (5–15 μM) of either CCCP or FCCP to eliminate the accumulation of the dyes by the

*Binding to the internal components*. This factor is impossible to eliminate, and it is the actual basis of the analysis of the PMP by fluorescence, since as already mentioned, the monitors used in these studies show very low fluorescence values in water and require their interaction with the internal components of the cells. However, as it will be seen ahead, binding values can be

Changes of fluorescence of different monitors are an excellent way to follow not only the qualitative variations of the PMP in yeast, depending on the dye and conditions used. This has been shown with ethidium bromide [13, 27]. Regarding the claims that follow the displacement of the maximal fluorescence, peaks can provide the way to determine the actual value of the PMP and should be taken with caution [19]. Two reasons allow to affirm this (a) as mentioned before, the values obtained are too low to explain the large accumulation of K+ of around 300 mM inside the cells in the presence of micromolar concentrations outside, even considering that part of it is within the vacuole [24], or partially neutralized by the simultaneous accumulation of bicarbonate [16], and (b) although the authors state that the PMP can be fully collapsed by the addition of 10–20 μM of the usual uncouplers, CCCP or FCCP, results show that the concentrations required to stimulate respiration are 5–10 μM and those

uptake are more than 20 μM [16]. Moreover, this anomalous incapacity of uncou-

the internal pH of yeast cells by following the distribution of 2,4-dinitrophenol [2, 29]. When using acridine yellow [15], the fluorescence changes observed when deenergizing the mitochondria are particularly large. This means that this dye may be particularly useful to monitor

Initial attempts to measure the PMP based on the accumulation of cationic agents [6, 7] were unreliable because of the slow entrance of the cationic agents used and the apparently incomplete equilibrium reached between the inside and outside of the cells. Previous calculations

16], gave results that appeared too high, and the variations under the different conditions too small. As already pointed out, more recently [15], acridine yellow was found to require

(**Figure 2**). Its behavior was also similar to that reported for other indicators when observed under the microscope (**Figure 3**). In summary, its activity was typical of that observed with

with the simple accumulation of different dyes, either ethidium bromide [13], or DiSC<sup>3</sup>

through the yeast plasma membrane has been used to measure

(3) to observe the fluorescence changes usually described

(3) [14,

**6. Conclusions from the fluorescence changes**

the changes of the electric potential difference of mitochondria.

**7. Actual measurement of the PMP by the probe accumulation**

mitochondria.

10 Old Yeasts - New Questions

measured.

to inhibit K+

plers to freely transport H+

higher concentrations than DiSC<sup>3</sup>

Following a similar procedure to that used with other dyes, its accumulation was measured to calculate the PMP under different conditions. In these experiments, from the amount of dye remaining in the supernatant after centrifuging the cells, we could calculate its internal amount, and from the value of the internal water content, its internal concentration, which allowed to calculate the apparent noncorrected Δψ values that are shown in **Table 1**.

These results were already encouraging and different to those obtained before with DiSC<sup>3</sup> (3) [16], but we still had to consider that at least part of the dye was not free, but bound to the internal components of the cells. Values were also interesting regarding their magnitude and reproducibility. With glucose alone, the highest accumulation was observed present in both the cytoplasm and the mitochondria. CCCP addition produced a decrease because the dye was no longer concentrated in the mitochondria, reaching a new equilibrium with the external concentration. As expected, adding 5 mM KCl, which should at least partially collapse the PMP, produced another large decrease. It is also important to emphasize that the addition of NaCl produced only a small change.

#### **7.2. Binding of the dye inside the cells, a possible solution**

Considering that we had no way to produce the efflux of the dye bound to the internal components of the cell, we decided to use another approach, trying to measure it. Chitosan, a cationic polymer mostly composed of glucosamine is very effective to permeabilize the plasma membrane of *Candida albicans* [30], but actually this effect was found before on *S. cerevisiae* (unpublished). We therefore used the cells incubated with glucose plus CCCP and added to


Experiments were conducted using the typical incubation mixture with 10 mM MES-TEA buffer, pH 6.0, 10 μM BaCl<sup>2</sup> , and 20 mM glucose, with 50 μM acridine yellow and 250 mg of cells (wet weight) in a final volume of 10.0 mL.Absorbance spectra of the samples, and the mean readings between 409 and 411 nm were obtained. From these readings and the linear part of a standard curve, the external concentrations were calculated. Accumulated dye was measured 5 min after the indicated successive additions by determining the concentration, and from it the amount of dye in the supernatant after centrifuging the cells. The internal amount of dye was obtained by subtracting that amount from that originally added. Its concentration was calculated considering the value of internal water of yeast, which has been measured and equivalent to 0.47 mL g−1, wet weight (33). The values were obtained in each case 5 min after the successive addition of (a) cells; (b) CCCP (10 μM), and (c) either KCl or NaCl (5 mM). Δψ was calculated from the Nernst equation, considering the log of the quotient of the internal/external concentrations.

**Table 1.** Raw calculations of Δψ from the measured accumulation of acridine yellow in yeast.

them 100 μg of chitosan. After centrifuging the cells, it was possible to calculate the amount of the dye leaking out, and of course, that remaining inside bound to the internal components. In this way the amount of dye that remained in the cells after the addition of chitosan could be subtracted from that remaining under the different conditions. Results of one typical of those experiments are the following:

Chitosan produces the permeabilization of the plasma membrane of the cells, producing the efflux of the dye, but still part of it remains inside, bound by its cationic nature. This is an interesting approach to subtract the contribution of the internal binding of the dye in the calculations of Δψ, at least in this yeast. However, still one problem exists: the values of the dye remaining inside the cells when KCl was added were found to be lower than those obtained with chitosan. This may be because K+ may produce an additional displacement of the also cationic dye from its binding sites.

#### **7.3. The exclusion of the dye from the vacuole**

Results from the accumulation of the dye considered a uniform distribution inside the cells, whose total water content has been measured and estimated at 47% of water per g of wet weight [31]. Microscope images show that after the uncoupler, the dye is no longer in the mitochondria and distributes in the cytoplasm, but it is absent from the vacuole [15]. This implies that the distribution volume of the dye is smaller than that in the total cell water. Considering this, the internal water in which the dye distributes is not the total value of 0.47 mL g−1 [31], but 0.355 mL g−1 of cytoplasmic water, excluding the vacuole. Using this new volume, the values shown in **Table 2** were obtained, indicating a still higher value of the PMP, shown in parenthesis in **Table 2**.


The experiment was performed as described in **Table 1**, but where indicated, 100 μg of chitosan was included, and the last sample was taken 5 min later. The amount of the dye that had not entered the cells, subtracted from the total added, allowed to get the amount that entered the cells. To obtain the internal concentration of the dye, this latter amount was divided by the internal water content of the cells, which was estimated before for this yeast strain of 0.47 mL g−1 of cells (33). From the internal and external concentrations, the quotient and its log were obtained, and using the Nernst equation, Δψ was calculated. Values in parenthesis were obtained considering that if the dye does not enter the vacuole, the total water in which the dye was distributed was only 0.355 mL g−1. Question marks indicate that the values were not calculated because the efflux of the dye with KCl was larger than that with chitosan.

**Table 2.** Values of the accumulation of acridine yellow obtained in a representative experiment under different conditions.

#### **8. Concluding remarks**

them 100 μg of chitosan. After centrifuging the cells, it was possible to calculate the amount of the dye leaking out, and of course, that remaining inside bound to the internal components. In this way the amount of dye that remained in the cells after the addition of chitosan could be subtracted from that remaining under the different conditions. Results of one typical of those

Chitosan produces the permeabilization of the plasma membrane of the cells, producing the efflux of the dye, but still part of it remains inside, bound by its cationic nature. This is an interesting approach to subtract the contribution of the internal binding of the dye in the calculations of Δψ, at least in this yeast. However, still one problem exists: the values of the dye remaining inside the cells when KCl was added were found to be lower than those obtained

Results from the accumulation of the dye considered a uniform distribution inside the cells, whose total water content has been measured and estimated at 47% of water per g of wet weight [31]. Microscope images show that after the uncoupler, the dye is no longer in the mitochondria and distributes in the cytoplasm, but it is absent from the vacuole [15]. This implies that the distribution volume of the dye is smaller than that in the total cell water. Considering this, the internal water in which the dye distributes is not the total value of 0.47 mL g−1 [31], but 0.355 mL g−1 of cytoplasmic water, excluding the vacuole. Using this new volume, the values shown in **Table 2** were obtained, indicating a still higher value of the PMP, shown in

Control 1953 4.1 476 2.67 −161 CCCP 1244 20.8 60 1.77 −107 KCl 284 64.4 4.4 0.64 −39 NaCl 1023 25.9 39 1.60 −96

Control 1562 4.1 380 2.6 −155 (211) CCCP 853 20.8 41 1.6 −97 (196) KCl 0.7 43.3 ? ? ?

The experiment was performed as described in **Table 1**, but where indicated, 100 μg of chitosan was included, and the last sample was taken 5 min later. The amount of the dye that had not entered the cells, subtracted from the total added, allowed to get the amount that entered the cells. To obtain the internal concentration of the dye, this latter amount was divided by the internal water content of the cells, which was estimated before for this yeast strain of 0.47 mL g−1 of cells (33). From the internal and external concentrations, the quotient and its log were obtained, and using the Nernst equation, Δψ was calculated. Values in parenthesis were obtained considering that if the dye does not enter the vacuole, the total water in which the dye was distributed was only 0.355 mL g−1. Question marks indicate that the values were not

**Table 2.** Values of the accumulation of acridine yellow obtained in a representative experiment under different conditions.

may produce an additional displacement of the also

**[Int] nmoles/mL [Ext] nmoles/mL Ratio Log mV**

experiments are the following:

12 Old Yeasts - New Questions

with chitosan. This may be because K+

**7.3. The exclusion of the dye from the vacuole**

Chitosan 391 40.4

calculated because the efflux of the dye with KCl was larger than that with chitosan.

cationic dye from its binding sites.

parenthesis in **Table 2**.

**Correction for internal binding**

Our work is a constant attempt to clarify a long time controversy, first about the estimation of the fluorescence changes of different monitors with different methods. We could add to the list acridine yellow, long known, but also quite inexpensive, which can be used to that purpose. With this dye larger concentrations are required, which may be probably due to the fact that it has a lower binding affinity to the internal components of the cells. This can be inferred already by comparing the results of its uncorrected uptake values as shown in **Table 3**.

With these corrections, larger differences under the conditions tested could be obtained with acridine yellow, as compared to those obtained with other agents, but much higher than those reported by other authors [19]. The simple accumulation in the presence of glucose already resulted in values lower than those reported before. Then, the addition of CCCP produced an apparent large decrease of the calculated PMP, because of the release of the large accumulation by the mitochondria and then outside the cells. Interesting also are the lower values obtained in the presence of K+ , whose uptake should lower the PMP.

From previous and recent work, we can summarize the basic conditions needed to obtain reliable results, such as the buffer used, which must not contain cations or organic molecules that may interfere or modify the PMP of the cells. When using different monitors, different conditions should be tested, mainly the concentration of the dye to adjust it depending on the yeast used. Changes due to binding to the surface of the cells can be minimized by the addition of a low concentration (10 μM) BaCl<sup>2</sup> . The accumulation of the monitor by mitochondria can be avoided by the addition of around 10 μM CCCP or FCCP. Finally, corrections can be applied by using the correct volumes for the distribution of the dye, as well as its efflux with a permeabilizing agent.

There is another factor influencing the results obtained. The efflux of the dye produced by chitosan was lower than that found after the addition of K+ . When the dye concentration after the addition of the monovalent cation is subtracted from that obtained after the addition of chitosan, a negative net accumulation results, meaning that the cells would have a positive PMP value, which is hard to accept. The most probable explanation is that when chitosan is present, the dye goes out of the cells, leaving inside that bound, and the dye binds inside because of its hydrophobic and also its cationic nature. So the addition of K+ not only reduces the PMP but also produces the liberation of the dye particularly from its internal binding due


**Table 3.** Comparison of PMP values (in mV) obtained with different dyes.

to its cationic nature. The conclusion then is that, although the accumulation of acridine yellow provides an adequate method to measure the PMP of yeast, the values obtained after the addition of positively charged ions that accumulate in large concentrations within the cell are distorted because of the displacement of the dye from what most probably are anionic sites inside the cell. It is possible that part of the K<sup>+</sup> taken up by the cells may be bound to their negative internal components. If this were so, one would expect acridine yellow to produce an efflux of K<sup>+</sup> . However, in other experiments (unpublished) we have found that the dye at concentrations of 60 and 120 μM, higher than those used in those reported here does not produce the efflux of the monovalent cation. It must also be considered that although the uptake of K+ is expected to decrease the PMP, the values obtained after the addition of this cation are too low. It has to be considered that after its addition, and because of the decrease of the PMP, this results in the stimulation of the plasma membrane H+ -ATPase, originating a transient increase of ADP, that is then compensated by the acceleration of glycolysis [32], all of which must at least partially restore the PMP values. In this sense, it appears that results following the accumulation of DiSC<sup>3</sup> (3) are more in agreement with these facts.

### **Appendix**

In more recent experiments (unpublished), in order to correct as much as possible the values of the binding of the dye to the internal cell components, we incubated the cells under the same conditions, always in the presence of 10 μM CCCP to avoid its accumulation by the mitochondria. Previously, we used 50 μM acridine yellow. When incubation was performed with glucose alone, practically all of the dye was taken up by the cells and made difficult to distinguish between the dye bound inside and that taken up driven by the PMP. Because of this, in these experiments we used the dye at a 150 μM concentration. The conditions were the following:


The Plasma Membrane Electric Potential in Yeast: Probes, Results, Problems, and Solutions:... http://dx.doi.org/10.5772/intechopen.70403 15


The experiment was conducted as described in **Table 1**, but to those treated with 100 μg of chitosan, after 10 min, 200 mM KCl was added, and after 5 more min, they were centrifuged. Also, in these experiments, a higher acridine yellow concentration (150 μM) was used. Results from a typical experiment.

**Table 4.** Accumulation values of acridine yellow in yeast cells and calculations to obtain the apparent values of the PMP.

The experiment was conducted as described for **Table 1**, but to those treated with 100 μg of chitosan, after 10 min, 200 mM KCl was added, and after 5 more min, they were centrifuged. Also, in these experiments, a higher acridine yellow concentration (150 μM) was used, and in all cases, 10 μM CCCP was present. Results from a typical experiment.

The accumulation values using 150 μM acridine yellow concentration were much higher, similar to previous experiments, and values of the PMP without any corrections were −219 and −169 mV, respectively, for the cells incubated only with glucose and with glucose plus 10 mM KCl.

From the values obtained with chitosan, we found that a total concentration of 50,554 nmoles/ mL still remained inside the cells, independent from the PMP, presumably bound, both because of the cationic and hydrophobic nature of acridine yellow. The internal concentration, 50,554, was reduced to 20,585 by the addition of 200 mM KCl. This amount remaining in the cells after the addition of chitosan and KCl is that bound due to its hydrophobic nature. The difference (50,554 − 20,585) can be considered that bound because of its cationic nature and is equal to 29,269. Al values are given in nmoles/mL.

These results then, allow the following corrections:

**1.** With glucose.

to its cationic nature. The conclusion then is that, although the accumulation of acridine yellow provides an adequate method to measure the PMP of yeast, the values obtained after the addition of positively charged ions that accumulate in large concentrations within the cell are distorted because of the displacement of the dye from what most probably are anionic sites

negative internal components. If this were so, one would expect acridine yellow to produce

concentrations of 60 and 120 μM, higher than those used in those reported here does not produce the efflux of the monovalent cation. It must also be considered that although the uptake

increase of ADP, that is then compensated by the acceleration of glycolysis [32], all of which must at least partially restore the PMP values. In this sense, it appears that results following

(3) are more in agreement with these facts.

In more recent experiments (unpublished), in order to correct as much as possible the values of the binding of the dye to the internal cell components, we incubated the cells under the same conditions, always in the presence of 10 μM CCCP to avoid its accumulation by the mitochondria. Previously, we used 50 μM acridine yellow. When incubation was performed with glucose alone, practically all of the dye was taken up by the cells and made difficult to distinguish between the dye bound inside and that taken up driven by the PMP. Because of this, in these experiments we used the dye at a 150 μM concentration. The conditions were

**A.** Cells with glucose in which the dye is taken up and accumulated by the cells due partly to

**B.** Cells incubated first with glucose for 10 min, adding then 10 mM KCl, in which a large efflux of the dye is observed due in part to the decrease of the PMP, but also to a large K<sup>+</sup> accumulation, around 200–300 mM, that produces its liberation from the anionic sites of

**C.** Cells permeabilized with 100 μg of chitosan for the total 3.0 mL of the incubation mixture.

**D.** Cells with glucose and with the same concentration of chitosan, but after their permeabilization adding 200 mM KCl. This concentration should displace the dye from the anionic sites to which it supposedly binds because of its cationic nature, but requires a large K+ concentration to be displaced. Then, the remaining dye inside is that due to its hydropho-

the cell. Not considering that this results in values lower than real for the PMP.

the PMP but also to its binding to their internal components.

Chitosan liberates the free dye and that bound remains inside.

 is expected to decrease the PMP, the values obtained after the addition of this cation are too low. It has to be considered that after its addition, and because of the decrease of the PMP,

. However, in other experiments (unpublished) we have found that the dye at

taken up by the cells may be bound to their


inside the cell. It is possible that part of the K<sup>+</sup>

this results in the stimulation of the plasma membrane H+

an efflux of K<sup>+</sup>

14 Old Yeasts - New Questions

**Appendix**

the following:

bicity (**Table 4**).

the accumulation of DiSC<sup>3</sup>

of K+


actual internal concentration of KCl plus that displaced would be 85,541 nmoles/mL. Then, the internal/external concentration ratio would be 1006, and the PMP would change to −180.2.

With these corrections the values obtained in three experiments (means ± std. dev) were −219.5 ± 1.8, with glucose, and −163.3 ± 1.4 with glucose plus 10 mM KCl. The values corrected for the binding due to the cationic nature of the dye were −205.3 ± 3.2 with glucose, and −183.2 ± 3.1 for glucose plus KCl.

As expected, values with glucose, when corrected for the amount of dye bound because of its cationic nature, are somewhat higher than those shown in **Table 1**. The value with glucose plus KCl is much higher, also as expected, because to the amount of dye remaining inside, that displaced by the large accumulation of K+ was added.

These results confirm that the PMP values obtained are higher than those suggested by other authors. In fact, only with glucose, corrected values are only around 15 mV higher; in the presence of K+ , the values are even higher. This in fact is not unexpected, because, although the addition of K+ , due to its transport mechanism should produce a decrease of the PMP, since our old studies [2, 32] it is known that K+ , by decreasing the PMP, accelerates the plasma membrane H+ -ATPase, which transiently increases the ADP levels, but this increase is rapidly compensated by accelerating glycolysis and respiration. This series of events, but mainly the acceleration of proton pumping, should compensate for the PMP decrease produced by the uptake of K+ . To our knowledge, these measurements with the shown corrections are the most accurate measurements of the PMP in *S. cerevisiae*.

### **Author details**

Antonio Peña\*, Norma Silvia Sánchez and Martha Calahorra

\*Address all correspondence to: apd@ifc.unam.mx

Department of Molecular Genetics, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Mexico City, Mexico

#### **References**


[4] Gaber RF, Styles CA, Fink GR. TRK1 encodes a plasma membrane protein required for high-affinity potassium transport in *Saccharomyces cerevisiae*. Molecular and Cellular Biology. 1988;**8**:2848-2858. DOI: 10.1128/MCB.8.7.2848

actual internal concentration of KCl plus that displaced would be 85,541 nmoles/mL. Then, the internal/external concentration ratio would be 1006, and the PMP would change to −180.2.

With these corrections the values obtained in three experiments (means ± std. dev) were −219.5 ± 1.8, with glucose, and −163.3 ± 1.4 with glucose plus 10 mM KCl. The values corrected for the binding due to the cationic nature of the dye were −205.3 ± 3.2 with glucose, and −183.2

As expected, values with glucose, when corrected for the amount of dye bound because of its cationic nature, are somewhat higher than those shown in **Table 1**. The value with glucose plus KCl is much higher, also as expected, because to the amount of dye remaining inside,

These results confirm that the PMP values obtained are higher than those suggested by other authors. In fact, only with glucose, corrected values are only around 15 mV higher; in the

compensated by accelerating glycolysis and respiration. This series of events, but mainly the acceleration of proton pumping, should compensate for the PMP decrease produced by the

Department of Molecular Genetics, Instituto de Fisiología Celular, Universidad Nacional

[1] Conway E, Brady TG, Carton E. Biological production of acid and alkali. II. A redox theory for the process in yeast with application to the production of gastric acidity. The

[2] Peña A, Cinco G, Gómez-Puyou A, Tuena M. Effect of the pH of the incubation medium on glycolysis and respiration in *Saccharomyces cerevisiae*. Archives of Biochemistry and

Biophysics. 1972;**153**:413-425. DOI: doi.org/10.1016/0003-9861(72)90359-1

Biophysics. 1975;**167**:397-409. DOI: doi.org/10.1016/0003-9861(75)90480-4

was added.

, the values are even higher. This in fact is not unexpected, because, although


. To our knowledge, these measurements with the shown corrections are the most

, due to its transport mechanism should produce a decrease of the PMP,

, by decreasing the PMP, accelerates the plasma

transport in yeast. Archives of Biochemistry and

± 3.1 for glucose plus KCl.

16 Old Yeasts - New Questions

presence of K+

membrane H+

uptake of K+

**Author details**

**References**

the addition of K+

that displaced by the large accumulation of K+

since our old studies [2, 32] it is known that K+

accurate measurements of the PMP in *S. cerevisiae*.

\*Address all correspondence to: apd@ifc.unam.mx

Autónoma de México, Mexico City, Mexico

[3] Peña A. Studies on the mechanism of K+

Antonio Peña\*, Norma Silvia Sánchez and Martha Calahorra

Biochemical Journal. 1950;**47**:369-374. PMID:14800895


[30] Peña A, Sánchez NS, Calahorra M. Effects of chitosan on *Candida albicans*. Conditions for its antifungal activity. BioMed Research International. 2013;**2013**:1-15. DOI: 10.1155/ 2013/527549

[17] Madrid R, Gómez MJ, Ramos J, Rodríguez-Navarro A. Ectopic potassium uptake in *trk1 trk2* mutants of *Saccharomyces cerevisiae* correlates with a highly hyperpolarized membrane potential. The Journal of Biological Chemistry. 1998;**273**:14838-14844. DOI:

[18] Gaskova D, Brodska B, Herman P, Vecer J, Malinsky J, Sigler K, Benada O, Plasek J. Fluorescent probing of membrane potential in walled cells: diS-C3(3) assay in

[19] Plášek J, Gášková D, Lichtenberg-Fraté H, Ludwig J, Höfer M. Monitoring of real changes of plasma membrane potential by diS-C(3)(3) fluorescence in yeast cell suspensions. Journal of Bioenergetics and Biomembranes. 2012;**44**:559-69. DOI: 10.1007/

and plasma-membrane potential control in *Saccharomyces cerevisiae*. Folia Microbiologia

ences the plasma membrane potential of *Saccharomyces cerevisiae*. FEMS Yeast Research.

[22] Maresova L, Muend S, Zhang Y-Q, Sychrova H, Rao R. Membrane hyperpolarization drives cation influx and fungicidal activity of amiodarone. The Journal of Biological

[23] Petrezselyova S, Zahradka J, Sychrova H. *Saccharomyces cerevisiae* BY4741 and W303-1A laboratory strains differ in salt tolerance. Fungal Biology. 2010;**114**:144-150. DOI: doi.

[24] Montiel V, Ramos J. Intracellular Na and K distribution in *Debaryomyces hansenii*. Cloning and expression in *Saccharomyces cerevisiae* of DhNHX1. FEMS Yeast Research. 2007;**7**:102-109.

[25] Peña A, Sánchez NS, Álvarez H, Calahorra M, Ramírez J. Effects of high medium pH on growth, metabolism and transport in *Saccharomyces cerevisiae*. FEMS Yeast Research.

[26] Slavik J. Fluorescent Probes in Cellular and Molecular Biology. CRC Press, Inc. Boca

[27] Peña A and Ramírez G. Interaction of ethidium bromide with the transport system for monovalent cations in yeast. The Journal of Membrane Biology. 1975;**22**:369-384. DOI:

[28] Peña A, Carrasco N, Mora MA. Uptake and effects of several cationic dyes on yeast. The

[29] Kotyk A. Intracellular pH of Baker's yeast. Folia Microbiologia (Praha). 1963;**8**:27-31.

Journal of Membrane Biology. 1979;**47**:261-284. DOI: 10.1007/BF01869081

supply

antiporter influ-

, K+ /H+

[20] Petrezsélyová S, Ramos J, Sychrová H. Trk2 transporter is a relevant player in K<sup>+</sup>

*Saccharomyces cerevisiae*. Yeast. 1998;**14:**1189-1197. PMID: 9791890

(Praha). 2011;56:23-28. DOI: 10.1007/s12223-011-0009-1

2006;**6**:792-800. DOI: 10.1111/j.1567-1364.2006.00062.x

org/10.1016/j.funbio.2009.11.002

DOI:10.1111/j.1567-1364.2006.00115.x

2015;**15**(2). DOI: 10.1093/femsyr/fou005

Raton, Ann Arbor, London, Tokyo. 1994

10.1007/BF01868181

DOI: 10.1007/BF02868762

[21] Kinclova-Zimmermannova O, Gaskova D, Sychrova H. The Na+

Chemistry. 2009;**284**:2795-2802. DOI: 10.1074/jbc.M806693200

10.1074/jbc.273.24.14838

18 Old Yeasts - New Questions

s10863-012-9458-8


**Provisional chapter**

#### **Metallothioneins,** *Saccharomyces cerevisiae***, and Heavy Metals: A Biotechnology Triad? Metals: A Biotechnology Triad?**

**Metallothioneins,** *Saccharomyces cerevisiae***, and Heavy** 

DOI: 10.5772/intechopen.70340

Ileana Cornelia Farcasanu and Lavinia Liliana Ruta Ileana Cornelia Farcasanu and Lavinia Liliana Ruta Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.70340

#### **Abstract**

Metal ions are the least sophisticated chemical species that interact or bind to biomolecules. The yeast Saccharomyces cerevisiae represents a versatile model organisms used in both basic and applicative research, and one of the main contributors to the understanding of the molecular mechanisms involved in the transport, accumulation, and homeostasis of heavy metals. With a negatively charged wall, the yeast cells are very good biosorbents for heavy metals. In addition to biosorption, the metabolically active cells take up heavy metals via the normal membrane transport systems. Once in the cell, the toxicity of the heavy metals is controlled by various mechanisms, including sequestration by metal-binding proteins, such as the metallothioneins. Metallothioneins are cysteine-rich proteins involved in the buffering of excess heavy metals, both essential (Cu and Zn) and nonessential (Cd, Ag, and Hg). S. cerevisiae has two innate metallothioneins, Cup1 and Crs5, intensively investigated. Additionally, S. cerevisiae served as a host for the heterologous expression of a variety of metallothioneins from different species. This review focuses on the technological implications of expressing metallothioneins in yeast and on the possibility to use these transgenic cells in heavy metal-related biotechnologies: bioremediation, recovery of rare metals, or obtaining clonable tags for protein imaging.

**Keywords:** metallothionein, *Saccharomyces cerevisiae*, heavy metal, bioremediation

#### **1. Introduction**

Biotechnology, which makes use of living organisms for technological purposes, is one of the applied fields that constantly benefited from the rapid advancements made in understanding life at molecular level. It is undoubtedly that the budding yeast *Saccharomyces cerevisiae* is one of the biotechnology's most versatile tools. Used since ancient times in bakery, brewery, and

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

of the biotechnology's most versatile tools. Used since ancient times in bakery, brewery, and wine making, the advent of molecular biology brought new glitter to this eukaryotic microorganism, and *S. cerevisiae* was practically reinvented. With an easy-to-manipulate genomics, with elegant and simple genetics, hosting molecules and biochemical processes well conserved along evolution, this microorganism served as a model organism for the discovery and understanding of numerous essential life mechanisms. Apart from playing an unique and undisputed role in basic research of life science, *S. cerevisiae* kept on adding value to its role in biotechnology, especially due to its capacity to heterologously express proteins, with various scopes: large-scale production of recombinant proteins of technological significance (enzymes, antibodies, and hormones); selection of strains with targeted characteristics and performances (metabolic engineering); environmental biotechnology (yeast surface display); and scientific reasons, such as elucidating the role of proteins from various organisms organism (yeast two-hybrid technique, expression of homologous counterparts from higher organisms in yeast, design yeast as model for human diseases, etc.) [1].

Metallothioneins (MTs) represent one of the numerous examples of proteins whose functions were investigated by heterologous expression in *S. cerevisiae* cells. MTs are low-molecular weight proteins that exist in most organisms from bacteria to humans, including yeasts [2]. MTs constitute an extremely heterogeneous family of cysteine-rich proteins (close to 30% of their amino acid content) that give rise to metal-thiolate complexes ensuing on metal ion coordination. MTs are considered to have many roles, being involved in protection against toxic metals, homeostasis/chaperoning of physiological metals, free-radical scavenging and antioxidative stress protection, control of oxidative state of the cell, antiapoptotic defense, etc. While some roles still remain obscure, it is widely accepted that all MTs have an undisputed capacity to buffer intracellular metal ions, especially Zn(II) and Cu(I) [3].

Heavy metals belong to a group of nondegradable chemicals naturally present in the environment. Numerous anthropogenic activities, especially the ones related to massive industrialization, intensive agriculture, or rapid urbanization led to important perturbations (accumulation, or in some cases, depletion) in heavy metal balance, with ecological, nutritional, and environmental impacts [4–10]. Some of the heavy metals (Co, Cu, Fe, Mn, Ni, and Zn) are essential for life in trace amounts, playing a pivotal role in the structure of enzymes and other proteins. Other heavy metals (Cd, Sb, Cr, Pb, As, Co, Ag, Se, and Hg) albeit not essential, interfere easily with the metabolism of essential heavy metals, competing for the various physiological transport systems as well as for the biomolecules they bind to. Essential or not, when present in high concentrations, heavy metals are strongly deleterious to living organisms due to nonspecific binding to proteins, often inducing oxidative stress, or disrupting biological membranes. Defense mechanisms against nonphysiological concentrations of heavy metal ions include excretion, compartmentalization in cell organelles, or increased synthesis of metal-buffering molecules, such as the MTs.

*S. cerevisiae* has been thoroughly investigated, and many mechanism involved in heavy metal transport and homeostasis have been elucidated in this organism [11–16], preparing the grounds for development of techniques used to engineer *S. cerevisiae* cells for increased heavy metal accumulation and improved tolerance. The present review focuses on the studies that relate heterologous expression of MTs in *S. cerevisiae* to the metal-binding characteristics observed and to the possibility to use them for biotechnological purposes.

#### **2. Innate and heterologous expression of MTs in** *S. cerevisiae*

of the biotechnology's most versatile tools. Used since ancient times in bakery, brewery, and wine making, the advent of molecular biology brought new glitter to this eukaryotic microorganism, and *S. cerevisiae* was practically reinvented. With an easy-to-manipulate genomics, with elegant and simple genetics, hosting molecules and biochemical processes well conserved along evolution, this microorganism served as a model organism for the discovery and understanding of numerous essential life mechanisms. Apart from playing an unique and undisputed role in basic research of life science, *S. cerevisiae* kept on adding value to its role in biotechnology, especially due to its capacity to heterologously express proteins, with various scopes: large-scale production of recombinant proteins of technological significance (enzymes, antibodies, and hormones); selection of strains with targeted characteristics and performances (metabolic engineering); environmental biotechnology (yeast surface display); and scientific reasons, such as elucidating the role of proteins from various organisms organism (yeast two-hybrid technique, expression of homologous counterparts from higher organ-

Metallothioneins (MTs) represent one of the numerous examples of proteins whose functions were investigated by heterologous expression in *S. cerevisiae* cells. MTs are low-molecular weight proteins that exist in most organisms from bacteria to humans, including yeasts [2]. MTs constitute an extremely heterogeneous family of cysteine-rich proteins (close to 30% of their amino acid content) that give rise to metal-thiolate complexes ensuing on metal ion coordination. MTs are considered to have many roles, being involved in protection against toxic metals, homeostasis/chaperoning of physiological metals, free-radical scavenging and antioxidative stress protection, control of oxidative state of the cell, antiapoptotic defense, etc. While some roles still remain obscure, it is widely accepted that all MTs have an undisputed capacity to buffer intracellular metal ions, especially Zn(II) and

Heavy metals belong to a group of nondegradable chemicals naturally present in the environment. Numerous anthropogenic activities, especially the ones related to massive industrialization, intensive agriculture, or rapid urbanization led to important perturbations (accumulation, or in some cases, depletion) in heavy metal balance, with ecological, nutritional, and environmental impacts [4–10]. Some of the heavy metals (Co, Cu, Fe, Mn, Ni, and Zn) are essential for life in trace amounts, playing a pivotal role in the structure of enzymes and other proteins. Other heavy metals (Cd, Sb, Cr, Pb, As, Co, Ag, Se, and Hg) albeit not essential, interfere easily with the metabolism of essential heavy metals, competing for the various physiological transport systems as well as for the biomolecules they bind to. Essential or not, when present in high concentrations, heavy metals are strongly deleterious to living organisms due to nonspecific binding to proteins, often inducing oxidative stress, or disrupting biological membranes. Defense mechanisms against nonphysiological concentrations of heavy metal ions include excretion, compartmentalization in cell organelles, or increased synthesis of metal-buffering

*S. cerevisiae* has been thoroughly investigated, and many mechanism involved in heavy metal transport and homeostasis have been elucidated in this organism [11–16], preparing

isms in yeast, design yeast as model for human diseases, etc.) [1].

Cu(I) [3].

22 Old Yeasts - New Questions

molecules, such as the MTs.

Apart from being classified on the basis of their structural homology or on taxonomic criteria, MTs are also classified on molecular functionality grounds, starting from their innate metalbinding abilities, into Cu(I)- and Zn(II)-thioneins, with the representative nonessential models Ag(I) and Cd(II), respectively [3, 17]. This is based on the formation of homometallic MT species when they are produced in metal-enriched media; this classification is not regarded as absolute, since cross-affinity is often noticed for Zn(II)-thioneins binding Cu(I) and vice versa [3].

*S. cerevisiae* has two structurally different MTs, Cup1, and Crs5. Cup1 has been classified as the strictest Cu(I)-isoform (genuine Cu(I)-thionein) [3]. Cup1 biosynthesis is copper-activated at transcriptional level via the copper-binding transcription factor Ace1/Cup2 [18–21] providing the principal method of cellular removal and sequestering the extremely toxic Cu(I) [21–23]. Although substantially divergent from vertebrate and plant MTs, the arrangement of 12 cysteine residues, which is a hallmark of metal-binding proteins, is partially conserved. In contrast to the MTs from higher eukaryotes, Cup1 is responsible only for Cu(I) and Cd(II) ion tolerance *in vivo* [24, 25], albeit capable of binding other metal ions *in vitro* [26]. This is in contrast to the MTs found in higher eukaryotes, which are typically capable of detoxifying an array of metal ions.

Considered a secondary copper-resistant agent in *S. cerevisiae*, Crs5 is nonhomologous to the paradigmatic Cup1, determining survival under Zn(II) overload in a *CUP1*-null background. Its overexpression prevents the deleterious effects exhibited on the *cup1Δ crs5Δ* double knock-out mutant by exposure to combined Zn(II)/Cu(II), similarly to mouse MT1 Zn-thionein, but not to Cup1. Numerous similar observations denoted that Crs5 has a dual metal-binding behavior, being significantly closer to Zn(II)-thioneins than to Cu(I)-thioneins [23, 27, 28].

Following the discovery and characterization of Cup1, many newly discovered MTs were characterized by heterologous expression in *S. cerevisiae* (**Table 1**).

In plants, the first evidence for the role of MTs in Cu(II) and Cd(II) tolerance was provided by expressing two *Arabidopsis thaliana* MT genes in MT-deficient yeast cells. For example, when expressed in *cup1Δ* knock-out mutant, both *At*MT1 and *At*MT2 complemented the *cup1Δ* mutation by providing a high level of resistance to CuSO4 and moderate resistance to CdSO4 [29]. Later, all four types of plant MTs were checked as metal chelators by expressing *A. thaliana* MT cDNAs (*At*MT1a, *At*MT2a, *At*MT2b, *At*MT3, *At*MT4a, and *At*MT4b) in the Cu(II) and



**Table 1.** Heterologous expression of MTs in *S. cerevisiae*.

**MT expressed Source organism Behavior in** *S. cerevisiae* **Reference**

Complement Cu(II) and Cd(II) sensitivity of a *cup1Δ* mutant; *At*MT4a, and *At*MT4b conferred greater Zn(II) tolerance and higher accumulation of Zn(II) than other MTs to the *zrc1Δ* 

Complements Cu(II) and Cd(II) sensitivity of a *cup1* mutant

Complement Cu (II) (all), Cd(II) (*Ha*MT4-1), and Zn(II) (*Ha*MT3,

*Nc*MT1, and to a lesser extent *Nc*MT2 complement Cu(II), Cd (II), and Zn(II)

Restore Cd(II) and Cu(II) tolerance to

, FeCl2

O2

Tolerance to salinity, alkaline conditions, and oxidative stress

Complements Cu(II) and Cd(II) sensitivity of a *cup1* mutant

Complement Cu(II) and Cd(II) sensitivity of *cup1* and *yap1* mutants

Zn(II), Fe(II), Fe(III), Cd(II), Cr(VI), and Ag(I); sensitivity to Mn(II), Co(II),

Tolerance to Cd(II), Zn(II), Cu(II), and NaCl stresses; increased accumulation of Cd(II), Zn(II), NaCl, but not of

O2 , and

, NaCl, NaHCO3

, NaCl, NaHCO3

*Ha*MT4-1) sensitivity

yeast sensitive strains

sensitivity

ethanol

and H2 O2

Cu(II)

surplus CuCl2

Tolerance to H2

Cu(II), Ni(II)

[29, 30]

[31]

[32, 33]

[35]

[36, 37]

[38]

[40]

[41]

[42]

[43]

[44]

[45]

,

,

*cot1Δ* mutant

*Arabidopsis thaliana*

nonaccumulator

production

*Nc*MT1, *Nc*MT2 *Noccaea* (*Thlaspi*) *caerulescens*

*Sv*MT2b, *Sv*MT3 *Silene vulgaris* (Moench) Garcke

*Put*MT2 *Puccinellia tenuiflora*

*Cv*MT1 *Chloris virgata* Swartz

*Th*MT3 *Tamarix hispida*

*Gint*MT1 *Glomus intraradices*

*Hc*MT1 and *Hc*MT2 *Hebeloma cylindrosporum*

plants

*Helianthus annuus*

Pb(II), and Hg(II)

Technical plant: biofuel

Technical plant: nutritional oil. Seeds tend to accumulate Cd(II),

Cd(II)/Zn(II) hyperaccumulator

*Os*MT1-1b *Oryza sativa* Confers tolerance to Cd(II), H2

*rg*MT *Oryza sativa* Confers vigorous growth under

*Os*MT1a *Oryza sativa* L. cv. Iapar 9 Confers tolerance to Zn(II) [39]

Cu(II)-hypertolerant plant

Alkaline/saline tolerant grass

Alkaline/saline tolerant plant

Arbuscular mycorrhizal fungus; confers heavy metal tolerance to

Ectomycorrhizal fungus; confers heavy metal tolerance to exposed

Alkaline tolerant grass

exposed plants

*Jc*MT2a *Jatropha curcas* L.

Model plant organisms, metal

*At*MT1a, *At*MT2a, *At*MT2b, *At*MT3, *At*MT4a, and *At*MT4b

24 Old Yeasts - New Questions

*Ha*MT1, *Ha*MT2, *Ha*MT3, *Ha*MT4

> Zn(II)-sensitive yeast mutants, *cup1Δ* and *zrc1Δ cot1Δ*, respectively. All four types of *At*MTs provided similar levels of Cu(II) tolerance and accumulation to the *cup1Δ* mutant, while the type-4 *At*MTs (*At*MT4a and *At*MT4b) conferred greater Zn(II) tolerance and higher Zn(II) accumulation to the *zrc1Δ cot1Δ* mutant [30]. Metal-gained tolerance was also tested in yeast mutants expressing MTs from technical plants. Thus, the Cu(II) and Cd(II) sensitivity of yeast mutants was complemented by expression of MT2a isolated from *Jatropha curcas* L., a technical plant used for biofuel production [31]. In a different study, expression of MTs from *Helianthus annuus* (sunflower) overcame the Cu(II), Zn(II), or Cd(II) sensitivity, depending on the MT type expressed ([32], **Table 1**). Along with high nutritional value and significant oil content, the seeds of *H. annuus* tend to accumulate Cd(II), Pb(II), and Hg(II) [33], and *Ha*MTs are major candidates to be one of the determinants for the high metal accumulation properties of this plant.

> Other MTs studied in yeast were isolated from heavy metal hypertolerant or hyperaccumulating plants. Hyperaccumulating plants belong to a small group of species capable of growing on metalliferous soils without developing toxicity symptoms [34]. The MTs from the intensively studied hyperaccumulator *Noccaea* (*Thlaspi*) *caerulescens* were expressed in yeast, and it was revealed that *Nc*MT1, and to a lesser extent *Nc*MT2, complemented the Cu(II), Cd(II), and Zn(II) sensitive phenotypes [35]. The *Silene vulgaris* (Moench) Garcke population with high levels of copper tolerance was shown to owe this hypertolerance to increased transcripts of *SvMT2b* gene; expression of *Sv*MT2b in yeast restored Cd(II) and Cu(II) tolerance in different hypersensitive strains [36]. In a different study, *Sv*MT3, whose gene has been locally duplicated in a tandem arrangement in *S. vulgaris* genome was shown to restore the Cu(II) tolerance along with increased Cu(II) accumulation in a Cu(II)-sensitive yeast mutant, and that both duplicated genes were functional [37].

Expression of plant MTs in *S. cerevisiae* cells sometimes determined other MTs-related phenotypes, besides metal tolerance and accumulation, indicating that heterologous MTs can be fully functional in yeast cells. Thus a heterologous expression in *S. cerevisiae* of *Os*MTI-1b, a MT isoform from *Oryza sativa* (rice), enhanced Cd(II), H2 O2, and ethanol tolerance [38], while *Os*MT-1a from a Brazilian variety of rice conferred Zn(II) tolerance [39]; *rg*MT from the same species conferred vigorous growth to transgenic yeast cells when exposed to surplus CuCl2 , FeCl2 , NaCl (salinity), NaHCO3 (alkalinity), or H2 O2 (exogenous oxidative stress) [40]. Encompassing a wider range of stresses, expression of *Put*MT2 from the saline/alkaline grass *Puccinellia tenuiflora* increased the tolerance of transgenic yeast cells to H2 O2 , NaCl, NaHCO3 , and also to a series of metal ions: Zn(II), Fe(II), Fe(III), Cd(II), Cr(VI), and Ag(I), while conferring sensitivity to Mn(II), Co(II), Cu(II), and Ni(II) [41]. Expression of *Cv*MT1 from the alkaline grass *Chloris virgata* Swartz significantly increased the yeast cell tolerance to salinity, alkaline conditions, and oxidative stress [42]. In the same line of studies, *Th*MT3 isolated from the alkaline/saline-resistant plant *Tamarix hispida* conferred the transgenic yeast cells increase tolerance to Cd(II), Zn(II), Cu(II), and NaCl stresses, triggering increased accumulation of Cd(II), Zn(II), NaCl, but not Cu(II) [43].

Often, plants acquire heavy metal tolerance when growing on contaminated sites due to symbiosis with the radicular, arbuscular mycorrhizal fungi that penetrate the cortical cells of the roots of a vascular plant; one MT isolated from such fungus, *Glomus intraradices*, was also shown to complement the Cu(II) and Cd(II) sensitivity of a *cup1* mutant [44], while MT1 and MT2 from the ectomycorrhizal fungi *Hebeloma cylindrosporum* and *Paxillus involutus* functionally complemented the Cu(II) and Cd(II) sensitivity of yeast mutants [45, 46].

Studies on animal MTs expressed in yeast are less numerous [33, 47, 48, 72] and are used mainly for technical purposes. One notable example though is mouse *Mm*MT1a, a canonical Zn(II)-thionein (yeast Cup1 is considered a canonical Cu(I)-thionein) [3] shown to confer tolerance when expressed in Zn(II)-sensitive yeast mutants [31]. *S. cerevisiae* was also used to express human MTs, but mainly as a host for large-scale production of hMTs [49–51], for which the more productive methylotrophic yeast *Pichia pastoris* is currently preferred [52].

#### **3. Biotechnological relevance of MTs expression in** *S. cerevisiae*

The main function of MTs resides in their structure: small proteins with a significant number of cysteine residues (15–30% of the total amino acid number) [53], a characteristic that confers them a remarkable capacity to bind heavy metal ions by forming metal-thiolate clusters. MTs are natively bound to Cu(I) or Zn(II), exhibiting various affinities for the two metals, in between the canonical Cu(I)-thionein (*S. cerevisiae* Cup1) and canonical Zn(II)-thionein (*C. elegans* MT1) [3]. Ag(I) and Cd(II) can be used as respective models of Cu(II) and Zn(II) for the study of the metal-binding sites of MTs, particularly in those techniques that require isotopically active nuclei (note that copper is in the cuprous form Cu(I) when bound to MT, but the environment contains the more stable cupric ion Cu(II); when taken up by the cell, Cu(II) can be reduced to Cu(I) by Fe(III)/Cu(II)-reductases, or simply by the reductive milieu of the cell).

With high thermodynamic stability combined with kinetic lability, MTs are important candidates for biotechnology applications. In the nonmetalate form, MTs are highly reactive and can virtually bind to any d10 metal [53], a trait that makes them interesting candidates for biotechnology. In this case, two aspects of MT reactivity are highly relevant: (1) metal uptake and release and (2) metal exchange [54]. Due to the polydentate thiolate nature of all MTs and their high affinity for most heavy metal ions, there are data available for binding of Cu(I), Cu(II), Cd(II), Hg(II), Ag(I), Au(I), Bi(III), As(III), Co(II), Fe(II), Pb(II), Pt(II), and Tc(IV) [55]. Another important feature of MT reactivity is the dynamic behavior, with metal uptake and release between species of different degrees of metalation. It is widely accepted that the binding of metal ions to MTs occurs rapidly, between 10 and 30 min, although longer stabilization times are required for certain ions, such as Hg(II) or Pb(II) [3].

Studies on metal exchange in MTs have also been done (usually with either Zn(II)- or Cu(I) thioneins), starting with a metal-loaded MT forced to exchange its initially bound metal ions with other ions. Considering the series of affinity order of heavy metal ions for the thiolate ligands: Fe(II) ≈ Zn(II) ≈ Co(II) < Pb(II) < Cd(II) < Cu(I) < Au(I) ≈ Ag(I) < Hg(II) < Bi(III) [56], the Zn(II)-loaded MTs would be more reactive than Cu(I)-loaded MTs. It was noted that metal exchange occurs at a much slower pace than metal binding to apo-MTs. For example, it was revealed that binding of four equivalents of Cu(I) to Zn(II)-Cup1 required a stabilization time of 24 h to produce a mixture of Cu4-Cup1 and Cu8-Cup1 species by total displacement of the initially bound Zn(II) [3, 57]. It is interesting to note that many xenobiotic metal ions (Cd(II), Pb(II), and Hg(II)) show higher affinity for thiolate ligands than Zn(II) or Cu(I) does, and thus, in case of intoxication, MTs can work as detoxifying agents [53]. This is highly relevant especially when designing a biotechnology system aimed for removal of toxic ions, as in the case of bioremediation. In the following paragraph, studies on metal accumulation by MTs expressing *S. cerevisiae* cells are presented, and also summarized in **Table 2**.

#### **3.1. Display of MTs on the surface of** *S. cerevisiae* **cells**

Expression of plant MTs in *S. cerevisiae* cells sometimes determined other MTs-related phenotypes, besides metal tolerance and accumulation, indicating that heterologous MTs can be fully functional in yeast cells. Thus a heterologous expression in *S. cerevisiae* of *Os*MTI-1b,

while *Os*MT-1a from a Brazilian variety of rice conferred Zn(II) tolerance [39]; *rg*MT from the same species conferred vigorous growth to transgenic yeast cells when exposed to surplus

(alkalinity), or H2

Encompassing a wider range of stresses, expression of *Put*MT2 from the saline/alkaline grass

and also to a series of metal ions: Zn(II), Fe(II), Fe(III), Cd(II), Cr(VI), and Ag(I), while conferring sensitivity to Mn(II), Co(II), Cu(II), and Ni(II) [41]. Expression of *Cv*MT1 from the alkaline grass *Chloris virgata* Swartz significantly increased the yeast cell tolerance to salinity, alkaline conditions, and oxidative stress [42]. In the same line of studies, *Th*MT3 isolated from the alkaline/saline-resistant plant *Tamarix hispida* conferred the transgenic yeast cells increase tolerance to Cd(II), Zn(II), Cu(II), and NaCl stresses, triggering increased accumulation of

Often, plants acquire heavy metal tolerance when growing on contaminated sites due to symbiosis with the radicular, arbuscular mycorrhizal fungi that penetrate the cortical cells of the roots of a vascular plant; one MT isolated from such fungus, *Glomus intraradices*, was also shown to complement the Cu(II) and Cd(II) sensitivity of a *cup1* mutant [44], while MT1 and MT2 from the ectomycorrhizal fungi *Hebeloma cylindrosporum* and *Paxillus involutus* function-

Studies on animal MTs expressed in yeast are less numerous [33, 47, 48, 72] and are used mainly for technical purposes. One notable example though is mouse *Mm*MT1a, a canonical Zn(II)-thionein (yeast Cup1 is considered a canonical Cu(I)-thionein) [3] shown to confer tolerance when expressed in Zn(II)-sensitive yeast mutants [31]. *S. cerevisiae* was also used to express human MTs, but mainly as a host for large-scale production of hMTs [49–51], for which the more productive methylotrophic yeast *Pichia pastoris* is currently preferred [52].

The main function of MTs resides in their structure: small proteins with a significant number of cysteine residues (15–30% of the total amino acid number) [53], a characteristic that confers them a remarkable capacity to bind heavy metal ions by forming metal-thiolate clusters. MTs are natively bound to Cu(I) or Zn(II), exhibiting various affinities for the two metals, in between the canonical Cu(I)-thionein (*S. cerevisiae* Cup1) and canonical Zn(II)-thionein (*C. elegans* MT1) [3]. Ag(I) and Cd(II) can be used as respective models of Cu(II) and Zn(II) for the study of the metal-binding sites of MTs, particularly in those techniques that require isotopically active nuclei (note that copper is in the cuprous form Cu(I) when bound to MT, but the environment contains the more stable cupric ion Cu(II); when taken up by the cell, Cu(II) can be reduced to Cu(I) by Fe(III)/Cu(II)-reductases, or simply by the reductive milieu of the cell).

O2

O2, and ethanol tolerance [38],

(exogenous oxidative stress) [40].

, NaCl, NaHCO3

,

O2

a MT isoform from *Oryza sativa* (rice), enhanced Cd(II), H2

*Puccinellia tenuiflora* increased the tolerance of transgenic yeast cells to H2

ally complemented the Cu(II) and Cd(II) sensitivity of yeast mutants [45, 46].

**3. Biotechnological relevance of MTs expression in** *S. cerevisiae*

, NaCl (salinity), NaHCO3

Cd(II), Zn(II), NaCl, but not Cu(II) [43].

CuCl2

, FeCl2

26 Old Yeasts - New Questions

Cell surface engineering has wide applicability due to the fact that virtually any protein can be produced and autoimmobilized on the cell exterior of an engineered cell (usually a microorganism). *S. cerevisiae* is suitable for this technique by which functional heterologous proteins/peptides can be displayed on cell surface by fusion with parts of cell wall- or cell membrane-anchoring proteins [58–62]. *S. cerevisiae*, generally regarded as safe (GRAS), is a more suitable host for cell surface engineering than other microorganisms in which the cell surface display system has been established, because yeast possesses a quality-control system for proteins and glycosylation systems of secreted proteins. In addition to the general advantages, high-molecular-mass proteins or proteins that require glycosylation modification can be displayed on yeast cell surface with maintenance of their activities, unlike when displaying them on bacteria [63]. Surface engineered cells can be subsequently treated as microparticles covered with the targeted protein [64].

*S. cerevisiae* cells are very good biosorbents for heavy metal ions due to the cell wall constituents, which readily sequester heavy metals once they encounter them. These constituents possess numerous metal-loving functional groups, including carboxylate, phosphate, sulfate, and


**Table 2.** MTs heterologously expressed in yeast that determine increased accumulation of metal ions.

sulfhydryl, which decorate the outer mannan-protein layer of the wall [65]. The metal-binding innate capacity of the cell wall can be substantially increased by expressing metal-binding peptides/proteins at the cell surface [59, 61]. Using this technique, yeast cells were modified for bioremediation of Cd(II) using a cell-surface display system of its own MT, Cup1, fused with a hexahistidyl residue, by using an α-agglutinin-based display system [66]. Surface-engineered yeast cells with Cup1 and hexa-His fused in tandem (Cup1-His6, originally named YMThexaHis) showed superior cell-surface adsorption and recovery of Cd(II) under EDTA treatment on the cell surface compared to the His6-displaying cells, through an additive effect on chelating ability. Remarkably, the expression of Cup1-His6 did not have a strong effect on the adsorption of Cu(II). The same study revealed that yeast cells displaying Cup1-His6 exhibited a higher potential for the adsorption of Cd(II) than *Escherichia coli* cells displaying the same constructs. Additionally, cells displaying tandem Cup1-His6 showed increased resistance to Cd(II) through active and enhanced adsorption of the toxic ion, indicating that Cup1-His6-displaying yeast cells are unique biosorbents with a superior functional chelating ability.

Adsorption of heavy metal ions at the cell surface has certain advantages compared to intracellular accumulation. First, surface adsorption allows recycling of the adsorbed ions, whereas intracellular accumulation necessitates disintegration of the cell for extraction. Second, surface adsorption is possible even in nonviable cells, providing that sufficient biomass can be produced. This is particularly important when cells are used to remove heavy metals from contaminated waters, and the conditions necessary to sustain living cells are difficult to achieve. And third, surface-engineered yeast cells can be used repeatedly as bioadsorbents since the recovery and treatment of the heavy metal ions does not greatly damage the cells [66]. In a sequel study, Cup1 was expressed as tandem head-to-tail repeats of the yeast MT lacking the first 8 amino acids (known to be nonsignificant for metal binding). Three types of constructs that were surface displayed contained 1, 4, and 8 tandem MT repeats [67].

The transgenic cells obtained were tested against excess Cd(II), and it was revealed that the adsorption and recovery of Cd(II) on the cell surface was increasingly enhanced with increasing the number of tandem repeats under conditions that allowed complete occupation of the Cd(II)-binding sites in the MT tandem repeats. Considering the relationship between cell-surface adsorption and protection against heavy metal ion toxicity, the tolerance of these surface-engineered yeasts to Cd(II) was found to be also dependent on the number of displayed MT tandem repeats, indicating that the characteristics of surface-engineered yeasts as a bioadsorbents correlated with the ability of the displayed proteins to bind metal ions [67]. Unfortunately, these promising studies soon came to a halt and no other metal ions or other MTs were taken into consideration to be used in this technique. It took ten years before another group displayed at the surface of yeast cells four type-2 MTs from *Solanum nigrum* (*Sn*MT): *Sn*MT2a, *Sn*MT2c, *Sn*MT2d, and *Sn*MT2e [68]. *S. nigrum* is an ornamental shrub (nightshade) and a Cd(II)/Zn(II) hyperaccumulator, apparently due to the four *Sn*MTs subtypes (*Sn*MT2a, *Sn*MT2c, *Sn*MT2d, and *Sn*MT2e) shown to have an important role in metal detoxification [69]. Yeast strains displaying the *Sn*MTs specified above on the cell surface were obtained, and these strains were shown to develop both Cd(II) tolerance and increased Cd(II) adsorption, exhibiting a higher affinity for Cd(II) than for Cu(II) or Hg(II) [68]. Notably, these displaying strains could effectively adsorb ultra-trace Cd(II) and accumulate it under a wide range of pHs (between 3 and 7), without disturbing the co-exising Cu(II) and Hg(II) [68]. Moreover, apart from showing a high potential for removing Cd(II) from contaminated waters, the yeastsurface engineered strains expressing *Sn*MT showed a remarkable resistance to Cd(II): while the nonengineered cells were stopped from dividing by 80 μM Cd(II), the engineered strains could live in 500 μM Cd(II) [68], a very high concentration for aqueous environments. While the study does not present accumulation data on other heavy metal ions, it is notable that the engineered strains expressing *Sn*MT could concentrate ultra-trace Cd(II) on the cell surface, encouraging further attempts to display other MTs on yeast surface (from hyperaccumulating species) with the final scope of concentrating rare metal ions from ultra-traces environments.

#### **3.2. MT-expressing** *S. cerevisiae* **cells for bioremediation**

sulfhydryl, which decorate the outer mannan-protein layer of the wall [65]. The metal-binding innate capacity of the cell wall can be substantially increased by expressing metal-binding peptides/proteins at the cell surface [59, 61]. Using this technique, yeast cells were modified for bioremediation of Cd(II) using a cell-surface display system of its own MT, Cup1, fused with a hexahistidyl residue, by using an α-agglutinin-based display system [66]. Surface-engineered yeast cells with Cup1 and hexa-His fused in tandem (Cup1-His6, originally named YMThexaHis) showed superior cell-surface adsorption and recovery of Cd(II) under EDTA treatment on the cell surface compared to the His6-displaying cells, through an additive effect on chelating ability. Remarkably, the expression of Cup1-His6 did not have a strong effect on the adsorption of Cu(II). The same study revealed that yeast cells displaying Cup1-His6 exhibited a higher potential for the adsorption of Cd(II) than *Escherichia coli* cells displaying the same constructs. Additionally, cells displaying tandem Cup1-His6 showed increased resistance to Cd(II) through active and enhanced adsorption of the toxic ion, indicating that Cup1-His6-displaying

**Expressed MT MT provenience Yeast gained characteristics due to** 

*Solanum nigrum* (Cd(II)/Zn(II) hyperaccumulator)

hyperaccumulator)

Cu(II) hMT2, GFP-hMT2 *Homo sapiens* Increased Cu(II) tolerance and capacity

Zn(II) *At*MT4a and *At*MT4b *Arabidopsis thaliana* Increased accumulation of Zn(II) [30]

Alkaline/saline tolerant plant

Alkaline/saline tolerant plant

**Table 2.** MTs heterologously expressed in yeast that determine increased accumulation of metal ions.

(Cd/Zn

**expression of MT**

adsorption; selectivity against Cu(II)

dependent on the number of tandem repeats; 4 and 8 repeats determined increased Cd(II) adsorption/recovery 5.9 and 8.9 times, respectively

Increased Cd(II) tolerance and adsorption; concentration of Cd(II) from ultra-trace media; selectivity to Cd(II) against Cu(II) and Hg(II)

Increased Cd(II) tolerance and

Increased Cd(II) tolerance and

Increased Zn(II) tolerance and

to remove Cu(II) when expressed from

accumulation

accumulation

accumulation

yeast *CUP1* promoter

*S. cerevisiae* Cd(II) tolerance, Cd(II) increased

*S. cerevisiae* Cd(II) tolerance and adsorption were

**Reference**

[66]

[67]

[69]

[76]

[43]

[72]

[43]

yeast cells are unique biosorbents with a superior functional chelating ability.

**Metal investigated**

Cd(II) Cup1/His6

28 Old Yeasts - New Questions

Cd(II) Δ1–8Cup1

Yeast surface display

*Sn*MT2d, and *Sn*MT2e Yeast surface display

Cd(II) *Th*MT3 *Tamarix hispida*

Zn(II) *Th*MT3 *Tamarix hispida*

Cd(II) *Sa*MT2 *Sedum alfredii* Hance

(Δ1–8Cup1)4 (Δ1–8Cup1)8 Surface display of tandem repeats of head-to-tail yeast MT lacking the first 8 amino acids

Cd(II) *Sn*MT2a*, Sn*MT2c,

Heavy metal bioremediation is an appealing approach for decontaminating polluted environments, especially because standard physico-chemical methods are ineffective and very often a source of pollution themselves [5]. An ideal heavy metal bioremediator would have certain metal-related characteristics: tolerance to high concentrations, increased accumulation, and substantial biomass production for effective removal of heavy metal ions from the contaminated sites. These traits fall into the characteristics of the heavy metal hyperaccumulating plants, with the exception that they usually do not produce sufficient biomass [70]. *S. cerevisiae* is an example of an organism that could be engineered for bioremediation purposes. The cell surface display of metal-binding peptides/proteins presented above may not be the best approach for obtaining hyperaccumulating yeasts, since the metal binding is restricted to cell surface. Rather, (over)expressing nontoxic metal-binding proteins within yeast cell may increase the chances of obtaining hyperaccumulating strains fit for bioremediation purposes. *S. cerevisiae* is not a heavy metal accumulator due to a very active excretion system which extrudes excess metal ions from the cell [71, 72]. However, the excess free ions could be retained within the cell in a nontoxic form through sequestration by an abundant metal-binding protein, such as an overexpressed MT. Recently, an increased Cu(II) bioremediation ability of new transgenic and adapted *S. cerevisiae* strains was described [73]. In this study, *S. cerevisiae* cells were manipulated to integrate human MT2 (hMT2) and GFP-hMT2, expressed from either the constitutively p*ADH1* yeast promoter or the Cu(II)-inducible p*CUP1* yeast promoter. It was shown that only cells that expressed hMT2 from the *CUP1* promoter exhibited both increased Cu(II) tolerance and capacity to remove Cu(II) ions from growth media [73].

#### **3.3. Heterologous expression of MTs from heavy metal hyperaccumulators**

The natural heavy metal hyperaccumulators, mostly belonging to a small group of plants [34, 70], are the species whose metal-related characteristics initially prompted the ideas of bioremediation, biomining, and bioextraction. To accumulate heavy metals without developing toxicity symptoms, these organisms utilize a variety of chemical ligands capable of coordinating the metal ions in a nontoxic form. Although MTs are important candidates for sequestering heavy metal ions, the studies relevant for correlating MT expression with heavy metal accumulating phenotype are scarce and hardly encouraging [74, 75]. The examples of MT from hyperaccumulating organisms expressed in yeast are few, and they mainly focus on functional complementation tests [33, 36–38, 69, 76]. One example is worth mentioning here though, as it deals with an unusual hyperaccumulating phenotype: Ag(I)-hyperaccumulation due to three distinct MT genes of the ectomycorrhizal fungus *Amanita strobiliformis* [77, 78]. Although expressed in *S. cerevisiae* only to test the restoration of Cd(II), Cu(II), and Zn(II) tolerance to sensitive mutants, further employment of *As*MT for cellular handling of Ag(I) is worth considering.

#### **3.4. Metallothionein as clonable tags**

Due to their small size and metal-binding capacity, metallothioneins may be interesting candidates for tagging proteins for imaging, especially by electron microscopy (EM) [79–81]. Localization of proteins in cells or complexes using EM relies upon the use of heavy metal clusters, which can be difficult to direct to sites of interest. For this reason, a metal-binding clonable tag, such as it is green fluorescent protein (GFP) for light microscopy, has been pursued for a long time, and would be unvaluable for imaging by EM techniques. In this respect, MT is a very good candidate, because instead of fluorescing like GFP, it would initiate formation of a heavy metal cluster adjacent to the protein to be analyzed. A suitable clonable tag for EM is expected to have certain properties: small size and low molecular weight, so as not to disrupt protein kinetics/function *in vivo*. Using MTs as clonable tag implies working on either macromolecular assemblies or cells, but avoiding issues of heavy metal toxicity by delaying the addition of metal until the samples that include a protein-MTH chimera are in preparation for EM. Two successful procedures: (1) adding heavy metal to sections of samples that have already been rapidly frozen, fixed by freeze substitution, and embedded in a hydrophilic plastic and (2) adding metal during the process of freeze substitution have been described [82]. Using *S cerevisiae* as an expression system, it was shown that MT can be localized in the complex environment of a cell, and with a very good signal-to-noise ratio [83]. Thus, mouse MT1 used to tag the yeast centrosomal protein Spc42 allowed the localization the MT-tagged Spc42 in the outer layer of the central plaque of the mature yeast spindle pole body. Nevertheless, although very promising, MT tagging for protein localization may not be universally applicable as this approach did not work with protein components of the nuclear pore complex [82]. Another potential use of MT as clonable tag for imaging would be the yellow luminescence observed for Cu(I)-MT complexes [84, 85].

#### **4. Concluding remarks**

substantial biomass production for effective removal of heavy metal ions from the contaminated sites. These traits fall into the characteristics of the heavy metal hyperaccumulating plants, with the exception that they usually do not produce sufficient biomass [70]. *S. cerevisiae* is an example of an organism that could be engineered for bioremediation purposes. The cell surface display of metal-binding peptides/proteins presented above may not be the best approach for obtaining hyperaccumulating yeasts, since the metal binding is restricted to cell surface. Rather, (over)expressing nontoxic metal-binding proteins within yeast cell may increase the chances of obtaining hyperaccumulating strains fit for bioremediation purposes. *S. cerevisiae* is not a heavy metal accumulator due to a very active excretion system which extrudes excess metal ions from the cell [71, 72]. However, the excess free ions could be retained within the cell in a nontoxic form through sequestration by an abundant metal-binding protein, such as an overexpressed MT. Recently, an increased Cu(II) bioremediation ability of new transgenic and adapted *S. cerevisiae* strains was described [73]. In this study, *S. cerevisiae* cells were manipulated to integrate human MT2 (hMT2) and GFP-hMT2, expressed from either the constitutively p*ADH1* yeast promoter or the Cu(II)-inducible p*CUP1* yeast promoter. It was shown that only cells that expressed hMT2 from the *CUP1* promoter exhibited both increased Cu(II) tolerance

and capacity to remove Cu(II) ions from growth media [73].

30 Old Yeasts - New Questions

lular handling of Ag(I) is worth considering.

**3.4. Metallothionein as clonable tags**

**3.3. Heterologous expression of MTs from heavy metal hyperaccumulators**

The natural heavy metal hyperaccumulators, mostly belonging to a small group of plants [34, 70], are the species whose metal-related characteristics initially prompted the ideas of bioremediation, biomining, and bioextraction. To accumulate heavy metals without developing toxicity symptoms, these organisms utilize a variety of chemical ligands capable of coordinating the metal ions in a nontoxic form. Although MTs are important candidates for sequestering heavy metal ions, the studies relevant for correlating MT expression with heavy metal accumulating phenotype are scarce and hardly encouraging [74, 75]. The examples of MT from hyperaccumulating organisms expressed in yeast are few, and they mainly focus on functional complementation tests [33, 36–38, 69, 76]. One example is worth mentioning here though, as it deals with an unusual hyperaccumulating phenotype: Ag(I)-hyperaccumulation due to three distinct MT genes of the ectomycorrhizal fungus *Amanita strobiliformis* [77, 78]. Although expressed in *S. cerevisiae* only to test the restoration of Cd(II), Cu(II), and Zn(II) tolerance to sensitive mutants, further employment of *As*MT for cel-

Due to their small size and metal-binding capacity, metallothioneins may be interesting candidates for tagging proteins for imaging, especially by electron microscopy (EM) [79–81]. Localization of proteins in cells or complexes using EM relies upon the use of heavy metal clusters, which can be difficult to direct to sites of interest. For this reason, a metal-binding clonable tag, such as it is green fluorescent protein (GFP) for light microscopy, has been pursued for a long time, and would be unvaluable for imaging by EM techniques. In this respect, MT is a very good candidate, because instead of fluorescing like GFP, it would initiate formation of a heavy metal cluster adjacent to the protein to be analyzed. A suitable clonable tag for EM is expected to have certain properties: small size and low molecular weight, so The numerous studies on MTs stand for the uniqueness of these small proteins whose undisputed trait is binding to heavy metal ions. This is evidently due to the cysteinyl residues, which represent more than 20% of the total number of MT amino acids, whereas the usual percentage of cysteinyl residues seldom surpasses 5% in most proteins. The intrinsic characteristic of sequestering metal ions in thiolate clusters make MTs very interesting biomolecules for various biotechnological application. Since *S. cerevisiae* represents a very good cellular system for heterologous expression of MTs from virtually any species (including itself), the use of MT-(over)expressing yeasts is a promising starting point for biotechniques such as heavy metal bioremediation and bioextraction. The data summarized in **Table 1** reveal that until now MT-expressing *S. cerevisiae* cells have been used for functional complementation studies (mainly MTs from plants and mycorrhizal fungi) rather than to investigate their biotechnological potential. Moreover, most of the studies concern the ions that naturally bind to MTs, Cu(I) and Zn(II), and their nonessential counterparts Ag(I) and Cd(II), along with few studies on sulfur-loving metal ions such as Hg(II) and Pb(II). Although other interesting noncanonical metal ions such as Mn(II), Ni(II), and Co(II) have been shown to strongly bind to MTs [55, 56], very few studies actually determined the MT binding to these ions *in vivo* (**Table 2**), and MT-expressing yeast cells would be very good models for filling this gap. Especially, obtaining heavy metal hyperaccumulating yeast cells by heterologous (over)expression of recombinant MTs would open new opportunities for bioremediation, bioextraction, and for emerging techniques, such as synthesis of clonable heavy metal nanoparticles [86]. Another promising biotechnique involving MTs is obtaining new clonable tags for cell imaging. While some timid progress has been reported on imaging by EM of proteins tagged with MT in yeast cells [82], the possibility to use the yellow luminescence of Cu(I)-MT for imaging MT-tagged proteins is largely unexplored. In this direction, construction of a systematic collection of *S. cerevisiae* strains that express *all* the MTs identified so far would be not only a challenge but also a prerequisite for systematic investigation of MTs for various biotechnology purposes.

#### **Acknowledgements**

The authors received funding from the Romanian—EEA Research Programme operated by the Ministry of National Education under the EEA Financial Mechanism 2009–2014 and Project Contract No 21 SEE/30.06.2014.

#### **Author details**

Ileana Cornelia Farcasanu\* and Lavinia Liliana Ruta

\*Address all correspondence to: ileana.farcasanu@chimie.unibuc.ro

Department of Organic Chemistry, Biochemistry and Catalysis, Faculty of Chemistry, Bucharest University, Romania

#### **References**


[9] Caito S, Aschner M. Neurotoxicity of metals. Handbook of Clinical Neurology. 2015;**131**:169-189. DOI: 10.1016/B978-0-444-62627-1.00011-1

**Acknowledgements**

32 Old Yeasts - New Questions

**Author details**

**References**

0827-2

07743-6

10.1016/j.temb.2005.02.010

10.15430/JCP.2015.20.4.232

Project Contract No 21 SEE/30.06.2014.

Bucharest University, Romania

978-3-527-64486-5

Ileana Cornelia Farcasanu\* and Lavinia Liliana Ruta

\*Address all correspondence to: ileana.farcasanu@chimie.unibuc.ro

The authors received funding from the Romanian—EEA Research Programme operated by the Ministry of National Education under the EEA Financial Mechanism 2009–2014 and

Department of Organic Chemistry, Biochemistry and Catalysis, Faculty of Chemistry,

[1] Feldmann H. Yeasts in biotechnology. In: Feldmann H, editor. Yeast: Molecular and Cell Biology. 2nd ed. Wiley-Blackwell, Weinheim, Germany; 2012. pp. 347-371. ISBN:

[2] Capdevila M, Atrian S. Metallothionein protein evolution: A mini assay. Journal of Biological Inorganic Chemistry. 2011;**16**:977-989. DOI: 10.1007/s00775-011-0798-3

[3] Palacios O, Atrian S, Capdevila M. Zn- and Cu-thioneins: A functional classification. Journal of Biological Inorganic Chemistry. 2011;**16**:991-1009. DOI: 10.1007/s00775-011-

[4] Prasad MNV, editor. Heavy Metal Stress in Plants. From Biomolecules to Ecosystems. Berlin, Heidelberg: Springer Berlin Heidelberg; 2004. p. 454. DOI: 10.1007/978-3-662-

[5] Bradl H. Heavy Metals in the Environment: Origin, Interaction and Remediation. Vol. 6.

[6] He ZL, Yang XE, Stoffella PJ. Trace elements in agroecosystems and impacts on the environment. Journal of Trace Elements in Medicine and Biology. 2005;**19**:125-140. DOI:

[7] Kim HS, Kim YJ, Seo YR. An overview of carcinogenic heavy metal: Molecular toxicity mechanism and prevention. Journal of Cancer Prevention. 2015;**20**:232-240. DOI:

[8] Jan AT, Azam M, Siddiqui K, Ali A, Choi I, Haq QM. Heavy metals and human health: Mechanistic insight into toxicity and counter defense system of antioxidants. International

Journal of Molecular Sciences. 2015;**16**:29592-29630. DOI: 10.3390/ijms161226183

1st ed. London: Academic Press; 2005. p. 282. ISBN: 9780120883813


Garcke populations from copper mines is associated with increased transcript levels of a 2b-type metallothionein gene. Plant Physiology. 2001;**126**:1519-1526. DOI: 10.1104/ pp.126.4.1519

[37] Nevrtalova E, Baloun J, Hudzieczek V, Cegan R, Vyskot B, Dolezel J, Safar J, Milde D, Hobza R. Expression response of duplicated metallothionein 3 gene to copper stress in *Silene vulgaris* ecotypes. Protoplasma. 2014;**251**:1427-1439. DOI: 10.1007/s00709-014-0644-x

[23] Jensen LT, Howard WR, Strain JJ, Winge DR, Culotta VC. Enhanced effectiveness of copper ion buffering by *CUP1* metallothionein compared with *CRS5* metallothionein in *Saccharomyces cerevisiae*. The Journal of Biological Chemistry. 1996;**271**:18514-18519. DOI:

[24] Ecker DJ, Butt TR, Sternberg EJ, Neeper MP, Debouck C, Gorman JA, Crooke ST. Yeast metallothionein function in metal ion detoxification. The Journal of Biological Chemistry.

[25] Jeyaprakash A, Welch JW, Fogel S. Multicopy *CUP1* plasmids enhance cadmium and copper resistance levels in yeast. Molecular & General Genetics. 1991;**225**:363-368 [26] Winge DR, Nielson KB, Gray WR, Hamer DH. Yeast metallothionein. Sequence and metal-binding properties. The Journal of Biological Chemistry. 1985;**260**:14464-14470 [27] Culotta VC, Howard WR, Liu XF. CRS5 encodes a metallothionein-like protein in *Saccharomyces cerevisiae*. The Journal of Biological Chemistry. 1994;**269**:25295-25302 [28] Pagani A, Villarreal L, Capdevila M, Atrian S. The *Saccharomyces cerevisiae* Crs5 Metallothionein metal-binding abilities and its role in the response to zinc overload.

Molecular Microbiology. 2007;**63**:256-269. DOI: 10.1111/j.1365-2958.2006.05510.x

*Arabidopsis*. Plant Cell. 1994;**6**:875-884. DOI: 10.1105/tpc.6.6.875

Physiology. 2008;**146**:1697-1706. DOI: 10.1104/pp.108.115782

2010;**61**:517-534. DOI: 10.1146/annurev-arplant-042809-112156

2005;**222**:716-729. DOI: 10.1007/s00425-005-0006-1

[29] Zhou J, Goldsbrough PB. Functional homologs of fungal metallothionein genes from

[30] Guo WJ, Meetam M, Goldsbrough PB. Examining the specific contributions of individual *Arabidopsis* metallothioneins to copper distribution and metal tolerance. Plant

[31] Mudalkar S, Golla R, Sengupta D, Ghatty S, Reddy AR. Molecular cloning and characterisation of metallothionein type 2a gene from *Jatropha curcas* L., a promising biofuel plant. Molecular Biology Reports. 2014;**41**:113-124. DOI: 10.1007/s11033-013-2843-5 [32] Tomas M, Pagani MA, Andreo CS, Capdevila M, Atrian S, Bofill R. Sunflower metallothionein family characterisation. Study of the Zn(II)- and Cd(II)-binding abilities of the HaMT1 and HaMT2 isoforms. Journal of Inorganic Biochemistry. 2015;**48**:35-48. DOI:

[33] Madejón P, Murillo JM, Marañón T, Cabrera F, Soriano MA. Trace element and nutrient accumulation in sunflower plants two years after the Aznalcóllar mine spill. The Science

[34] Krämer U. Metal hyperaccumulation in plants. Annual Review of Plant Biology.

[35] Roosens NH, Leplae R, Bernard C, Verbruggen N. Variations in plant metallothioneins: The heavy metal hyperaccumulator *Thlaspi caerulescens* as a study case. Planta.

[36] van Hoof NA, Hassinen VH, Hakvoort HW, Ballintijn KF, Schat H, Verkleij JA, Ernst WH, Karenlampi SO, Tervahauta AI. Enhanced copper tolerance in *Silene vulgaris* (Moench)

of the Total Environment. 2003;**307**:239-257. DOI: 10.1016/S0048-9697(02)00609-5

10.1074/jbc.271.31.18514

34 Old Yeasts - New Questions

1986;**261**:16895-168900

10.1016/j.jinorgbio.2015.02.016


[61] Liu Z, Ho SH, Hasunuma T, Chang JS, Ren NQ, Kondo A. Recent advances in yeast cell-surface display technologies for waste biorefineries. Bioresource Technology. 2016;**215**:324-333. DOI: 10.1016/j.biortech.2016.03.132

[48] Wang SH, Chang CY, Chen CF, Tam MF, Shih YH, Lin LY. Cloning of porcine neuron growth inhibitory factor (metallothionein III) cDNA and expression of the gene in *Saccharomyces cerevisiae*. Gene. 1997;**203**:189-197. DOI: 10.1016/S0378-1119(97)00513-1

[49] Cismowski MJ, Huang PC. Purification of mammalian metallothionein from recombi-

[50] Cismowski MJ, Huang PC. Effect of cysteine replacements at positions 13 and 50 on

[51] Cismowski MJ, Narula SS, Armitage IM, Chernaik ML, Huang PC. Mutation of invariant cysteines of mammalian metallothionein alters metal binding capacity, cadmium resistance, and 113Cd NMR spectrum. The Journal of Biological Chemistry.

[52] Wu Q, Li B, Wu F, Yang L, Li S, Li H, Wu D, Cui T, Tang D. High-level expression, efficient purification, and bioactivity of recombinant human metallothionein 3 (rhMT3) from methylotrophic yeast *Pichia pastoris*. Protein Expression and Purification. 2014;**101**:121-126.

[53] Capdevilaa M, Bofilla R, Palaciosa O, Atrianb S. State-of-the-art of metallothioneins at the beginning of the 21st century. Coordination Chemistry Reviews. 2012;**256**:46-62.

[54] Blindauer CA, Leszczyszyn OI. Metallothioneins: Unparalleled diversity in structures and functions for metal ion homeostasis and more. Natural Product Reports. 2010;27:720-

[55] Bell SG, Vallee BL. The metallothionein/thionein system: An oxidoreductive metabolic

[56] Vasak M. Metal removal and substitution in vertebrate and invertebrate metallothioneins. In: Riordan JF, Vallee BL, editors. Metallobiochemistry Part B, Metallothionein and Related Molecules. Vol. 205. 1st ed. San Diego: Academic Press; 1991. pp. 452-458.

[57] Orihuela R, Monteiro F, Pagani A, Capdevila M, Atrian S. Evidence of native metal-S(2-) metallothionein complexes confirmed by the analysis of Cup1 divalent-metal-ion binding

[58] Shibasaki S, Ueda M. Bioadsorption strategies with yeast molecular display technology.

[59] Li PS, Tao HC. Cell surface engineering of microorganisms towards adsorption of heavy metals. Critical Reviews in Microbiology. 2015;**41**:140-149. DOI: 10.3109/1040841X.2013.

[60] Cherf GM, Cochran JR. Applications of yeast surface display for protein engineering. Methods in Molecular Biology. 2015;**1319**:155-175. DOI: 10.1007/978-1-4939-2748-7\_8

properties. Chemistry. 2010;**16**:12363-12372. DOI: 10.1002/chem.201001125

Biocontrol Science. 2014;**19**:157-164. DOI: 10.4265/bio.19.157

zinc link. Chembiochem. 2009;**10**:55-62. DOI: 10.1002/cbic.200800511

nant systems. Methods in Enzymology. 1991;**205**:312-319

metallothionein structure. Biochemistry. 1991;**30**:6626-6632

1991;**266**:24390-24397

36 Old Yeasts - New Questions

DOI: 10.1016/j.pep.2014.06.009

DOI: 10.1016/j.ccr.2011.07.006

741. DOI: 10.1039/b906685n

ISBN: 9780121821067

813898


Environmental Science and Pollution Research International. 2016;**23**:19613-19625. DOI: 10.1007/s11356-016-7157-4


[85] McNulty M, Puljung M, Jefford G, Dubreuil RR. Evidence that a copper-metallothionein complex is responsible for fluorescence in acid-secreting cells of the *Drosophila* stomach. Cell and Tissue Research. 2001;**304**:383-389. DOI: 10.1007/s004410100371

Environmental Science and Pollution Research International. 2016;**23**:19613-19625. DOI:

[74] Küpper H, Götz B, Mijovilovich A, Küpper FC, Meyer-Klaucke W. Complexation and toxicity of copper in higher plants. I. Characterization of copper accumulation, speciation, and toxicity in *Crassula helmsii* as a new copper accumulator. Plant Physiology.

[75] Mijovilovich A, Leitenmaier B, Meyer-Klaucke W, Kroneck PMH, Goötz B, Küpper H. Complexation and toxicity of copper in higher plants. II. Different mechanisms for copper versus cadmium detoxification in the copper-sensitive cadmium/zinc hyperaccumulator *Thlaspi caerulescens* (Ganges ecotype). Plant Physiology. 2009;**151**:715-731. DOI:

[76] Zhang J, Zhang M, Tian S, Lu L, Shohag MJ, Yang X. Metallothionein 2 (SaMT2) from *Sedum alfredii* Hance confers increased Cd tolerance and accumulation in yeast and

[77] Beneš V, Hložková K, Matěnová M, Borovička J, Kotrba P. Accumulation of Ag and Cu in *Amanita strobiliformis* and characterization of its Cu and Ag uptake transporter genes *AsCTR2* and *AsCTR3*. Biometals. 2016;**29**:249-264. DOI: 10.1007/s10534-016-9912-x [78] Hložková K, Matěnová M, Žáčková P, Strnad H, Hršelová H, Hroudová M, Kotrba P. Characterization of three distinct metallothionein genes of the Ag-hyperaccumulating ectomycorrhizal fungus *Amanita strobiliformis*. Fungal Biology. 2016;**120**:358-369. DOI:

[79] Mercogliano CP, DeRosier DJ. Gold nanocluster formation using metallothionein: mass spectrometry and electron microscopy. Journal of Molecular Biology. 2006;**355**:211-223.

[80] Mercogliano CP, DeRosier DJ. Concatenated metallothionein as a clonable gold label for electron microscopy. Journal of Structural Biology. 2007;**160**:70-82. DOI: 10.1016/j.

[81] Fernández de Castro I, Sanz-Sánchez L, Risco C. Metallothioneins for correlative light and electron microscopy. Methods in Cell Biology. 2014;**124**:55-70. DOI: 10.1016/

[82] Morphew MK, O'Toole ET, Page CL, Pagratis M, Meehl J, Giddings T, Gardner JM, Ackerson C, Jaspersen SL, Winey M, Hoenger A, McIntosh JR. Metallothionein as a clonable tag for protein localization by electron microscopy of cells. Journal of Microscopy.

[83] Presta A, Stillman MJ. Incorporation of copper into the yeast *Saccharomyces cerevisiae*. Identification of Cu(I)—metallothionein in intact yeast cells. Journal of Inorganic

[84] Nakayama K, Okabe M, Aoyagi K, Yamanoshita O, Okui T, Ohyama T, Kasai N. Visualization of yellowish-orange luminescence from cuprous metallothioneins in liver

of Long-Evans Cinnamon rat. Biochimica et Biophysica Acta. 1996;**1289**:150-158

Biochemistry. 1997;**66**:231-240. DOI: 10.1016/S0162-0134(96)00216-4

tobacco. PloS One. 2014;**9**:e102750. DOI: 10.1371/journal.pone.0102750

10.1007/s11356-016-7157-4

38 Old Yeasts - New Questions

10.1104/pp.109.144675

10.1016/j.funbio.2015.11.007.

DOI: 10.1016/j.jmb.2005.10.026

B978-0-12-801075-4.00003-3

2015;**260**:20-29. DOI: 10.1111/jmi.12262

jsb.2007.06.010

2009;**151**:702-714. DOI: 10.1104/pp.109.139717

[86] Ni TW, Staicu LC, Nemeth RS, Schwartz CL, Crawford D, Seligman JD, Hunter WJ, Pilon-Smits EA, Ackerson CJ. Progress toward clonable inorganic nanoparticles. Nanoscale. 2015;**7**:17320-17327. DOI: 10.1039/c5nr04097c

**Provisional chapter**

## **Yeast as a Biocatalyst in Microbial Fuel Cell**

**Yeast as a Biocatalyst in Microbial Fuel Cell**

DOI: 10.5772/intechopen.70402

Enas Taha Sayed and Mohammad Ali Abdelkareem Abdelkareem

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.70402

Enas Taha Sayed and Mohammad Ali

#### **Abstract**

Microbial fuel cells (MFCs) are fascinating bioelectrochemical devices that use the catalytic activity of living microorganisms to draw electric energy from organic matter present naturally in the environment or in the waste. Yeasts are eukaryotic microorganisms, classified as members of the fungus kingdom. Several yeast strains have been studied as biocatalysts in MFC with or without external mediator such as *Saccharomyces cerevisiae, Candida melibiosica, Hansenula anomala, Hansenula polymorpha, Arxula adeninvorans and Kluyveromyces marxianus*. In this chapter, we will focus on the use of yeast as a biocatalyst in the anode of microbial fuel cells (MFCs). How different yeast strains transfer electrons to the anode of the microbial fuel cells, advantages and challenges of the use of yeasts in MFCs, how to improve the performance and sustainability of the yeast-based MFCs through the modification of the anode electrode surface, and the application of the yeastbased MFCs in continuous wastewater treatment were discussed.

**Keywords:** yeast, microbial fuel cell, biocatalyst, electron transfer, mediator

#### **1. Introduction**

Microbial production of energy and/or chemicals from renewable carbohydrate feedstocks, and other organic-based wastes such as wastewater, is an attractive alternative to the current common fossil fuels. Microbial fuel cells (MFCs) are among the fast-growing microbial electrochemical systems (MESs) that offer a promising way for simultaneous wastewater treatment and electricity production [1–3]. Although MFCs showed promising features such as simultaneous wastewater treatment and electricity generation, low sludge production, wide range of substrates and operating at room temperature, the low power output and high cost especially that of the Pt cathode are the main challenges facing their commercialization [4–6].

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

In MFCs, the exo-electrogenic microorganisms act as biocatalysts in anaerobic oxidation of the organic materials that exist in different wastes, liberating electrons that can be collected by a conductive electrode, i.e., anode, generating an external power-producing circuit, and protons transferred through an electrolyte to a cathode surface. At the cathode, electrons react with protons and oxygen producing water [7–9]. The exo-electrogenic microorganisms that can be used in MFCs can be a prokaryote or eukaryote. Although prokaryotic microorganisms showed promising results in the MFCs and a lot of research has been carried out using them due to their ease in the electron transfer mechanism, yeast, as a eukaryote, attracted researchers' attention and was extensively studied as a biocatalyst in MFCs [4–6].

#### **2. Microbial fuel cells: structure, components and mechanism**

Microbial electrochemical systems (MESs) are innovative technology, recently implemented for numerous applications [10–15] such as (i) the simultaneous wastewater treatment and electricity production by MFCs, (ii) bio-hydrogen and/or other chemical production by microbial electrolysis cells (MECs), (iii) water desalination by microbial dialysis cells (MDCs) and (iv) electricity production in sediments or plant MFCs.

In case of MFCs, microorganisms oxidize organic matter, producing electrons that travel through a series of respiratory enzymes in the cell and make energy for the cell in the form of ATP. The electrons are then released to a terminal electron acceptor (TEA) that becomes reduced. Many TEAs such as oxygen, nitrate, sulfate and others readily diffuse into the cell where they accept electrons forming products that can diffuse out of the cell. However, it is now known that some microorganisms can transfer electrons exogenously (i.e., outside the cell) to a TEA such as metal oxides like iron oxide. This is the case of bacteria called exoelectrogens, which can be used to produce electricity in MFC [16].

**Figure 1** shows a schematic diagram of an air-cathode MFC that consists of anode and cathode electrodes separated by a separator (if needed). The anode compartment composed of anode and carbon source (organic materials), with or without exogenous mediator. At the cathode, an electron acceptor (O<sup>2</sup> from air) reacts with protons that pass from the anode to the cathode through the electrolyte, and the electrons produce water.

#### **2.1. Anode material**

Anode material is considered as an important parameter that affects the performance of MFCs. The anode of the MFCs should have high electrical, mechanical and chemical stability, be biocompatible and have high surface area [20]. Carbon materials (conventional and nonconventional) are the best materials that are applied as anode in the MFCs showing high power output. The conventional carbon materials such as carbon paper, carbon cloth, carbon brush and carbon felt, and the nonconventional ones such as carbon nanotubes (CNTs), carbon nanofibers and graphene have been extensively applied in MFCs. Little work have been carried out using noncarbonaceous materials such as stainless steel, gold and titanium [17–19], which showed a lower performance compared to that obtained in case of using carbon.

**Figure 1.** A schematic diagram showing the main components of an air-cathode MFC.

#### **2.2. Cathode material**

In MFCs, the exo-electrogenic microorganisms act as biocatalysts in anaerobic oxidation of the organic materials that exist in different wastes, liberating electrons that can be collected by a conductive electrode, i.e., anode, generating an external power-producing circuit, and protons transferred through an electrolyte to a cathode surface. At the cathode, electrons react with protons and oxygen producing water [7–9]. The exo-electrogenic microorganisms that can be used in MFCs can be a prokaryote or eukaryote. Although prokaryotic microorganisms showed promising results in the MFCs and a lot of research has been carried out using them due to their ease in the electron transfer mechanism, yeast, as a eukaryote, attracted research-

Microbial electrochemical systems (MESs) are innovative technology, recently implemented for numerous applications [10–15] such as (i) the simultaneous wastewater treatment and electricity production by MFCs, (ii) bio-hydrogen and/or other chemical production by microbial electrolysis cells (MECs), (iii) water desalination by microbial dialysis cells (MDCs) and

In case of MFCs, microorganisms oxidize organic matter, producing electrons that travel through a series of respiratory enzymes in the cell and make energy for the cell in the form of ATP. The electrons are then released to a terminal electron acceptor (TEA) that becomes reduced. Many TEAs such as oxygen, nitrate, sulfate and others readily diffuse into the cell where they accept electrons forming products that can diffuse out of the cell. However, it is now known that some microorganisms can transfer electrons exogenously (i.e., outside the cell) to a TEA such as metal oxides like iron oxide. This is the case of bacteria called exo-

**Figure 1** shows a schematic diagram of an air-cathode MFC that consists of anode and cathode electrodes separated by a separator (if needed). The anode compartment composed of anode and carbon source (organic materials), with or without exogenous mediator. At the

Anode material is considered as an important parameter that affects the performance of MFCs. The anode of the MFCs should have high electrical, mechanical and chemical stability, be biocompatible and have high surface area [20]. Carbon materials (conventional and nonconventional) are the best materials that are applied as anode in the MFCs showing high power output. The conventional carbon materials such as carbon paper, carbon cloth, carbon brush and carbon felt, and the nonconventional ones such as carbon nanotubes (CNTs), carbon nanofibers and graphene have been extensively applied in MFCs. Little work have been carried out using noncarbonaceous materials such as stainless steel, gold and titanium [17–19], which showed a lower performance compared to that obtained in

from air) reacts with protons that pass from the anode to the

ers' attention and was extensively studied as a biocatalyst in MFCs [4–6].

(iv) electricity production in sediments or plant MFCs.

cathode, an electron acceptor (O<sup>2</sup>

**2.1. Anode material**

42 Old Yeasts - New Questions

case of using carbon.

electrogens, which can be used to produce electricity in MFC [16].

cathode through the electrolyte, and the electrons produce water.

**2. Microbial fuel cells: structure, components and mechanism**

Cathode material has a significant impact on the overall cell voltage and it should have a high redox potential. Carbon materials such as carbon paper and carbon cloth modified with high active catalyst such as Pt catalyst are among the most common cathodes of the MFCs [20]. Although modifying the carbon cloth and/or carbon paper with Pt significantly decreased the oxygen reduction activation energy and increased the reaction rate, the high cost and scarcity of the Pt are the main challenges facing the application of such cathode. Recently, a wide range of non–Pt-based catalysts were investigated as cathodes in MFCs and showed promising results that gave them a potential to replace Pt catalyst in the near future such as carbon nitrogen alloys and metal carbides [18, 20–29].

#### **2.3. Separator**

As anode is working under anaerobic conditions, while cathode is working under aerobic conditions, the addition of separator with high ionic conductivity and low permeability could improve the MFC performance [30]. A large number of separators have been extensively studied in MFCs such as anion and cation exchange membranes, salt bridge, glass fibers, microfiltration membrane, porous fabrics, and coarse-pore filters [31–37]. It is worth mentioning that some MFCs showed better performance even without using the separator [3].

#### **2.4. Microbes and electron transfer in microbial fuel cells**

Microorganisms are generally divided into two main categories, prokaryotes and eukaryotes. Prokaryotes are simpler (no distinct nucleus) and smaller in size (around 1 μ in diameter) compared to eukaryotes that have larger size (5–10 μ or more) and are complex (possessing a distinct nucleus and subcellular organelles such as plastids and mitochondria) [4, 6]. All microorganisms that are capable of exo-cellular electron transfer (exo-electrogens) can be effectively used in MFCs without adding soluble exogenous mediators [4, 22, 30–38].

The possible electron transfer mechanisms in MFCs are shown in **Figure 2** and can be summarized in the following:

**1.** Direct electron transfer (DET) whether by direct cell attachment or through nanowires (pili)

DET requires a direct contact between the anode surface and the outer membrane of the microorganism. Pili are nanowires that are formed out to connect the microorganism's membrane to the anode surface. The merits of the pili formation that multiple layers biofilm microorganisms can participate in the electron transfer while bulk ones do not participate in the electron transfer [4, 39–43].

**2.** Indirect electron transfer through external or internal mediators

In this type, a redox active material (mediator) is responsible for the electron transfer between the microorganism and the anode surface. This redox can either be exerted naturally by the

**Figure 2.** Schematic diagram showing different electron transfer mechanisms in MFCs.

microorganisms (internal) or can be added from outside (external). These mediators whether internal or external will be responsible for the electron transfer from the bulk microorganisms to the anode surface. The electron transfer in the mediated electron transfer is higher than that in the DET [4, 44–51].

Internal mediators have several advantages over the external ones such as they are cheap as they are exerted by the microorganism and have no toxic effect on the microorganism. **Figure 3** shows a schematic diagram of the disadvantages of external mediators and some types of the internal and external mediators.

Several external mediators have been investigated in MFCs such as methylene blue (MB), methyl red, methanyl yellow, methyl orange, bromocresol purple, bromocresol green (BcG), romothymol blue, bromophenol blue, Congo red, cresol red, eriochrome black T, murexide, neutral red (NR), yeast extract, etc.

**Figure 3.** External and internal mediators in MFCs.

**2.4. Microbes and electron transfer in microbial fuel cells**

marized in the following:

44 Old Yeasts - New Questions

pate in the electron transfer [4, 39–43].

**2.** Indirect electron transfer through external or internal mediators

**Figure 2.** Schematic diagram showing different electron transfer mechanisms in MFCs.

(pili)

Microorganisms are generally divided into two main categories, prokaryotes and eukaryotes. Prokaryotes are simpler (no distinct nucleus) and smaller in size (around 1 μ in diameter) compared to eukaryotes that have larger size (5–10 μ or more) and are complex (possessing a distinct nucleus and subcellular organelles such as plastids and mitochondria) [4, 6]. All microorganisms that are capable of exo-cellular electron transfer (exo-electrogens) can be

The possible electron transfer mechanisms in MFCs are shown in **Figure 2** and can be sum-

**1.** Direct electron transfer (DET) whether by direct cell attachment or through nanowires

DET requires a direct contact between the anode surface and the outer membrane of the microorganism. Pili are nanowires that are formed out to connect the microorganism's membrane to the anode surface. The merits of the pili formation that multiple layers biofilm microorganisms can participate in the electron transfer while bulk ones do not partici-

In this type, a redox active material (mediator) is responsible for the electron transfer between the microorganism and the anode surface. This redox can either be exerted naturally by the

effectively used in MFCs without adding soluble exogenous mediators [4, 22, 30–38].

#### **3. Yeast as a biocatalyst in MFCs**

Yeast is a eukaryote with cell compartmentalization and has more complicated architecture compared to prokaryotes. Yeast is considered as an ideal biocatalyst for microbial fuel cell applications as most strains are nonpathogens, can metabolize wide range of substrates, are robust, and are easily handled. The bio-catalytic activity of the yeast would be related to the existence of different natural electron shuttles, mediators, such as azurin, ferredoxin and cytochromes, which could be used by redox enzymes for electron transfer from the yeast cells to the anode surface. This is in addition to the high extent of proteins in the yeast cell membrane, which is an important characteristic of electroactive species [4, 6]. Yeast cells also have a thick (100–200 nm) cell wall constructed of polysaccharides and proteins [43, 52]. Yeast cytochromes are located in the mitochondria, and transmembrane proteins (tPMETs) are located in the cell membrane, which are enclosed by the cell wall. Hence, to obtain an electrochemical response from the yeast cells, it has been assumed that a mediator must traverse the cell wall and interact with the membrane and/or internal redox sites such as NAD+/NADH [41, 42], or that the response originates from the soluble electroactive species exported from the cell [4, 45].

The electron transfer during the metabolism of the organic materials in the yeast cell is shown in **Figure 4**. Electrons liberate during the oxidation of the substrate into pyruvate in the glycolysis process, which takes place in the cytosol of the cell. These electrons received by the NAD+ forming NADH, which is recycled through its oxidation by the liberation of the electrons to the anode surface whether directly through the tPMETs or through the mediator to form NAD+ again — cycle of NADH to NAD+ . In mitochondria, oxidation of pyruvate into organic acids is associated with the liberation of the electrons that are received by the NAD+ forming NADH, which in turn are oxidized by releasing electrons to the mediator to form the NAD+ again. The reduced form of the mediator lost electrons to the anode surface to complete the cycle [38, 46].

Several yeast strains have been studied as biocatalysts in MFC with or without external mediator such as *Saccharomyces cerevisiae* (*S. cerevisiae*) [41–52], *Candida melibiosica* 2491 (*C. melibiosica*) [53–56], *Hansenula anomala* (*H. anomala*) [40], *Hansenula polymorpha* (*Hansenula polymorpha*) [57], *Arxula adeninivorans* (*A. adeninivorans*) [58] and *Kluyveromyces marxianus* (*K. marxianus*) [59].

#### **3.1.** *S. cerevisiae*

Baker's yeast (*S. cerevisiae*) is a single cell-based organism used in bread-making and beer production industry. *S. cerevisiae* is a simple eukaryotic cell, which serves as a model system

**Figure 4.** Schematic diagram shows the possible electrons' origin and transfer of yeast cells to MFC.

[59, 60] for many eukaryotes, including human cells, for the study of fundamental cellular processes such as the cell cycle, DNA replication, recombination, cell division and metabolism [60, 61]. *S. cerevisiae* is considered to be a good biocatalyst in MFC due to its broad substrate spectrum, easy and fast mass cultivation, nonpathogenic, cheap and can be maintained for a long time in the dried state [9, 60–62]. Due to these features, *S. cerevisiae* was recently used in a large-scale MFC [63].

#### *3.1.1. Mediator-less MFC*

located in the cell membrane, which are enclosed by the cell wall. Hence, to obtain an electrochemical response from the yeast cells, it has been assumed that a mediator must traverse the cell wall and interact with the membrane and/or internal redox sites such as NAD+/NADH [41, 42], or that the response originates from the soluble electroactive species exported from

The electron transfer during the metabolism of the organic materials in the yeast cell is shown in **Figure 4**. Electrons liberate during the oxidation of the substrate into pyruvate in the glycolysis process, which takes place in the cytosol of the cell. These electrons received by the NAD+ forming NADH, which is recycled through its oxidation by the liberation of the electrons to the anode surface whether directly through the tPMETs or through the mediator to

organic acids is associated with the liberation of the electrons that are received by the NAD+ forming NADH, which in turn are oxidized by releasing electrons to the mediator to form the NAD+ again. The reduced form of the mediator lost electrons to the anode surface to complete

Several yeast strains have been studied as biocatalysts in MFC with or without external mediator such as *Saccharomyces cerevisiae* (*S. cerevisiae*) [41–52], *Candida melibiosica* 2491 (*C. melibiosica*) [53–56], *Hansenula anomala* (*H. anomala*) [40], *Hansenula polymorpha* (*Hansenula polymorpha*) [57], *Arxula adeninivorans* (*A. adeninivorans*) [58] and *Kluyveromyces marxianus* (*K. marxianus*) [59].

Baker's yeast (*S. cerevisiae*) is a single cell-based organism used in bread-making and beer production industry. *S. cerevisiae* is a simple eukaryotic cell, which serves as a model system

**Figure 4.** Schematic diagram shows the possible electrons' origin and transfer of yeast cells to MFC.

. In mitochondria, oxidation of pyruvate into

the cell [4, 45].

46 Old Yeasts - New Questions

the cycle [38, 46].

**3.1.** *S. cerevisiae*

form NAD+ again — cycle of NADH to NAD+

Mediator-less MFCs are those that operate without the addition of any external mediator. Sayed et al. [6] studied the mechanism by which *S. cerevisiae* transfers the electrons to the anode surface whether through the solution species or through the surface-confined species in a mediator-less MFC. *S. cerevisiae* was cultivated outside the MFC and then applied in an air-cathode mediator-less MFC using glucose as a substrate. Carbon paper was used as an anode and carbon paper with Pt/C as a cathode. When the MFC was operated with the yeast cells, the anode potential decreased from 0.4 to 0.1 V (vs. NHE pH 7) during 45 h. At the same time, the open circuit voltage (OCV) increased from 0.25 to 0.65 V. A maximum power output above 3 mW/m<sup>2</sup> was attained during the linear sweep voltammetry (LSV). At the end of the MFC operation, when the anolyte was replaced with a fresh one without yeast cells, i.e., just glucose, into the anode chamber, the cell attained the same maximum cell voltage within 1 h of cell operation. The same maximum power generation during the LSV was also attained. On the other hand, when another MFC using a fresh anode was operated with the filtered anolyte solution, i.e., no yeast cells, neither cell voltage nor anode potential changed, **Figures 5** and **6**. The ex-situ cyclic voltammetry of the filtered anolyte at the end of the experiments showed no redox peaks; i.e., no mediator existed in the anolyte. These measurements showed that the electron transfer was done through the surface-confined species, and there was no role of the solution species in it.

**Figure 5.** The OCV and the electrode potentials vs. time of the MFC using carbon paper (CP) as the anode material. (a) Without filtration and (b) with filtration [6].

**Figure 6.** The i-V and i-p curves measured before and after the replacements of the anolyte solution [6].

The same conclusions for the direct electron transfer and no role of the mediator in the electron transfer of the *S. cerevisiae* were confirmed by Rawson et al. [41] who studied the direct electron transfer from the *S. cerevisiae* cells attached to the anode surface. The authors modified the anode surface with a mediator, osmium bipyridine complex, layer that hindered the mediator from penetrating the cell wall and reacting with the internal redox species. Results showed that the electron transfered from the yeast cells to the electrode surface through the yeast cell wall and no involvement of the endogenous mediator in this electron transfer.

In another study, the performance of air-cathode MFC using *S. cerevisiae* as an anodic biocatalyst under different redox conditions and organic loading was investigated [38]. The MFC was operated with synthetic wastewater at organic loading rate (OLR) of 0.91 kg COD/m<sup>3</sup> -day and the performance of yeast-based MFC along with wastewater treatment was investigated at different feeding pH of 5.0, 6.0 and 7.0. Using cyclic voltammetry, which is an effective tool to identify the electron transfer mechanism in MFCs [64], the MFC performance was dependent on the OLR and the pH. Cyclic voltammetry confirmed the existence of the NADH/NAD+ and FADH/FAD+.

Although *S. cerevisiae* could be effectively used as a biocatalyst in mediator-less MFC, the power output was limited by the low electron transfer rate from the microorganism to the anode surface. The performance of the *S. cerevisiae*-based MFC could be improved by enhancing the rate of electron from the yeast cell to the anode surface by one or more of the following techniques:


#### *3.1.1.1. Enhancement of electron transfer in a mediator-less MFC*

The same conclusions for the direct electron transfer and no role of the mediator in the electron transfer of the *S. cerevisiae* were confirmed by Rawson et al. [41] who studied the direct electron transfer from the *S. cerevisiae* cells attached to the anode surface. The authors modified the anode surface with a mediator, osmium bipyridine complex, layer that hindered the mediator from penetrating the cell wall and reacting with the internal redox species. Results showed that the electron transfered from the yeast cells to the electrode surface through the yeast cell wall and no involvement of the endogenous mediator in this electron transfer.

**Figure 6.** The i-V and i-p curves measured before and after the replacements of the anolyte solution [6].

In another study, the performance of air-cathode MFC using *S. cerevisiae* as an anodic biocatalyst under different redox conditions and organic loading was investigated [38]. The MFC was

the performance of yeast-based MFC along with wastewater treatment was investigated at different feeding pH of 5.0, 6.0 and 7.0. Using cyclic voltammetry, which is an effective tool to identify the electron transfer mechanism in MFCs [64], the MFC performance was dependent on the OLR and the pH. Cyclic voltammetry confirmed the existence of the NADH/NAD+

Although *S. cerevisiae* could be effectively used as a biocatalyst in mediator-less MFC, the power output was limited by the low electron transfer rate from the microorganism to the anode surface. The performance of the *S. cerevisiae*-based MFC could be improved by enhancing the rate of electron from the yeast cell to the anode surface by one or more of the following


operated with synthetic wastewater at organic loading rate (OLR) of 0.91 kg COD/m<sup>3</sup>

and FADH/FAD+.

48 Old Yeasts - New Questions

techniques:

**i.** Anode modification [42].

**ii.** Immobilization of the yeast cells on carbon nanotube [43].

**iii.** Yeast surface display of dehydrogenases [52].

**iv.** Addition of exogenous mediators [44–51].

The electrical conductivity of the anode plays an important role in the performance of the MFCs. The effect of the modification of carbon paper with thin layer of different transition metals, i.e., cobalt and gold, on the performance of air-cathode MFCs using *S. cerevisiae* as a biocatalyst was investigated [42]. Sputtering technique was used for preparing different thin layers of Co and Au with thicknesses of 5 and 30 nm on the surface of carbon electrodes. The 5-nm layer showed no significant effect on the cell performance, and this was related to the rare existence of the metals detected by the energy dispersive x-ray (EDX) measurements. On the other hand, 30 nm of Co significantly improved the performance where the power output increased from 12.8 to 20.2 mW/m<sup>2</sup> while the steady current discharge at 0.2 V increased from 8 to 27 mA/m<sup>2</sup> , **Figure 7**. On the other hand, 30 nm of Au-modified electrode showed a negative effect on the cell performance. The positive effect of the Co on the performance was related to the enhancement of the electron transfer by the Co and the stimulation of the yeast growth on the modified electrode surface as confirmed by the SEM images. While Au suppressed the growth of the yeast cells as proved from the SEM images due to its poisoning effect, decreasing the performance (**Table 1**) [42].

The electron transfer of *S. cerevisiae* based MFC was enhanced by immobilizing *S. cerevisiae* on carbon nanotube (yeast/CNT) to be used as a catalyst in a membrane-less MFC [43]. The effect of the entrapping polymer (EP) and cross-linker (glutaraldehyde, GA) addition on the performance and stability of the MFC using *laccase* as cathodic catalyst was investigated. GA was selected as cross-liner due to its ability to promote cross-linking between yeast cells and poly(ethylenimine) (PEI), which used as the entrapping polymer due to its positive charge property. Bare CNT showed only C═C (sp<sup>2</sup> ) bonds indicating that CNT had not any functional group. In case of the immobilized yeast cells, C─N (C═N) bond peak appeared indicating that yeast cell and CNT were properly bonded. The immobilization of the yeast enhanced the power by 150% where it increased from 138 to 344 mW/m<sup>2</sup> .

**Figure 7.** The i-t measurements at 0.2 V for a mediator-less yeast-based MFC using nonmodified (NME) carbon paper and Co, 30 nm, modified one [42].


**Table 1.** Summary of the studies done on the mediator-less *S. cerevisiae* yeast-based MFC. The performance of *S. cerevisiae* based MFC was improved by displaying dehydrogenases, cellobiose dehydrogenase from *Corynascus thermophilus* (CtCDH) on the surface of *S. cerevisiae* using the yeast surface display system [52]. The surface displayed dehydrogenases were used in mediator-less two compartments MFCs. The MFCs were operated using unmodified *S. cerevisiae*, CtCDH-displaying *S. cerevisiae* and glucose oxidase (GOx) was used for comparison. Graphite plates modified with multi-walled carbon nanotubes (MWCNT) were used as electrodes in the anode and cathode compartments that were separated by Nafion 117. A maximum power output of CtCDH-displaying *S. cerevisiae* MFC was 33 mW/m<sup>2</sup> which was around 12 times higher than those obtained in case of GOx, and unmodified *S. cerevisiae*, 2.8 and, 2.7 mW/m<sup>2</sup> , respectively.

#### *3.1.2. Mediated yeast-based MFC*

**Ref.**

**Max. power** **mW/m2 mW/m3**

6 3. 12.9

42

2

> 38

52

2.8

33

> 46

47 28 **Table 1.**

Summary of the studies done on the mediator-less *S. cerevisiae* yeast-based MFC.

850 mL (760 mL WV)

Nafion 117


Graphite plates

Graphite plates

Glucose

Dual chamber

40

500 mL

Nafion 117

Potassium ferricyanide

Reticulated Vitreous carbon

Reticulated Vitreous carbon

Glucose

Dual chamber

8–10 mL (5 mL

Nafion

117

O2 (air)

A graphite plate

A graphite plate/

MWCNT

d-glucose

lactose

WV)

2.7

25.51

350 mL (320 mL WV)

Nafion 117

O2 (air)

Graphite plate

Graphite plate

Synthetic wastewater

Lactose

Dual

chamber

Air cathode

20.2

(70

mL WV)

Nafion 117

O2 (air)

Pt/C over carbon paper

Glucose

Co sputtered carbon paper

Au-sputtered carbon

paper

Air cathode

17

84 mL (70

mL WV)

NRE 212

O2 (air)

**Anode chamber** 

**Separator**

**Cathode** **Electron acceptor**

**Electrode**

Pt/C over carbon paper

Carbon paper Carbon paper

Glucose

Air cathode

50 Old Yeasts - New Questions

**Anode material**

**Carbon source**

**MFC type**

**(WV)**

Several studies have been carried out to enhance the electron transfer through the addition of an external mediator. A candidate external mediator must satisfy several requirements such as being electrochemically active, fast release of electrons on the electrode surface, biocompatible to the microorganisms, soluble and chemically stable in the anolyte media, easily penetrate the cell membrane, and has a prober redox potential that is sufficiently positive to provide fast electron transfer from microorganisms to the anode while not too strong to avoid a big loss of potential [2, 14, 16]. Different mediators such as MB, NR, thionine, yeast extract, and others enhanced the electron transfer in *S. cerevisiae* yeast-based MFCs, and their power output are shown in **Table 2**.

Using copper electrodes and a sulfonated polyether ether ketone (SPEEK) as proton exchange membrane, Permana et al. [48] studied the performance of dual chamber *S. cerevisiae* yeastbased MFCs with and without MB using glucose as substrate. The MFC operated with MB showed higher cell voltage, higher power and energy outputs, and slightly lower glucose consumption without affecting the bioethanol production compared to the mediator-less MFC. Using rotating disc electrodes (RDEs), Ganguli and Dunn [45] were able to simultaneously determine the catalytic current under quiescent conditions along with the reduced mediator concentration that not adsorbed by the yeast. Based on the results from the anode kinetics study, a yeast powered microbial fuel cell successfully produced power density of ∼1500 mW/m<sup>2</sup> once the reduced mediator concentration stabilized.

The effect of the anode modification on the performance of the mediated *S. cerevisiae* yeastbased MFC that used glucose as a substrate and MB as a mediator was investigated [50]. The anode carbon paper was sputtered with a thin layer of 30 nm of Co (Co30) or Au (Au30). The modification of the anode significantly improved the performance from 80 to 148 mW/m<sup>2</sup> and 120 mW/m<sup>2</sup> in case of Co30 and Au30, respectively, as shown in **Figure 8**. Although the cell resistance in case of Au is lower than that in Co, the performance of the latter was better and this was related to the poisons effect of the Au on the growth of the yeast cell on the anode surface; therefore, only the yeast in the anolyte took part in the performance, while in case of the Co, the yeast cells in the anolyte and those formed as biofilm on the anode surface took part in the electron transfer. The cell resistance decreased from 25 μΩ cm<sup>2</sup> in the case of nonmodified (NME) anode to 4 and 3 μΩ cm<sup>2</sup> in case of Co30 and Au30, respectively. The better performance



**Ref.**

**mW/m2**

> 59

50

120

> 45

46 39

31

52

32 22 14 400

MB

32 mL

NR

Nafion 115

Reticulated vitreous carbon

Reticulated vitreous carbon

Dextrose

Dual chamber

44

80 500

MB &NR

MB (0.1 M)

25 mL

O2 (air)

Pt/C over carbon cloth

Graphite plate

Air cathode

l-arabinose

d-cellobiose

d-galactose

146.71 ± 7.7 MB

500 mL

Nafion 117

Potassium ferricyanide

Reticulated vitreous carbon

Reticulated vitreous carbon

Glucose d-xylose

d-glucose

Dual chamber

150

MB

10 mL

Nafion

Potassium ferricyanide

Carbon felt

Carbon felt

Glucose

Dual chamber

148

MB

(70 mL WV)

Nafion 117

O2 (air)

Pt/C over carbon paper

Glucose

Co-sputtered carbon paper

Au-sputtered carbon

paper

Air cathode

22 80

850 × 103

2-hydroxy-1,4-

 naphthoquinone

WV, 7.5 cm3

Gore-Tex, 30 μm

K3[Fe(CN)6]

**mW/m3**

**Mediator**

**Anode** 

**Cathode**

**Anode material**

**Carbon** 

**source**

**MFC type**

**chamber**

**Separator**

**Electron acceptor**

**Electrode**

Carbon rods

Carbon rods and carbon fiber bundles

Carbon paper

Glucose

Dual- chamber

52 Old Yeasts - New Questions

**(WV)**

**Max. power**

**Table 2.** Summary of the studies done on the mediated *S. cerevisiae* yeast-based MFC.

**Figure 8.** The i-V and i-p curves of the yeast-based MFC with 0.1 mM MB using nonmodified carbon paper, and Co30 and Au30 as anodes [50].

in both cases was related to the metal-modified surface that significantly enhanced the electron transfer via the exogenous mediator. It was also considered that the highly conductive surface of the Co or Au on the anode surface increased the efficiency of the electron transfer by contacting a part of the mediator with an electric charge on the anode.

MB was also used in air-cathode MFC that used modified *S. cerevisiae* using yeast surface display system [52]. Pyranose dehydrogenase from *Agaricus meleagri*s (AmPDH) was displayed on the surface of *S. cerevisiae*. The MFCs were operated using unmodified *S. cerevisiae* or AmPDH-displaying *S. cerevisiae* with various fuels, d-xylose, d-glucose, l-arabinose, d-cellobiose and d-galactose using 0.1 mM MB. AmPDH displaying *S. cerevisiae* generated high power outputs using the different substrates, 3.1, 3.9, 3.2, 2.2, and 1.4 μW/cm<sup>2</sup> in case of using d-glucose, d-xylose, l-arabinose, d-cellobiose and d-galactose, respectively, compared with a maximum power output of 0.8 μW/cm<sup>2</sup> in case of the unmodified *S. cerevisiae* using d-xylose as a fuel [52].

Compared to MB, NR showed promising results in a two-compartment *S. cerevisiae* yeast-based MFC for degradation of whey. With a fixed concentration of the two mediators of 100 μmol/l, the maximum power and current densities increased from 1.43 μW and 11.5 μA to 50 μW and 470 μA in case of the NR compared to 11.3 μW and 120 μA in case of MB. These results showed that NR served as a suitable mediator and enhanced the electrical energy by 5 folds compared to that of MB [49]. When NR (0.5 mM) was added to the MB (0.5 mM), the *S. cerevisiae* yeastbased MFC showed a maximum power output of 500 mW/m<sup>2</sup> compared to 400 mW/m<sup>2</sup> in case of 1 mM of MB [44]. This increase in the performance was related to the role of the MB in the enhancement of the anaerobic respiration, while NR involved with fermentation only. This study [44] showed that the addition of the MB was effective than NR, which is in contradiction to that reported by Najafpour et al. [49]. This might be related to the difference in the operation conditions, and/or any other reasons that is not clear for the authors right now.

Thionine is another mediator that worked effectively in *S. cerevisiae* yeast-based MFC [47]. Thionine addition significantly increased the performance from 3 to 28 mW/m<sup>2</sup> . An optimum concentration of thionine was 500 mM, giving a maximum voltage of 420 mV and a maximum current of 700 mA/m<sup>2</sup> . Cyclic voltammetry measurements showed a redox peak of −0.1 V vs. Ag/AgCl.

Yeast extract, which is one of the main components of the biological cultivating media, was effectively used as a mediator in *S. cerevisiae* yeast-based MFC [51]. Using two different anodes, plain carbon paper and gold-plated carbon paper, the current density increased from 94 and to 190 and 300 mA/m<sup>2</sup> , respectively, by yeast extract addition as shown in **Figures 9a** and **10a**. While the power density increased from 12.9 and 2 to 32.6 and 70 mW/m<sup>2</sup> with the yeast extract addition for the plain and the gold-plated electrodes, respectively, as shown in **Figures 9b** and **10b**. The role of the yeast extract as an electron transfer mediator was confirmed using the gold-plated carbon paper where no cells were detected on its surface (as confirmed from the scanning electron microscopic [SEM] images); therefore, the role of the surface-confined species in the cell performance was denied.

#### **3.2.** *C. melibiosica*

in both cases was related to the metal-modified surface that significantly enhanced the electron transfer via the exogenous mediator. It was also considered that the highly conductive surface of the Co or Au on the anode surface increased the efficiency of the electron transfer by contact-

**Figure 8.** The i-V and i-p curves of the yeast-based MFC with 0.1 mM MB using nonmodified carbon paper, and Co30

MB was also used in air-cathode MFC that used modified *S. cerevisiae* using yeast surface display system [52]. Pyranose dehydrogenase from *Agaricus meleagri*s (AmPDH) was displayed on the surface of *S. cerevisiae*. The MFCs were operated using unmodified *S. cerevisiae* or AmPDH-displaying *S. cerevisiae* with various fuels, d-xylose, d-glucose, l-arabinose, d-cellobiose and d-galactose using 0.1 mM MB. AmPDH displaying *S. cerevisiae* generated

using d-glucose, d-xylose, l-arabinose, d-cellobiose and d-galactose, respectively, compared

Compared to MB, NR showed promising results in a two-compartment *S. cerevisiae* yeast-based MFC for degradation of whey. With a fixed concentration of the two mediators of 100 μmol/l, the maximum power and current densities increased from 1.43 μW and 11.5 μA to 50 μW and 470 μA in case of the NR compared to 11.3 μW and 120 μA in case of MB. These results showed that NR served as a suitable mediator and enhanced the electrical energy by 5 folds compared to that of MB [49]. When NR (0.5 mM) was added to the MB (0.5 mM), the *S. cerevisiae* yeast-

of 1 mM of MB [44]. This increase in the performance was related to the role of the MB in the enhancement of the anaerobic respiration, while NR involved with fermentation only. This study [44] showed that the addition of the MB was effective than NR, which is in contradiction to that reported by Najafpour et al. [49]. This might be related to the difference in the operation

conditions, and/or any other reasons that is not clear for the authors right now.

in case of

in case

in case of the unmodified *S. cerevisiae* using

compared to 400 mW/m<sup>2</sup>

high power outputs using the different substrates, 3.1, 3.9, 3.2, 2.2, and 1.4 μW/cm<sup>2</sup>

ing a part of the mediator with an electric charge on the anode.

based MFC showed a maximum power output of 500 mW/m<sup>2</sup>

with a maximum power output of 0.8 μW/cm<sup>2</sup>

d-xylose as a fuel [52].

and Au30 as anodes [50].

54 Old Yeasts - New Questions

*C. melibiosica* is a yeast strain that possess high phytase activity, which existed in plant wastes. This yeast strain was used in numerous studies as a biocatalyst in MFC with and without mediator as can be seen in **Table 3**. The catalytic activity of *C. melibiosica* was studied in a dual chamber MFC with and without the addition of MB using different carbon sources, i.e., fructose, glucose and sucrose [53]. Results showed that *C. melibiosica* could be used as a biocatalyst in a mediator-less MFC giving a maximum power output of 60 mW/m<sup>3</sup> in case of fructose. This power increased three times either by the addition of yeast extract and peptone or by the MB addition [53].

**Figure 9.** The effect of the yeast extract (YE) addition to *S. cerevisiae* (DY)-based MFC using nonmodified carbon paper as anode on (a) the electrode potentials, and (b) the current-voltage and current-power curves [51].

**Figure 10.** The effect of the yeast extract (YE) addition on *S. cerevisiae* (DY)-based MFC in case of using gold-sputtered carbon paper as anode on (a) the electrode potentials, and (b) the current-voltage and current-power curves [51].

The effect of the mediator type, i.e., bromocresol green (BcG), bromocresol purple, romothymol blue, bromophenol blue, Congo red, cresol red, eosin, eriochrome black T, methyl red, methanyl yellow, MB, methyl orange, murexide and NR on the performance of *C. melibiosica*based MFC was investigated [54]. Results showed that among the investigated mediators, MB, methyl orange, methyl red and NR increased the performance compared to the mediatorless MFC. MB showed the best among all of them where the performance increased from 20 to 640 mW/m<sup>2</sup> with MB concentration of 0.8 mM. This was related to its ability not only to increase the electron transfer rate but also forcing the living cells to switch on various catabolic pathways and divert electrons from different energetic levels, thus increasing the energy production. This had been confirmed by measuring the ethanol production. where the MFC that operated using MO and MR produced trace amounts of ethanol, while in case of MB, ethanol was not detected. These indicated that the aerobic respiration processes were predominant in these cases. On the other hand, ethanol was produced in large quantities when NR and BcG were used, demonstrating that these mediators stopped the respiratory processes and displaced them with alcoholic fermentation.

The performance of *C. melibiosica*-based MFC was investigated using modified and nonmodified (NME) carbon felt [55]. The carbon felt was modified by Ni using two different techniques, i.e., galvanostatic pulse (GME) and potentiostatic pulse (PME). Carbon felt was used as the cathode, Nafion 117 as the separator, fructose, yeast extract and peptone (YPfru) as the anolyte, and potassium ferricyanide as the catholyte. The power output of the MFC significantly increased using the modified electrodes where it increased from 36 mW/m<sup>2</sup> in case of the NME to 390 and 720 mW/m<sup>2</sup> in case of PME and GME, respectively. These values were even higher than that obtained in case of using the NME with addition of the external mediator, MB. The authors related the improvement in the cell performance to the existence of Ni ions, which acted as an electron acceptor and/or due to adaptive mechanism which enhanced electron transfer through the yeast membrane. In another study, the authors prepared carbon felt modified with NiFe and NiFeP using the same preparation method [56]. They found


The effect of the mediator type, i.e., bromocresol green (BcG), bromocresol purple, romothymol blue, bromophenol blue, Congo red, cresol red, eosin, eriochrome black T, methyl red, methanyl yellow, MB, methyl orange, murexide and NR on the performance of *C. melibiosica*based MFC was investigated [54]. Results showed that among the investigated mediators, MB, methyl orange, methyl red and NR increased the performance compared to the mediatorless MFC. MB showed the best among all of them where the performance increased from

**Figure 10.** The effect of the yeast extract (YE) addition on *S. cerevisiae* (DY)-based MFC in case of using gold-sputtered carbon paper as anode on (a) the electrode potentials, and (b) the current-voltage and current-power curves [51].

to increase the electron transfer rate but also forcing the living cells to switch on various catabolic pathways and divert electrons from different energetic levels, thus increasing the energy production. This had been confirmed by measuring the ethanol production. where the MFC that operated using MO and MR produced trace amounts of ethanol, while in case of MB, ethanol was not detected. These indicated that the aerobic respiration processes were predominant in these cases. On the other hand, ethanol was produced in large quantities when NR and BcG were used, demonstrating that these mediators stopped the respiratory

The performance of *C. melibiosica*-based MFC was investigated using modified and nonmodified (NME) carbon felt [55]. The carbon felt was modified by Ni using two different techniques, i.e., galvanostatic pulse (GME) and potentiostatic pulse (PME). Carbon felt was used as the cathode, Nafion 117 as the separator, fructose, yeast extract and peptone (YPfru) as the anolyte, and potassium ferricyanide as the catholyte. The power output of the MFC signifi-

even higher than that obtained in case of using the NME with addition of the external mediator, MB. The authors related the improvement in the cell performance to the existence of Ni ions, which acted as an electron acceptor and/or due to adaptive mechanism which enhanced electron transfer through the yeast membrane. In another study, the authors prepared carbon felt modified with NiFe and NiFeP using the same preparation method [56]. They found

cantly increased using the modified electrodes where it increased from 36 mW/m<sup>2</sup>

processes and displaced them with alcoholic fermentation.

the NME to 390 and 720 mW/m<sup>2</sup>

with MB concentration of 0.8 mM. This was related to its ability not only

in case of PME and GME, respectively. These values were

in case of

20 to 640 mW/m<sup>2</sup>

56 Old Yeasts - New Questions


Yeast as a Biocatalyst in Microbial Fuel Cell http://dx.doi.org/10.5772/intechopen.70402 57 that among the different tested electrodes, NiFeP-modified electrodes showed the best performance of 260 ± 8 and 155 ± 6 mW/m<sup>2</sup> prepared potentiostatically and galvanostatically, respectively. The authors related the improvement in the performance to same reasons that were described above [55].

#### **3.3. Other yeast strains**

#### *3.3.1. H. anomala*

The catalytic activity of *H. anomala* in a mediator-less MFC using glucose as the substrate was investigated [40]. The *H. anomala* cells were immobilized on the surface of the anode by physical adsorption and covalent linkage. The results showed that *H. anomala* could transfer the electrons through the redox proteins, i.e., *ferricyanide reductase* and *lactate dehydrogenase* exist in their outer membrane. Moreover, the MFC was operated using different anodes, i.e., graphite, graphite felt and polyaniline–Pt-composite-coated graphite. A maximum power output of 2.34, 2.9 and 0.69 W/m<sup>3</sup> was obtained in case of graphite felt, graphite modified with PANI and Pt, and graphite, respectively. The high performance was related to the high surface of the graphite felt and the presence of the catalytic active Pt in case of the graphite modified with PANI and Pt, respectively.

#### *3.3.2. H. polymorpha*

The electron transfer pathways between the cytosolic redox enzymes of *H. polymorpha*, overexpressing flavocytochrome b2 (FC b2), and the electrode surface was studied [57]. Both wild and genetic *H. polymorpha* yeast cells were entrapped in osmium-complex-modified redox polymers (OsRP), which are essential for the electron transfer communication, on the surface of graphite electrodes. With the addition of l-lactate, current generation was noticeable when genetic modified one was used and it was in direct contact with the redox polymer, i.e., OsRP. The results suggested that the overexpression of FC b2 and the related amplification of the FC b2/ l-lactate reaction cycle were essential to provide enough charge to the electron-exchange network in order to facilitate sufficient electrochemical coupling between the cells, via the redox polymer, and the electrode. Also they suggested that the intimate contact between the cell walls and the redox polymer is a prerequisite for electrically wiring the cytosolic FC b2/ l-lactate redox activity.

#### *3.3.3. A. adeninvorans*

The biocatalytic activity of the nonconventional yeast *A. adeninvorans* in a mediator-less dual chamber MFC was investigated [58]. Results showed that *A. adeninvorans* was effectively used as a biocatalyst ion in the MFC, generating a power of more than 0.025 W/m<sup>2</sup> . The electron transfer was confirmed to be through the secretion of an endogenous mediator in the solution. This was confirmed using cyclic voltammetry of the supernatant from the *A. adeninvorans*. An irreversible oxidation peak at +0.45 V appeared. An *A. adeninvorans* yeast-based MFC showed a better performance than that obtained in case of *S. cerevisiae* yeast-based MFC, and this was related to the exertion of endogenous mediator in case of *A. adeninvorans*.

#### *3.3.4. K. marxianus*

that among the different tested electrodes, NiFeP-modified electrodes showed the best per-

respectively. The authors related the improvement in the performance to same reasons that

The catalytic activity of *H. anomala* in a mediator-less MFC using glucose as the substrate was investigated [40]. The *H. anomala* cells were immobilized on the surface of the anode by physical adsorption and covalent linkage. The results showed that *H. anomala* could transfer the electrons through the redox proteins, i.e., *ferricyanide reductase* and *lactate dehydrogenase* exist in their outer membrane. Moreover, the MFC was operated using different anodes, i.e., graphite, graphite felt and polyaniline–Pt-composite-coated graphite. A maximum power output of

and Pt, and graphite, respectively. The high performance was related to the high surface of the graphite felt and the presence of the catalytic active Pt in case of the graphite modified

The electron transfer pathways between the cytosolic redox enzymes of *H. polymorpha*, overexpressing flavocytochrome b2 (FC b2), and the electrode surface was studied [57]. Both wild and genetic *H. polymorpha* yeast cells were entrapped in osmium-complex-modified redox polymers (OsRP), which are essential for the electron transfer communication, on the surface of graphite electrodes. With the addition of l-lactate, current generation was noticeable when genetic modified one was used and it was in direct contact with the redox polymer, i.e., OsRP. The results suggested that the overexpression of FC b2 and the related amplification of the FC b2/ l-lactate reaction cycle were essential to provide enough charge to the electron-exchange network in order to facilitate sufficient electrochemical coupling between the cells, via the redox polymer, and the electrode. Also they suggested that the intimate contact between the cell walls and the redox polymer is a prerequisite for electrically wiring the

The biocatalytic activity of the nonconventional yeast *A. adeninvorans* in a mediator-less dual chamber MFC was investigated [58]. Results showed that *A. adeninvorans* was effectively used

transfer was confirmed to be through the secretion of an endogenous mediator in the solution. This was confirmed using cyclic voltammetry of the supernatant from the *A. adeninvorans*. An irreversible oxidation peak at +0.45 V appeared. An *A. adeninvorans* yeast-based MFC showed a better performance than that obtained in case of *S. cerevisiae* yeast-based MFC, and this was

. The electron

as a biocatalyst ion in the MFC, generating a power of more than 0.025 W/m<sup>2</sup>

related to the exertion of endogenous mediator in case of *A. adeninvorans*.

was obtained in case of graphite felt, graphite modified with PANI

prepared potentiostatically and galvanostatically,

formance of 260 ± 8 and 155 ± 6 mW/m<sup>2</sup>

were described above [55].

**3.3. Other yeast strains**

2.34, 2.9 and 0.69 W/m<sup>3</sup>

*3.3.2. H. polymorpha*

*3.3.3. A. adeninvorans*

with PANI and Pt, respectively.

cytosolic FC b2/ l-lactate redox activity.

*3.3.1. H. anomala*

58 Old Yeasts - New Questions

Kaneshiro et al. [59] have investigated the catalytic activity of six different yeast strains in a dual chamber MFC with glucose as the substrate including *K. marxianus, S. cerevisiae*, *Pichia pastoris*, *H. polymorpha*, *Kluyveromyces lactis*, *Schizosaccharomyces pombe, Candida glabrata* and yeast strains isolated from soil [59]. Among the different tested yeast strains, *K. marxianus* showed the highest cell performance followed by *S. cerevisiae* and *P. pastoris*. Although *K. marxianus* showed the lowest glucose consumption, it showed the lowest ethanol production indicating highest efficiency. Furthermore, *K. marxianus* showed catalytic activity for the metabolism of fructose and xylose; therefore, the authors suggested that *K. marxianus* could be effectively used for of woody biomass. *K. marxianus* is one of the robust yeast strains that could be used at high temperature; therefore, the authors investigated its catalytic activity under different temperatures, 37, 45 and 50°C. The results showed that *K. marxianus* had the highest activity at 45°C. This could be used for the treatment of high-temperature effluents that are produced in some industries.

#### **4. Large-scale yeast-based MFC**

A novel yeast-based MFC stack that composed of 4 units of total capacity of 1840 mL was designed and operated using glucose as the carbon source, graphite plates as the electrodes and Nafion 117 as the separator [63]. The stack was operated under continuous mode with a hydraulic retention time of 6.7 h. Single cell and cells connected in parallel and/or series connections were investigated to achieve the best operating conditions. A maximum current of 6447 mA/m<sup>2</sup> and maximum power of 2003 mW/m<sup>2</sup> were obtained. A Columbic efficiency of 22% was obtained in the parallel connection. **Figure 11** showed that the stack could be operated for more than 3 days with stable voltage and power output. The results obtained in this study proved the potential of yeast for scaling up. **Table 4** showed summary of the materials and operating conditions used in the stack.

**Figure 11.** Close circuit voltage and produced power from staked MFC at parallel mode with 1 KΩ resistances in external circuit for 148 h [63].


**Table 4.** A summary of the stack materials and operating conditions.

#### **5. Conclusions and recommendations**

Yeast is successfully used as a biocatalyst in MFC, which exhibits different electron transfer mechanisms according to its strains. In *S. cerevisiae* and *H. anomala*, the electron transfer takes place through the surface-confined species; in *C. melibiosica, H. polymorpha and A. adeninivorans*, the transfer of electrons from yeast cells to the anode is both by the secretion of redox molecules and by the direct electron transfer. The modification of the anode and the addition of external mediator significantly enhanced the cell performance. *K. marxianus* is one of the most promising yeast strains as it could effectively metabolize the complex organic materials with high power output even under high operating temperature conditions; therefore, it could be a best choice for wastes with fluctuated temperature. Further studies on this type and other types are required. Moreover, the surface modification of the carbon material with graphene could improve the performance.

#### **Author details**

Enas Taha Sayed<sup>1</sup> \* and Mohammad Ali Abdelkareem1,2

\*Address all correspondence to: e.kasem@mu.edu.eg

1 Chemical Engineering Department, Faculty of Engineering, Minia University, El-Minya, Egypt

2 Department of Sustainable and Renewable Energy Engineering, University of Sharjah, Sharjah, United Arab Emirates

#### **References**

**5. Conclusions and recommendations**

**Table 4.** A summary of the stack materials and operating conditions.

Mode Continuous up flow mode

MFC material Plexiglas

60 Old Yeasts - New Questions

Membrane Nafion 117.32 cm<sup>2</sup>

Fuel Glucose, 30 g/L

Anode chamber (volume) 460 mL Working volume 350 mL Current collector Copper wire

HRT 6.7 h

graphene could improve the performance.

\*Address all correspondence to: e.kasem@mu.edu.eg

\* and Mohammad Ali Abdelkareem1,2

1 Chemical Engineering Department, Faculty of Engineering, Minia University, El-Minya,

2 Department of Sustainable and Renewable Energy Engineering, University of Sharjah,

**Author details**

Enas Taha Sayed<sup>1</sup>

Sharjah, United Arab Emirates

Egypt

Yeast is successfully used as a biocatalyst in MFC, which exhibits different electron transfer mechanisms according to its strains. In *S. cerevisiae* and *H. anomala*, the electron transfer takes place through the surface-confined species; in *C. melibiosica, H. polymorpha and A. adeninivorans*, the transfer of electrons from yeast cells to the anode is both by the secretion of redox molecules and by the direct electron transfer. The modification of the anode and the addition of external mediator significantly enhanced the cell performance. *K. marxianus* is one of the most promising yeast strains as it could effectively metabolize the complex organic materials with high power output even under high operating temperature conditions; therefore, it could be a best choice for wastes with fluctuated temperature. Further studies on this type and other types are required. Moreover, the surface modification of the carbon material with

MFC type MFCs stack composed of 4 anodes and 3 cathodes compartments

Anode Graphite plates, size of 40 × 60 × 1.2 mm Cathode Graphite plates, size of 40 × 60 × 1.2 mm

Catholyte Potassium permanganate (400 μmol/L)

Anode media Yeast (*S. cerevisiae* PTCC 5269). NR (200 μmol/L)


[30] Kim I, Chae K, Choi M, Verstraete W. Microbial fuel cells: Recent advances, bacterial communities and application beyond electricity generation. Environmental Engineering Research. 2008;**13**:51-65

[15] ElMekawy A, Hegab HM, Vanbroekhoven K, Pant D. Techno-productive potential of photosynthetic microbial fuel cells through different configurations. Renewable and

[17] Richter H, McCarthy K, Nevin K, Johnson J, Rotello V, Lovley D. Electricity generation by Geobacter sulfurreducens attached to gold electrodes. Langmuir. 2008;**24**:4376-4379

[18] Dumas C, Mollica A, Feron D, Basseguy R, Etcheverry L, Bergel A. Marine microbial fuel cell: Use of stainless steel electrodes as anode and cathode materials. Electrochimica

[19] Heijne A, Hamelers H, Saakes M, Buisman C. Performance of non-porous graphite and titanium-based anodes in microbial fuel cells. Electrochimica Acta. 2008;**53**:5697-5703 [20] Watanabe K. Recent developments in microbial fuel cell technologies for sustainable

[21] Wang B. Recent development of non-platinum catalysts for oxygen reduction reaction.

[22] Logan BE, Hamelers B, Rozendal R, Schro¨der U, Keller J, Freguia S, et al. Microbial fuel cells: Methodology and technology. Environmental Science & Technology. 2006;

[23] Chen Z, Higgins D. Nitrogen doped carbon nanotubes and their impact on the oxygen

[24] Roche I, Katuri K, Scott K. A microbial fuel cell using manganese oxide oxygen reduction

[25] Zhang L, Liu C, Zhuang L, Li W, Zhou S, Zhang J. Manganese dioxide as an alternative cathodic catalyst to platinum in microbial fuel cells. Biosensors & Bioelectronics.

gen reduction reaction and their applications in microbial fuel cells. Biosensors &

[27] Zhang Y, Hu Y, Li S, Sun J, Hou B. Manganese dioxide-coated carbon nanotubes as an improved cathodic catalyst for oxygen reduction in a microbial fuel cell. Journal of

[28] Liu XW, Sun XF, Huang YX, Sheng GP, Zhou K, Zeng RJ, et al. Nano-structured manganese oxide as a cathodic catalyst for enhanced oxygen reduction in a microbial fuel cell

native cathodic catalyst to platinum in microbial fuel cells. Journal of Power Sources.

catalysts for oxy-


bioenergy. Journal of Bioscience and Bioengineering. 2008;**6**:528-536

Sustainable Energy Reviews. 2014;**39**:617-627 [16] Logan BE, Microbial Fuel Cells. 1st ed. Wiley; 2007

Journal of Power Sources. 2005;**152**:1-15

reduction reaction in fuel cells. Carbon. 2010;**48**:57-65

catalysts. Journal of Applied Electrochemistry. 2010;**40**:13-21

[26] Lu M, Kharkwal S, Ng H, Li S. Carbon nanotube supported MnO<sup>2</sup>

fed with a synthetic wastewater. Water Research. 2010;**44**:5298-5305

[29] Wen Q, Wang S, Yan J, Cong L, Pan Z, Ren Y, et al. MnO<sup>2</sup>

Acta. 2007;**53**:468-473

62 Old Yeasts - New Questions

**40**(51):81-92

2009;**24**:2825-2829

2012;**216**:187-191

Bioelectronics. 2011;**26**:4728-4732

Power Sources. 2011;**196**:9284-9289


[57] Shkil H, Schulte A, Guschin D, Schuhmann W. Electron transfer between genetically modified *Hansenula polymorpha* yeast cells and electrode surfaces via Os-complex modified redox polymers. ChemPhysChem. 2011;**12**:806-813

[43] Christwardana M, Kwon Y. Yeast and carbon nanotube based biocatalyst developed by synergetic effects of covalent bonding and hydrophobic interaction for performance enhancement of membraneless microbial fuel cell. Bioresource Technology.

[44] Wilkinson S, Klar J, Applegarth S. Optimizing biofuel cell performance using a targeted

[45] Ganguli R, Dunn BS. Kinetics of anode reactions for a yeast-catalysed microbial fuel cell.

[46] Gunawardena A, Fernando S, To F. Performance of a yeast-mediated biological fuel cell.

[47] Rahimnejad M, Najafpour G, Ghoreyshi A, Talebnia F, Premier G, Bakeri G, Kim J, Oh S. Thionine increases electricity generation from microbial fuel cell using *S. cerevisiae* and

[48] Permana D, Rosdianti D, Ishmayana S, Rachman S, Putra H, Rahayuningwulan D, Hariyadi H. Preliminary investigation of electricity production using dual chamber microbial fuel cell (DCMFC) with *S. cerevisiae* as biocatalyst and methylene blue as an

[49] Najafpour G, Rahimnejad M, Mokhtarian N, Daud W, Ghoreyshi A. Bioconversion of whey to electrical energy in a biofuel cell using *S. cerevisiae*. World Applied Sciences

[50] Kasem E, Tsujiguchi T, Nakagawa N. Effect of metal modification to carbon paper anodes on the performance of yeast-based microbial fuel cells part II: In the case with exogenous

[51] Sayed ET, Barakat NAM, Abdelkareem MA, Fouad H, Nakagawa N. Yeast extract as an effective and safe mediator for the Baker's-yeast-based microbial fuel cell. Industrial and

[52] Gal I, Schlesinger O, Amir L, Alfonta L. Yeast surface display of dehydrogenases in

[53] Hubenova Y, Mitov M. Potential application of Candida melibiosica in biofuel cells.

[54] Babanova S, Hubenova Y, Mitov M. Influence of artificial mediators on yeast-based fuel cell performance. Journal of Bioscience and Bioengineering. 2011;**112**:379-387

[55] Hubenova Y, Rashkov R, Buchvarov V, Arnaudova M, Babanova S, Mitov M.Improvement of yeast-biofuel cell output by electrode modifications. Industrial and Engineering

[56] Hubenova Y, Rashkov R, Buchvarov V, Babanova S, Mitov M. Nanomodified NiFe- and NiFeP-carbon felt as anode electrocatalysts in yeast-biofuel cell. Journal of Materials

mediator, methylene blue. Key Engineering Materials. 2013;**534**:82-87

exoelectrogenic mixed culture. The Journal of Microbiology. 2012;**50**:575-580

mixed mediator combination. Electroanalysis. 2006;**18**:2001-2007

International Journal of Molecular Sciences. 2008;**9**:1893-1907

electron mediator. Procedia Chemistry. 2015;**17**:36-43

Engineering Chemistry Research. 2015;**54**:3116-3122

Bioelectrochemistry. 2010;**78**:57-61

Chemistry Research. 2011;**50**:557-564

Science. 2011;**46**:7074-7081

microbial fuel-cells. Bioelectrochemistry. 2016;**112**:53-60

2017;**225**:175-182

64 Old Yeasts - New Questions

Fuel Cell. 2009;**9**:44-52

Journal. 2010;**8**:01-05


**Provisional chapter**

#### **Advances in Metabolic Engineering of** *Saccharomyces cerevisiae* **for the Production of Industrially and Clinically Important Chemicals** *cerevisiae* **for the Production of Industrially and Clinically Important Chemicals**

**Advances in Metabolic Engineering of** *Saccharomyces* 

DOI: 10.5772/intechopen.70327

Burcu Turanlı-Yıldız, Burcu Hacısalihoğlu and Z. Petek Çakar Z. Petek Çakar Additional information is available at the end of the chapter

Burcu Turanlı-Yıldız, Burcu Hacısalihoğlu and

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.70327

#### **Abstract**

Sustainable production of chemicals is of increasing importance, due to depletion of petroleum and environmental concerns. In addition to its importance in basic research as a simple, eukaryotic model organism, *Saccharomyces cerevisiae* has long been exploited in industry because of its physiological properties. And today, the development in genetic engineering toolbox and genome-scale metabolic models of *S. cerevisiae* has extended its application range to new products and bioprocesses. In addition, evolutionary engineering strategies have been useful in improving cellular properties of *S. cerevisiae*, such as tolerance to product toxicity and inhibitors. In this chapter, recent metabolic and evolutionary engineering studies that involve *S. cerevisiae* for the production of bulk chemicals and fine chemicals including flavours and pharmaceuticals are reviewed. It was shown that metabolic engineering particularly allowed the improvement of pharmaceuticals production, which will enable economic and large-scale production of many valuable pharmaceuticals. It is clear that *S. cerevisiae* will continue to be an important host for future metabolic engineering and metabolic pathway engineering applications to produce a variety of industrially and clinically important chemicals.

**Keywords:** pharmaceuticals, adaptive evolution, bulk chemicals, evolutionary engineering, flavours, fine chemicals, glutathione, metabolic engineering, organic acids, *Saccharomyces cerevisiae*

#### **1. Introduction**

Metabolic engineering was defined by Bailey [1] as 'the improvement of cellular activities by manipulation of enzymatic, transport and regulatory functions of the cell with the use of

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

recombinant DNA technology'. More than 20 years after this first definition as a new scientific discipline, metabolic engineering has become an increasingly important research field of biotechnology. Today, metabolic engineering requires interdisciplinary work that includes molecular biology, applied microbiology, biochemical reaction engineering, biomedical research with the aid of high-throughput analytical tools in 'omics' research and bioinformatics [2].

There are two major approaches in metabolic engineering, as described by Bailey et al. [3], the rational metabolic engineering and inverse metabolic engineering. In rational metabolic engineering, extensive genetic and biochemical information is required on the metabolism or metabolic pathway of interest to make defined genetic manipulations. The cellular physiological responses are also complex. Thus, trying to re-engineer a cellular machine that is too complex and about which there is limited information is a major limitation in rational metabolic engineering. Difficulties in cloning in industrial strains due to the lack of relevant genetic tools, and GMO-concerns of the public regarding food industry are additional issues [2]. The inverse metabolic engineering approach was designed to avoid the above-mentioned limitations. Here, the desired phenotype is identified first, as a 'bottom-up' approach, and then its genetic and/or environmental basis is determined which is the most challenging step. However, owing to the powerful high-throughput analytical technologies in genomics, transcriptomics, proteomics and metabolomics, this step is becoming easier [2, 4]. Thus, without any need for extensive initial information on biochemistry, genetics and regulation on the organism of interest, the desired phenotype can be obtained. Adaptive evolution or evolutionary engineering, which is based on random mutation and selection by systematic cultivation of an initial microbial culture in the presence of a selective pressure to obtain desirable phenotypes [5], is a common inverse metabolic engineering strategy [2].

Metabolic engineering is a key strategy for harnessing microorganisms' ability to produce chemicals from renewable carbon sources. Microbial processes are attractive since they have significantly lower environmental impacts than the petroleum-based processes. However, the former is primarily an economic challenge. Therefore, it is vital to develop superior strains with improved yield, titer and productivity by engineering microbial physiology, stress response and metabolism [6]. Considering the market value of chemical products based on petroleum, the cost-competitive bio-based products, once achieved, would have significant economic value as replacements. It is estimated that the global market share of bio-based chemicals will rise from 2% in 2008 to 22% in 2025 [7].

In this chapter, we focused on the recent metabolic engineering studies that involve the baker's yeast, *Saccharomyces cerevisiae*, for the production of industrially and clinically important compounds. *S. cerevisiae* has many advantages to be used in metabolic engineering studies: it has 'Generally Recognized as Safe' (GRAS) status, and there is extensive information on its genetics, physiology and biochemistry. Besides being a common industrial microorganism for ethanol fermentation, baking, brewing, etc., *S. cerevisiae* has been regarded as a versatile cell factory for the production of a wide range of natural compounds by manipulation of the endogenous pathways and/or integration of heterologous pathways. In this review, metabolic engineering studies with *S. cerevisiae* are divided into two major categories: production of bulk chemicals, and production of fine chemicals including flavours and pharmaceuticals. Regarding the production of bulk chemicals, examples of organic acids that have potentials to be produced by fermentation at large-scale were discussed. As fine chemicals, glutathione and a variety of secondary metabolites used in food, cosmetic and health industries were discussed.

#### **2. Production of bulk chemicals**

recombinant DNA technology'. More than 20 years after this first definition as a new scientific discipline, metabolic engineering has become an increasingly important research field of biotechnology. Today, metabolic engineering requires interdisciplinary work that includes molecular biology, applied microbiology, biochemical reaction engineering, biomedical research with the aid of high-throughput analytical tools in 'omics' research and bioinformatics [2].

68 Old Yeasts - New Questions

There are two major approaches in metabolic engineering, as described by Bailey et al. [3], the rational metabolic engineering and inverse metabolic engineering. In rational metabolic engineering, extensive genetic and biochemical information is required on the metabolism or metabolic pathway of interest to make defined genetic manipulations. The cellular physiological responses are also complex. Thus, trying to re-engineer a cellular machine that is too complex and about which there is limited information is a major limitation in rational metabolic engineering. Difficulties in cloning in industrial strains due to the lack of relevant genetic tools, and GMO-concerns of the public regarding food industry are additional issues [2]. The inverse metabolic engineering approach was designed to avoid the above-mentioned limitations. Here, the desired phenotype is identified first, as a 'bottom-up' approach, and then its genetic and/or environmental basis is determined which is the most challenging step. However, owing to the powerful high-throughput analytical technologies in genomics, transcriptomics, proteomics and metabolomics, this step is becoming easier [2, 4]. Thus, without any need for extensive initial information on biochemistry, genetics and regulation on the organism of interest, the desired phenotype can be obtained. Adaptive evolution or evolutionary engineering, which is based on random mutation and selection by systematic cultivation of an initial microbial culture in the presence of a selective pressure to obtain desirable

Metabolic engineering is a key strategy for harnessing microorganisms' ability to produce chemicals from renewable carbon sources. Microbial processes are attractive since they have significantly lower environmental impacts than the petroleum-based processes. However, the former is primarily an economic challenge. Therefore, it is vital to develop superior strains with improved yield, titer and productivity by engineering microbial physiology, stress response and metabolism [6]. Considering the market value of chemical products based on petroleum, the cost-competitive bio-based products, once achieved, would have significant economic value as replacements. It is estimated that the global market share of bio-based

In this chapter, we focused on the recent metabolic engineering studies that involve the baker's yeast, *Saccharomyces cerevisiae*, for the production of industrially and clinically important compounds. *S. cerevisiae* has many advantages to be used in metabolic engineering studies: it has 'Generally Recognized as Safe' (GRAS) status, and there is extensive information on its genetics, physiology and biochemistry. Besides being a common industrial microorganism for ethanol fermentation, baking, brewing, etc., *S. cerevisiae* has been regarded as a versatile cell factory for the production of a wide range of natural compounds by manipulation of the endogenous pathways and/or integration of heterologous pathways. In this review, metabolic

phenotypes [5], is a common inverse metabolic engineering strategy [2].

chemicals will rise from 2% in 2008 to 22% in 2025 [7].

The oil refinery is currently the major source of bulk chemicals such as solvents and polymer precursors. A significant portion of petroleum is used in the chemical catalysis for the production of chemicals and plastics [8]. However, in recent years, microbial production of chemicals based on renewable sources, such as biomass, has become important as a part of the efforts to reduce demand on diminishing petroleum and to reduce hazardous wastes. In addition, biotechnology makes new chemical monomers accessible, which are otherwise inaccessible due to high production cost [9].

In bio-refineries, the biomass is the first converted into simple sugars and then to valuable chemicals. Microorganisms are the main players of the latter conversion. Therefore, the development of a suitable strain for the particular process is needed. As a model yeast, *S. cerevisiae* has been a focus of metabolic engineering studies for the bio-based production of chemicals. 1,4-Diacids (succinic, fumaric and malic), itaconic acid, 3-hydroxypropionic acid and lactic acid are organic acids listed among the high-potential targets for industrial biotechnology [10]. Representative examples for the production of these bulk chemicals by metabolically engineered *S. cerevisiae* are summarized in **Table 1**.

Succinic acid is used in a wide range of industries from food to agriculture. Also, it has been considered as a generic intermediate for the bio-based polymers and can be a substitute of petroleum-derived maleic anhydride, which has a huge market [11]. Therefore, an increasing demand of succinic acid is expected in the future. Currently, it is mainly produced by chemical syntheses, which are based on petrochemical precursors. Biotechnological routes are pursued to achieve a sustainable production of succinic acid. *Anaerobiospirillum succiniciproducens* and *Actinobacillus succinogenes* are natural succinic acid producers. However, these organisms are prokaryotes that favour neutral pH for growth and require neutralization and a cost-additive product recovery process. In addition, there is a lack of suitable genetic tools for these organisms [12]. Although *S. cerevisiae* is not a natural producer of succinic acid as an end product, there have been efforts to metabolically engineer *S. cerevisiae,* since it has favourable properties such as the ability to operate at low pH values [13]. In general, the tricarboxylic acid (TCA) cycle and glyoxylate shunt are the focus of these studies. In order to redirect oxidative TCA pathway, elimination of succinate and isocitrate dehydrogenases has been proposed as a strategy. A yeast strain with disturbed TCA cycle due to deletions of *SDH1, SDH2, IDH1, IDP1*, produced succinic acid at a yield of 0.11 mol/mol glucose in shake


**Table 1.** Bulk chemical production by metabolically engineered *S. cerevisiae*.

flask cultures [14]. A computational pathway prediction algorithm has been utilized to identify multiple gene deletion targets to redirect carbon fluxes towards succinic acid [15]. Three deletion targets, *SDH3*, *SER3* and *SER33*, were identified to couple succinic acid production to biomass formation. This strategy was based on the elimination of succinic acid consumption by the deletion of *SDH3* encoding cytochrome b subunit of succinate dehydrogenase. The serine biosynthesis was also disrupted by the deletions of *SER3* and *SER33*, which are paralogs encoding 3-phosphoglycerate dehydrogenase. Therefore, serine and glycine production were linked to succinic acid production via glyoxylate pathway. However, the engineered strain required glycine to be supplemented in the medium. Further, two successive laboratory evolution experiments for glycine prototrophy and faster growth were performed with this strain. Finally, overexpression of isocitrate lyase, Icl1p, in the evolved strain, resulted in a succinic acid yield of 0.07 mol/mol glucose under aerobic conditions without glycine addition. Metabolic profiling analysis of a succinic acid-producing recombinant *S. cerevisiae* hinted a metabolic engineering strategy involving expression of a malic acid transporter from *Schizosaccharomyces pombe* (*MAE1)* to export succinic acid out of cells [16].

Itaconic acid has currently application in the manufacture of pharmaceuticals, adhesives and resins. In addition, its polymerized form (polyitaconic acid) has potentials as a replacement of acrylic acid in the development of superabsorbents [17], and can be used in contact lenses, detergents and cleaners [18]. *Aspergillus terreus* is the present organism of choice for the industrial fermentation of itaconic acid. However, the process bears some constraints due to inherent characteristics of *A. terreus*, such as inhibition in the media and sensitivity to shear stress [19]. Kanamasa et al*.* isolated *cis*-aconitic acid decarboxylase (CAD), which is the key enzyme in the conversion of *cis*-aconitate to itaconic acid in *A. terreus*, and its heterologous expression in *S. cerevisiae* showed the possibility of itaconic acid production in yeast [20]. Blazeck et al*.* utilized a synthetic hybrid promoter carrying an enhancer and a core promoter module to optimize CAD expression in *S. cerevisiae* [19, 21]. A genome-wide metabolic model of the yeast was used to identify gene deletion targets to further increase the itaconic acid titer. Three sequential rounds of genome scan *in silico* highlighted three deletion targets; cytoplasmic trifunctional C1-tetrahydrofolate (THF) synthase, a putative tryptophan 2,3-dioxygenase or indoleamine 2,3-dioxygenase and a peroxisomal acyl-CoA thioesterase, encoded by *ADE3, BNA2* and *TES1*, respectively. The deletions rewired metabolic flux towards TCA cycle and enhanced itaconic acid titer (168 mg/L). However, further efforts are necessary to redirect carbon flux towards itaconic acid production in the yeast to approach titers obtained in *Aspergillus* species (>80 g/L).

3-Hydroxypropionic acid (3-HP) is another important platform chemical which can be produced from either sugars or glycerol and can be converted to 1,3-propanediol, acrylic acid, malonic acid, and acrylamide. 3-HP derivatives have a variety of applications in super absorbent polymers, surface coatings, adhesives and paints [11]. Although there are biological pathways to 3-HP via either glycerol, lactate, malonyl-CoA or β-alanine intermediates, no organism is known to produce it as an end product [22]. The pathways based on malonyl-CoA and β-alanine have been constructed in *S. cerevisiae* [23, 24]. Chen et al*.* evaluated different malonyl-CoA reductases. Malonyl-CoA reductase (MCRCa) from *Chloroflexus aurantiacus* was expressed in the yeast for the conversion of malonyl-CoA to 3-HP in a two-step reduction reaction. Further, carbon flux was redirected towards 3-HP through increasing the levels of malonyl-CoA and its immediate precursor, acetyl-CoA. For this purpose, native *ADH2* (alcohol dehydrogenase) and *ALD6* (NADP-dependent aldehyde dehydrogenase), and synthetic *acsL641P SE* (acetylation-insensitive acetyl-CoA synthetase from *Salmonella enterica*) were overexpressed to increase the level of acetyl-CoA. The cellular concentration of malonyl-CoA was increased by over-expression of *ACC1* (acetyl-CoA carboxylase), which is the sole enzyme in the conversion of acetyl-CoA to malonyl-CoA. Finally, 3-HP was produced at a titer of 463 mg/L when the production was coupled with enhanced supply of electron donor of MCRCa (NADPH) by heterologous expression of GAPNp (a non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase from *Streptococcus mutans*) [23]. In another study, a significant improvement of 3-HP production was achieved when multiple copies of MCR were integrated

flask cultures [14]. A computational pathway prediction algorithm has been utilized to identify multiple gene deletion targets to redirect carbon fluxes towards succinic acid [15]. Three deletion targets, *SDH3*, *SER3* and *SER33*, were identified to couple succinic acid production to biomass formation. This strategy was based on the elimination of succinic acid consumption by the deletion of *SDH3* encoding cytochrome b subunit of succinate dehydrogenase. The serine biosynthesis was also disrupted by the deletions of *SER3* and *SER33*, which are paralogs encoding 3-phosphoglycerate dehydrogenase. Therefore, serine and glycine production were linked to succinic acid production via glyoxylate pathway. However, the engineered strain required glycine to be supplemented in the medium. Further, two successive laboratory evolution experiments for glycine prototrophy and faster growth were performed with this strain. Finally, overexpression of isocitrate lyase, Icl1p, in the evolved strain, resulted

**Bulk chemical produced Representative studies and their strain improvement strategy [reference no]** Succinic acid Disturbance of the citric acid cycle by deleting *SDH1, SDH2, IDH1, IDP1* [14]

Itaconic acid Overexpression of *CAD* with a synthetic hybrid promoter and enhancement of flux

3-Hydroxypropionic acid Reconstruction of malonyl-CoA to 3-HP pathway via expression of *MCR* from

overexpressing *PAND* from *Tribolium castaneum* [24]

Lactic acid Expression of genome-integrated *L-LDH* from bovine under *PDC1* promoter and

*SER33* [15]

genes [19]

cofactor availability [23]

inactivation of *PDC1* [28]

DNA library [35]

**Table 1.** Bulk chemical production by metabolically engineered *S. cerevisiae*.

[16]

70 Old Yeasts - New Questions

[25]

Disabled serine synthesis from glycolysis through a triple deletion of *SDH1*, *SER3* and

*Schizosaccharomyces pombe* in *Saccharomyces cerevisiae SDH1*- and *SDH2*-disrupted strains

towards the citric acid cycle by the sequential deletion of the *ADE3*, *BNA2* and *TES1*

*Sulfolobus tokodaii* and *HPDH* from *Metallosphaera sedula* and increased precursor and

Reconstruction of β-alanine to 3-HP pathway via coexpression of *BAPAT* from *Bacillus cereus* and *HPDH* from *Escherichia coli* and redirection of flux towards β-alanine by

Reconstruction of malonyl-CoA to 3-HP pathway via coexpression of *MCR* from *Chloroflexus aurantiacus* and an inhibition-deficient *ACC1* and optimization of acetyl-CoA supply by overexpressing native *PDC1, ALD6, and ACS* from *Salmonella enterica*

Adaptive laboratory evolution for improved tolerance to 3-HP at pH 3.5 [45]

Expression of genome-integrated *D-LDH* from *Leuconostoc mesenteroides* subsp.

Deletion of *PDC1* and expression of multiple copies of *L-LDH* from bovine [30] Inhibition of L-LDH consumption by deletion of *DLD1* and *JEN1*, elimination of ethanol and glycerol production by deleting *PDC1, ADH1*, *GPD1* and *GPD2,* and improvement of lactic acid tolerance by adaptive evolution and overexpression of *HAA1* [31]

Repression of ethanol production by deleting *PDC1* and *ADH1* and enhanced acetyl-CoA supply by the introduction of the genes encoding acetylating acetaldehyde

Expression of *ESBP6*, a novel target isolated by screening a multi-copy yeast genomic

Enhancement of lactic acid transport by expressing *JEN1* and *ADY1* [34]

*mesenteroides* under *PDC1* promoter and inactivation of *PDC1* [29]

Overexpression of *HXT1* and *HXT7* hexose transporters [32]

dehydrogenase enzyme from *Escherichia coli* [33]

Enhanced succinic acid export via heterologous expression of *MAE1* from

into the yeast genome and a modified ACCp with phosphorylation deficiency was expressed. Finally, engineering of the redox metabolism of this strain produced 3-HP at a titer of 9.8 g/L in a glucose-limited, fed-batch system [25]. Borodina et al*.* utilized genome-scale modelling to compare the two biosynthetic routes in terms of maximum theoretical yields and identified β-alanine pathway as a more favourable route. They implemented the biosynthesis of 3-HP from glucose via β-alanine through coexpression of β-alanine-pyruvate aminotransferase from *Bacillus cereus* and 3-hydroxypropanoate dehydrogenase from *Escherichia coli*. Further, carbon-flux was redirected towards β-alanine by the supply of L-aspartate, the immediate precursor of β-alanine. The final strain yielded 3-HP at a titer of 13.7 g/L in glucose-limited fed-batch cultivation. In a similar fashion, production of 3-HP via both malonyl-CoA and β-alanine pathway was reported in a xylose-utilizing *S. cerevisiae* [24].

Lactic acid is a well-known fermentation product which is already widely used in food, cosmetics and pharmaceutical industries. Lactic acid derived from biomass is also valued as a monomer in the development of bioplastics [26]. Lactic acid bacteria, especially, *Lactobacillus* species, are often employed in lactic acid production. For large-scale lactic acid production, fermenting microorganisms with high acid tolerance, simple nutritional requirements and capability of growth at high cell density are pursued [27]. To this end, *S. cerevisiae* was engineered for lactic acid production by integrating lactate dehydrogenase (*LDH*) gene into its genome [28, 29]. Reduction in ethanol and glycerol production is desirable to direct metabolite fluxes to lactic acid production. Therefore, deletions of *PDH* encoding pyruvate dehydrogenase, *ADH* encoding alcohol dehydrogenase and *GPD1* encoding glycerol-3-phosphate dehydrogenase were reported to improve lactic acid production in *LDH*-expressing yeast strains [30, 31]. Another approach was the improvement of cell growth either by an increased glucose uptake via overexpression of hexose transporters (*HXT1* and *HXT7*) or an enhanced acetyl-CoA supply through implementing an acetyl-CoA synthesis pathway from *E. coli* in lactic acid-producing *S. cerevisiae* [32, 33]. In addition, elimination of NADH-consuming reactions through deletions of *NDE1* and *NDE2* encoding mitochondrial external NADH dehydrogenases was shown to improve lactic acid production due to increased cofactor availability. The yeast strains that expressed *JEN1* and *ADY2* encoding monocarboxylate permeases constitutively had improved lactic acid production due to higher efflux of lactic acid [34]. Recently, screening of a multi-copy genomic DNA library revealed a novel protein (ESBP6) involved in lactic acid adaptation response, although having a low similarity to monocarboxylate permeases [35]. Lactic acid accumulation under low pH conditions has detrimental effects on yeast cells. Therefore, tolerance to weak acids is another target to achieve high levels of organic acids like lactic acid. A recombinant *LDH*-expressing yeast strain was subjected to adaptive laboratory evolution in the presence of gradually increased D-lactic acid levels. A lactate over-producing strain was obtained with additional copies of *LDH* and *HAA1*, encoding a transcription activator involved in lactic acid stress, and a titer of 112 g/L was achieved in fed-batch cultivation [31].

Product toxicity is a major obstacle for achieving high titers of the target chemicals such as organic acids, aromatic substances and antibiotics [36]. There is limited knowledge about the molecular basis of the product toxicity and tolerance to enable a rational prediction of genetic changes [37]. David et al*.* developed, for the first time, a hierarchical dynamic pathway control system involving a two-stage fermentation concept and the use of a metabolic sensor in *S. cerevisiae* [38]. The growth and production phases were decoupled to allow sufficient biomass formation before accumulation of the product beyond toxic levels. In addition, they designed a metabolite sensor based on prokaryotic fapR-fapO system to regulate expression of pathway enzymes in relation to availability of metabolite pools during the production phase. Efficiency of this concept was demonstrated in 3-HP production, which was increased by 10-fold in titers. A more common, alternative approach used against product toxicity or toxic/inhibitory compounds is evolutionary engineering. It is particularly useful for obtaining genetically complex microbial phenotypes such as tolerance to inhibitors/toxic compounds or various stress types [39]. Successful results were obtained by our research group, regarding evolutionary engineering of multi-stress resistant [40], cobalt-resistant [41, 42], nickel-resistant [43], and ethanol-tolerant [44] *S. cerevisiae*. Another example for the use of evolutionary engineering against product toxicity involves adaptive evolution for lactic acid tolerance in *S. cerevisiae* [31]. Similarly, Kildegaard et al*.* isolated *S. cerevisiae* strains with resistance to 3-HP through laboratory evolution. Genome sequencing of the evolved strains and subsequent functional analyses identified a relevant mutation in *SFA1* gene (*S*-(hydroxymethyl) glutathione dehydrogenase) related to 3-HP tolerance [45].

#### **3. Production of fine chemicals**

Plant secondary metabolites hold the potential to be used as pharmaceuticals, cosmetic and food ingredients. However, the yield of these molecules when extracted from natural producers is not in sufficient amounts to meet industrial demands. In addition, chemical synthesis of these complex structures often requires multiple reaction steps and is not a commercially attractive route due to low product yields [46]. Currently, advances in metabolic engineering allowed commercial-scale microbial production of a number of fine chemicals [47–49]. Besides, there is an ongoing academic interest for reconstitution of biosynthetic pathways of several natural products, including complex pathways, in *S. cerevisiae.* Discovery of gene clusters involved in the biosynthesis of secondary metabolites have enhanced progress in microbial production of these molecules [50]. Computational studies have also been conducted to optimize heterologous production in a variety of industrial host microorganisms including *S. cerevisiae,* which involved application of flux balance analysis on genome-scale models for different hosts to identify the optimum host for production [51].

#### **3.1. Flavours**

into the yeast genome and a modified ACCp with phosphorylation deficiency was expressed. Finally, engineering of the redox metabolism of this strain produced 3-HP at a titer of 9.8 g/L in a glucose-limited, fed-batch system [25]. Borodina et al*.* utilized genome-scale modelling to compare the two biosynthetic routes in terms of maximum theoretical yields and identified β-alanine pathway as a more favourable route. They implemented the biosynthesis of 3-HP from glucose via β-alanine through coexpression of β-alanine-pyruvate aminotransferase from *Bacillus cereus* and 3-hydroxypropanoate dehydrogenase from *Escherichia coli*. Further, carbon-flux was redirected towards β-alanine by the supply of L-aspartate, the immediate precursor of β-alanine. The final strain yielded 3-HP at a titer of 13.7 g/L in glucose-limited fed-batch cultivation. In a similar fashion, production of 3-HP via both malonyl-CoA and

Lactic acid is a well-known fermentation product which is already widely used in food, cosmetics and pharmaceutical industries. Lactic acid derived from biomass is also valued as a monomer in the development of bioplastics [26]. Lactic acid bacteria, especially, *Lactobacillus* species, are often employed in lactic acid production. For large-scale lactic acid production, fermenting microorganisms with high acid tolerance, simple nutritional requirements and capability of growth at high cell density are pursued [27]. To this end, *S. cerevisiae* was engineered for lactic acid production by integrating lactate dehydrogenase (*LDH*) gene into its genome [28, 29]. Reduction in ethanol and glycerol production is desirable to direct metabolite fluxes to lactic acid production. Therefore, deletions of *PDH* encoding pyruvate dehydrogenase, *ADH* encoding alcohol dehydrogenase and *GPD1* encoding glycerol-3-phosphate dehydrogenase were reported to improve lactic acid production in *LDH*-expressing yeast strains [30, 31]. Another approach was the improvement of cell growth either by an increased glucose uptake via overexpression of hexose transporters (*HXT1* and *HXT7*) or an enhanced acetyl-CoA supply through implementing an acetyl-CoA synthesis pathway from *E. coli* in lactic acid-producing *S. cerevisiae* [32, 33]. In addition, elimination of NADH-consuming reactions through deletions of *NDE1* and *NDE2* encoding mitochondrial external NADH dehydrogenases was shown to improve lactic acid production due to increased cofactor availability. The yeast strains that expressed *JEN1* and *ADY2* encoding monocarboxylate permeases constitutively had improved lactic acid production due to higher efflux of lactic acid [34]. Recently, screening of a multi-copy genomic DNA library revealed a novel protein (ESBP6) involved in lactic acid adaptation response, although having a low similarity to monocarboxylate permeases [35]. Lactic acid accumulation under low pH conditions has detrimental effects on yeast cells. Therefore, tolerance to weak acids is another target to achieve high levels of organic acids like lactic acid. A recombinant *LDH*-expressing yeast strain was subjected to adaptive laboratory evolution in the presence of gradually increased D-lactic acid levels. A lactate over-producing strain was obtained with additional copies of *LDH* and *HAA1*, encoding a transcription activator involved in lactic acid stress, and a titer of 112 g/L was achieved

Product toxicity is a major obstacle for achieving high titers of the target chemicals such as organic acids, aromatic substances and antibiotics [36]. There is limited knowledge about the molecular basis of the product toxicity and tolerance to enable a rational prediction of genetic changes [37]. David et al*.* developed, for the first time, a hierarchical dynamic pathway control

β-alanine pathway was reported in a xylose-utilizing *S. cerevisiae* [24].

in fed-batch cultivation [31].

72 Old Yeasts - New Questions

Compounds belonging to isoprenoid and phenolics type of secondary metabolites are valued as natural fragrances and flavours. Flavour compounds can be produced from sugars (*de novo* synthesis) or from specific precursors (bioconversion) by using microorganisms.

Vanillin, a phenolic aldehyde, is one of the first flavour compounds produced in microbial hosts at commercial-scale. Current state of the microbial production of vanillin based on various precursors and the available production hosts have been recently reviewed by Gallage and Møller [52]. *De novo* biosynthesis of vanillin from glucose in *S. cerevisiae* has also been reported [53]. A multi-step conversion of a shikimate pathway intermediate (3-dehydroshikimate) to vanillin has been achieved through heterologous expression of four genes from *Podospora pauciseta, Nocardia iowensis, Corynebacterium glutamicum* and *Homo sapiens*. Once the vanillin biosynthesis was established, genome-scale metabolic modelling was used to identify gene deletion targets to improve vanillin production in *S. cerevisiae*. *PDC1* and *GDH1* deletions resulted in a five-fold increase in production (500 mg/L) [47].

*p*-Coumaric acid, a hydroxyl derivative of cinnamic acid, is a commercially attractive endproduct and a platform compound for flavonoids, polyphenols and polyketides, as well. Rodriguez et al*.* achieved high titers (2 g/L) of *p*-coumaric acid as the end-product in *S. cerevisiae,* through optimization of native aromatic amino acid biosynthesis [54]. The competing pathways were eliminated while enhancing production pathways by the expression of feedback resistant enzymes in combination with gene deletions and overexpression of analogue enzymes from *E. coli*.

β-Ionone is an apocarotenoid that is naturally present in raspberries. In *S. cerevisiae*, *de novo* synthesis of β-ionone was reported [55]. Beekwilder et al*.* constructed a β-carotene synthesis pathway via farnesyl diphosphate (FPP) intermediate through polycistronic expression of genes from *Xanthophyllomyces dendrorhous.* The pathway was further extended, for the first time, to produce β-ionone by the expression of a carotenoid-cleavage dioxygenase (*CCD1*) from raspberry.

2-Phenyl ethanol (2-PE) is another economically attractive flavour compound with a rose-like scent. Ehrlich pathway is involved in the bioconversion of phenylalanine to 2-phenyl ethanol within *S. cerevisiae*. Elimination of allosteric feedback regulation on the aromatic amino acid biosynthesis resulted in an increase of up to 200-fold in the production of aromatic compounds, including 2-PE. Romagnoli et al*.* constructed a deletion library of non-essential genes in *S. cerevisiae* by Synthetic Genetic Array (SGA) technology and identified that *ARO8* encoding an aromatic amino acid transaminase is a target to improve phenylethanol production from glucose [56]. Recently, Shen et al*.* identified *AAT2* encoding a cytosolic aspartate aminotransferase as another deletion target [57]. Deletion of these two genes in combination with the overexpression of Ehrlich pathway enzymes resulted in a significant improvement in 2-PE production from glucose, at a titer of 96 mg/L.

#### **3.2. Pharmaceuticals**

Another major area of metabolic engineering research is the production of clinically important compounds. In this section, examples will be given for the production of a variety of such compounds by metabolically engineered yeast. Representative examples for the production of pharmaceuticals by metabolically engineered *S. cerevisiae* are summarized in **Table 2**.

Glutathione, a naturally occurring tripeptide, is an important compound used in health and cosmetic industries. It is produced by using *S. cerevisiae* at commercial-scale. There has been a remarkable progress in glutathione production by metabolic engineering studies over the last few decades. Improved levels of glutathione production were achieved by *YAP1*


**Table 2.** Production of pharmaceuticals by metabolically engineered *S. cerevisiae*.

and Møller [52]. *De novo* biosynthesis of vanillin from glucose in *S. cerevisiae* has also been reported [53]. A multi-step conversion of a shikimate pathway intermediate (3-dehydroshikimate) to vanillin has been achieved through heterologous expression of four genes from *Podospora pauciseta, Nocardia iowensis, Corynebacterium glutamicum* and *Homo sapiens*. Once the vanillin biosynthesis was established, genome-scale metabolic modelling was used to identify gene deletion targets to improve vanillin production in *S. cerevisiae*. *PDC1* and *GDH1* dele-

*p*-Coumaric acid, a hydroxyl derivative of cinnamic acid, is a commercially attractive endproduct and a platform compound for flavonoids, polyphenols and polyketides, as well. Rodriguez et al*.* achieved high titers (2 g/L) of *p*-coumaric acid as the end-product in *S. cerevisiae,* through optimization of native aromatic amino acid biosynthesis [54]. The competing pathways were eliminated while enhancing production pathways by the expression of feedback resistant enzymes in combination with gene deletions and overexpression of analogue

β-Ionone is an apocarotenoid that is naturally present in raspberries. In *S. cerevisiae*, *de novo* synthesis of β-ionone was reported [55]. Beekwilder et al*.* constructed a β-carotene synthesis pathway via farnesyl diphosphate (FPP) intermediate through polycistronic expression of genes from *Xanthophyllomyces dendrorhous.* The pathway was further extended, for the first time, to produce β-ionone by the expression of a carotenoid-cleavage dioxygenase (*CCD1*)

2-Phenyl ethanol (2-PE) is another economically attractive flavour compound with a rose-like scent. Ehrlich pathway is involved in the bioconversion of phenylalanine to 2-phenyl ethanol within *S. cerevisiae*. Elimination of allosteric feedback regulation on the aromatic amino acid biosynthesis resulted in an increase of up to 200-fold in the production of aromatic compounds, including 2-PE. Romagnoli et al*.* constructed a deletion library of non-essential genes in *S. cerevisiae* by Synthetic Genetic Array (SGA) technology and identified that *ARO8* encoding an aromatic amino acid transaminase is a target to improve phenylethanol production from glucose [56]. Recently, Shen et al*.* identified *AAT2* encoding a cytosolic aspartate aminotransferase as another deletion target [57]. Deletion of these two genes in combination with the overexpression of Ehrlich pathway enzymes resulted in a significant improvement in 2-PE

Another major area of metabolic engineering research is the production of clinically important compounds. In this section, examples will be given for the production of a variety of such compounds by metabolically engineered yeast. Representative examples for the production of pharmaceuticals by metabolically engineered *S. cerevisiae* are summarized in **Table 2**.

Glutathione, a naturally occurring tripeptide, is an important compound used in health and cosmetic industries. It is produced by using *S. cerevisiae* at commercial-scale. There has been a remarkable progress in glutathione production by metabolic engineering studies over the last few decades. Improved levels of glutathione production were achieved by *YAP1*

tions resulted in a five-fold increase in production (500 mg/L) [47].

enzymes from *E. coli*.

74 Old Yeasts - New Questions

from raspberry.

**3.2. Pharmaceuticals**

production from glucose, at a titer of 96 mg/L.

overexpression [58], metabolic engineering of the yeast sulphate assimilation pathway and glutathione biosynthetic pathway [59]*,* overexpression of a novel glutathione export ABC protein (Adp1p, Gxa1p) and the engineered thiol redox metabolism [60]. Also, the inverse metabolic engineering approach was used to increase glutathione production in *S. cerevisiae* [61, 62]*.* In an evolutionary engineering study, acrolein, a toxic α,β-unsaturated aldehyde, was used as a selection agent. Two rounds of adaptive evolution in the presence of increasing levels of acrolein resulted in evolved strains with acrolein tolerance and up-to 3.3-fold higher glutathione accumulation in comparison to the parental strain [61]. Genome shuffling has also been applied to obtain yeast strains with increased glutathione content. Two rounds of recursive protoplast fusion were performed with the improved strains initially obtained from ultraviolet irradiation and chemical mutagenesis. The strain with highest glutathione content showed 9.9-fold transcriptional up-regulation of glutathione synthetase gene (*GSH-I*) [62].

Terpene derivatives are economically viable molecules that are used in the synthesis of drugs such as the antimalarial agent artemisinin, and the anticancer agent taxol [63]. Several terpenoids have been produced in *S. cerevisiae* by reconstitution of the relevant biosynthetic pathways. As part of efforts to establish a solid source of artemisinin, *S. cerevisiae* was metabolically engineered to produce artemisinic acid, which is an artemisinin precursor [48]. As the microbially produced artemisinic acid was converted to artemisinin by synthetic chemistry methods, that study was reported as a good example for combining biological production by metabolic engineering with production by synthetic chemistry [64]. Paddon et al. have, for the first time, designed a *S. cerevisiae* strain with the complete biosynthetic pathway of artemisinic acid, involving overexpression of the mevalonate pathway enzymes, and achieved commercial-scale titers (25 g/L) [48].

The well-known diterpenoid taxol is an anti-cancer agent [63, 65]. As a first step towards taxol production, *S. cerevisiae* was metabolically engineered for taxadiene biosynthesis [66]. For this purpose, heterologous genes encoding enzymes from the early steps of the taxoid biosynthesis pathway, isoprenoid pathway, were introduced, along with a regulatory factor to inhibit competing pathways. The results were promising enough for taxol production in recombinant microorganisms [66]. By using protein modelling and substrate docking, different geranylgeranyl diphosphate synthases were screened and expressed in a recombinant taxadiene-producing yeast. The yeast strains were compared in terms of their metabolism using metabolomics approach to identify an efficient host for taxadiene production [67].

Forskolin is a labdene diterpene with potentials to be used in the treatment of blood pressure, in weight-loss supplements and in the protection against congestive heart failure. Ignea et al*.* constructed a yeast platform to produce 11β-hydroxy-manoyl oxide, forskolin precursor. Although the forskolin biosynthetic pathway has not been completely discovered yet, a promiscuous cytochrome P450 from *Salvia pomifera* was identified as a replacement to achieve the synthesis of the forskolin precursor. This study can provide a basis for the biosynthesis of various tricyclic (8,13)-epoxy-labdanes [68].

Polyketides are also a major group of natural products with a wide range of applications as antibiotics, immunosuppressors, cholesterol lowering agents and other drugs [69]. *S. cerevisiae* is known as a suitable production host for simple polyketides. An earlier study demonstrated the production of a simple polyketide, 6-methylsalicylic acid, by heterologous expression of 6-methylsalicylic acid synthase in *S. cerevisiae* [69]. However, the major challenge in the synthesis of complex polyketides was the lack of polyketide precursor pathways in *S. cerevisiae*. To overcome this, a relevant pathway was introduced into *S. cerevisiae* to produce a precursor for complex polyketides, methylmalonyl-coenzyme A (CoA). This engineered yeast strain had the capability of the production of a triketide lactone, when supplemented with propyldiketide thioester [70]. Since polyketides are derived from acetyl-CoA and malonyl-CoA precursors, an increase in the acetyl-CoA and the cofactor (NADPH) in a yeast strain expressing 2-pyrone synthase (2-PS) from *Gerbera hybrida* led to 6.4-fold higher triacetic acid lactone production, compared to the reference strain [71].

overexpression [58], metabolic engineering of the yeast sulphate assimilation pathway and glutathione biosynthetic pathway [59]*,* overexpression of a novel glutathione export ABC protein (Adp1p, Gxa1p) and the engineered thiol redox metabolism [60]. Also, the inverse metabolic engineering approach was used to increase glutathione production in *S. cerevisiae* [61, 62]*.* In an evolutionary engineering study, acrolein, a toxic α,β-unsaturated aldehyde, was used as a selection agent. Two rounds of adaptive evolution in the presence of increasing levels of acrolein resulted in evolved strains with acrolein tolerance and up-to 3.3-fold higher glutathione accumulation in comparison to the parental strain [61]. Genome shuffling has also been applied to obtain yeast strains with increased glutathione content. Two rounds of recursive protoplast fusion were performed with the improved strains initially obtained from ultraviolet irradiation and chemical mutagenesis. The strain with highest glutathione content showed 9.9-fold transcriptional up-regulation of glutathione synthetase gene (*GSH-I*) [62]. Terpene derivatives are economically viable molecules that are used in the synthesis of drugs such as the antimalarial agent artemisinin, and the anticancer agent taxol [63]. Several terpenoids have been produced in *S. cerevisiae* by reconstitution of the relevant biosynthetic pathways. As part of efforts to establish a solid source of artemisinin, *S. cerevisiae* was metabolically engineered to produce artemisinic acid, which is an artemisinin precursor [48]. As the microbially produced artemisinic acid was converted to artemisinin by synthetic chemistry methods, that study was reported as a good example for combining biological production by metabolic engineering with production by synthetic chemistry [64]. Paddon et al. have, for the first time, designed a *S. cerevisiae* strain with the complete biosynthetic pathway of artemisinic acid, involving overexpression of the mevalonate pathway enzymes, and achieved

The well-known diterpenoid taxol is an anti-cancer agent [63, 65]. As a first step towards taxol production, *S. cerevisiae* was metabolically engineered for taxadiene biosynthesis [66]. For this purpose, heterologous genes encoding enzymes from the early steps of the taxoid biosynthesis pathway, isoprenoid pathway, were introduced, along with a regulatory factor to inhibit competing pathways. The results were promising enough for taxol production in recombinant microorganisms [66]. By using protein modelling and substrate docking, different geranylgeranyl diphosphate synthases were screened and expressed in a recombinant taxadiene-producing yeast. The yeast strains were compared in terms of their metabolism using metabolomics approach to identify an efficient host for taxadiene production [67].

Forskolin is a labdene diterpene with potentials to be used in the treatment of blood pressure, in weight-loss supplements and in the protection against congestive heart failure. Ignea et al*.* constructed a yeast platform to produce 11β-hydroxy-manoyl oxide, forskolin precursor. Although the forskolin biosynthetic pathway has not been completely discovered yet, a promiscuous cytochrome P450 from *Salvia pomifera* was identified as a replacement to achieve the synthesis of the forskolin precursor. This study can provide a basis for the biosynthesis of

Polyketides are also a major group of natural products with a wide range of applications as antibiotics, immunosuppressors, cholesterol lowering agents and other drugs [69]. *S. cerevisiae* is known as a suitable production host for simple polyketides. An earlier study demonstrated

commercial-scale titers (25 g/L) [48].

76 Old Yeasts - New Questions

various tricyclic (8,13)-epoxy-labdanes [68].

The strategy of engineered precursor pools has also been applied in the production of resveratrol. Resveratrol is a polyketide derivative with potent antioxidant properties and it has been recently brought to market as a bio-product [72]. Earlier reports on the production of resveratrol were based on bioconversion of aromatic precursors such as *p*-coumaric acid and tyrosine by engineered *S. cerevisiae* strains [73, 74]*.* The highest resveratrol titer achieved by using this approach was obtained by an engineered industrial Brazilian *S. cerevisiae* strain, at a titer of 391 mg/L resveratrol on complex medium supplemented with *p*-coumaric acid [75]. Recently, in order to produce resveratrol from cheaper carbon sources, *de novo* biosynthesis of resveratrol via tyrosine intermediate in *S. cerevisiae* has been established by constructing an engineered pathway, involving tyrosine ammonia-lyase from *Herpetosiphon aurantiacus*, 4-coumaryl-CoA ligase from *Arabidopsis thaliana* and resveratrol synthase from *Vitis vinifera* [49]. To direct flux towards tyrosine, feedback-insensitive *ARO4* encoding 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase and *ARO7* encoding a chorismate mutase were overexpressed. To increase the precursor malonyl-CoA, an inactivation-sensitive acetyl-CoA carboxylase was overexpressed. Resveratrol production was further improved by integration of multiple copies of pathway genes, and finally, a titer of 415.65 and 531.41 mg/L resveratrol was obtained in a fed-batch cultivation with glucose or ethanol as the carbon source, respectively [76]. Koopman et al. also focused on deregulation of feedback mechanism of aromatic amino acid biosynthesis for *de novo* production of naringenin, which is an important platform molecule for the production of flavonoids [77].

Dihydrochalcones (DHCs) such as nothofagin, phlorizin and naringin dihydrochalcone are another group of polyketide derivatives with commercial value as antioxidants, antidiabetics or sweeteners. Recently, *de novo* synthesis of DHCs via phloretin intermediate has been reported in *S. cerevisiae* [78]. First, phloretin biosynthesis was achieved with the aid of a side activity of an endogenous double-bond reductase, in combination with heterologous pathway enzymes. To eliminate by-product formation, a chalcone synthase with high substrate specificity was expressed from *Hordeum vulgare*. Commencing with phloretin, several DHC derivatives with antioxidant, antidiabetic and sweetener properties have been obtained through an extension pathway involving methylation or glycosylation by previously known enzymes.

Recently, there have also been many reports on the reconstitution of biosynthetic pathways of alkaloids in yeast. Alkaloids are nitrogen-containing complex molecules with potent biological activity. Currently, there are around 50 alkaloid-based drugs, including the anticancer drug vincristine, the antitussive agent noscapine and the analgesic codeine. Strictosidine was the first reported plant-derived alkaloid produced *de nov*o in *S. cerevisiae* [79]. Strictosidine is a common intermediate of a list of alkaloids derived from tryptophan in plants, including the antimalarial quinine and anticancer agent vincristine [79]. Brown et al*.* reconstituted its biosynthetic pathway in *S. cerevisiae*. To enable strictosidine production in yeast, 14 genes from *Catharanthus roseus* were expressed [79]. The flux through the pathway was further improved by integration of additional copies of the relevant endogenous genes and three gene deletions that eliminated competing pathways. *S. cerevisiae* has also been engineered for the production of (*S*)-reticuline, which is a key branch point intermediate in the biosynthesis of a variety of alkaloids, including well-known opioids such as morphine and thebaine [80]. Bioconversion of a commercial substrate norlaudanosoline to reticuline was reported in an engineered yeast strain expressing three different AdoMet-dependent methyltransferase enzymes (6-OMT, CNMT and 4'-OMT) from plant and human origin [81]. Trenchard et al. constructed a route to reticuline which enabled *de novo* synthesis of this molecule via norcoclaurine intermediate, the actual intermediate in plants. The pathway comprised of a modified yeast amino acid biosynthesis pathway, in combination with a heterologous pathway involving seven relevant enzymes [80]. In other studies, *S. cerevisiae* strains were engineered to produce berberine, dihydrosanguinarine and noscapine from norlaudanosoline via reticuline intermediate, through a 7-, 10- and 14-step pathway involving heterologous expression of plant enzymes, respectively [82–84]. Also, the production of codeine and morphine from (*R*)-reticuline was reported by reconstitution of a seven-gene pathway in *S. cerevisiae* [85]. These studies provided a basis towards designing yeast cell factories for *de novo* production of reticuline-derived molecules. Recently, a complete pathway of biosynthesis of opioid thebaine from sugar has been established in *S. cerevisiae* [86]*.* This work involved a combination of enzyme discovery, protein engineering of a key cytochrome P450 and pathway optimization. The thebaine-producing yeast strains required expression of 21 heterologous genes from plants, mammals, bacteria and yeast. The pathway was also extended through expression of two additional genes from bacteria and plant to produce hydrocodone, a widely prescribed opioid drug.

#### **4. Summary and outlook**

For fine chemicals such as amino acids, vitamins, flavours, nutraceuticals, organic acids and fragrances, profit margins are usually not high and could be affected by substrate availability and cost. However, metabolic engineering enabled improvements in production of both pharmaceuticals and fine chemicals which will allow economic and large-scale production of many valuable compounds in near future.

It is obvious that *S. cerevisiae* will continue to be an important host for future metabolic engineering applications. There will be more comprehensive future studies on the production of chemicals by metabolic engineering of *S. cerevisiae.* These metabolic engineering strategies will most likely involve combinations of rational and inverse metabolic engineering approaches by adaptive evolution of recombinant *S. cerevisiae* with engineered metabolic pathways for various substrate utilization. Additionally, more studies on adaptive evolution and molecular characterization of tolerance to toxic end-products are expected in the future. Similarly, metabolic pathway engineering of *S. cerevisiae* will allow efficient production of more clinically important compounds and fine chemicals. It can be predicted that the advances in systems biology and bioinformatics will make a significant contribution to yeast metabolic engineering.

#### **Acknowledgements**

drug vincristine, the antitussive agent noscapine and the analgesic codeine. Strictosidine was the first reported plant-derived alkaloid produced *de nov*o in *S. cerevisiae* [79]. Strictosidine is a common intermediate of a list of alkaloids derived from tryptophan in plants, including the antimalarial quinine and anticancer agent vincristine [79]. Brown et al*.* reconstituted its biosynthetic pathway in *S. cerevisiae*. To enable strictosidine production in yeast, 14 genes from *Catharanthus roseus* were expressed [79]. The flux through the pathway was further improved by integration of additional copies of the relevant endogenous genes and three gene deletions that eliminated competing pathways. *S. cerevisiae* has also been engineered for the production of (*S*)-reticuline, which is a key branch point intermediate in the biosynthesis of a variety of alkaloids, including well-known opioids such as morphine and thebaine [80]. Bioconversion of a commercial substrate norlaudanosoline to reticuline was reported in an engineered yeast strain expressing three different AdoMet-dependent methyltransferase enzymes (6-OMT, CNMT and 4'-OMT) from plant and human origin [81]. Trenchard et al. constructed a route to reticuline which enabled *de novo* synthesis of this molecule via norcoclaurine intermediate, the actual intermediate in plants. The pathway comprised of a modified yeast amino acid biosynthesis pathway, in combination with a heterologous pathway involving seven relevant enzymes [80]. In other studies, *S. cerevisiae* strains were engineered to produce berberine, dihydrosanguinarine and noscapine from norlaudanosoline via reticuline intermediate, through a 7-, 10- and 14-step pathway involving heterologous expression of plant enzymes, respectively [82–84]. Also, the production of codeine and morphine from (*R*)-reticuline was reported by reconstitution of a seven-gene pathway in *S. cerevisiae* [85]. These studies provided a basis towards designing yeast cell factories for *de novo* production of reticuline-derived molecules. Recently, a complete pathway of biosynthesis of opioid thebaine from sugar has been established in *S. cerevisiae* [86]*.* This work involved a combination of enzyme discovery, protein engineering of a key cytochrome P450 and pathway optimization. The thebaine-producing yeast strains required expression of 21 heterologous genes from plants, mammals, bacteria and yeast. The pathway was also extended through expression of two additional genes from

bacteria and plant to produce hydrocodone, a widely prescribed opioid drug.

For fine chemicals such as amino acids, vitamins, flavours, nutraceuticals, organic acids and fragrances, profit margins are usually not high and could be affected by substrate availability and cost. However, metabolic engineering enabled improvements in production of both pharmaceuticals and fine chemicals which will allow economic and large-scale production of

It is obvious that *S. cerevisiae* will continue to be an important host for future metabolic engineering applications. There will be more comprehensive future studies on the production of chemicals by metabolic engineering of *S. cerevisiae.* These metabolic engineering strategies will most likely involve combinations of rational and inverse metabolic engineering approaches by adaptive evolution of recombinant *S. cerevisiae* with engineered metabolic pathways for various substrate utilization. Additionally, more studies on adaptive

**4. Summary and outlook**

78 Old Yeasts - New Questions

many valuable compounds in near future.

We thank Arman Akşit for technical assistance. Our research presented in this review was supported by the Scientific and Technological Research Council of Turkey (TUBITAK) (project nos: 105T314 and 107T284, PI: ZPÇ), TUBITAK-COST (project no: 109T638, PI: ZPÇ, COST Action no: CM0902), Federation of European Microbiological Societies (FEMS) Research Fellowship (2009-2 to BTY), Turkish State Planning Organization (DPT) and Istanbul Technical University Research Funds (project nos: 30108, 33237, 34200; PI: ZPÇ). BH is financially supported by the Faculty Member Training Programme (ÖYP) of the Council of Higher Education (YÖK), Turkey. We also would like to thank former graduate students Urartu Ö.Ş. Şeker, Ceren Alkım, Tuğba Sezgin, Ülkü Yılmaz, Gökhan Küçükgöze and Berrak Gülçin Balaban, as well as our colleagues and collaborators Uwe Sauer, Jean Marie François, Laurent Benbadis, Süleyman Akman, Bülent Balta, Candan Tamerler and Mehmet Sarıkaya for their scientific contribution in our research presented in this review.

#### **Author details**

Burcu Turanlı-Yıldız1,2,† , Burcu Hacısalihoğlu1,2,† and Z. Petek Çakar1,2\*

\*Address all correspondence to: cakarp@itu.edu.tr

1 Department of Molecular Biology and Genetics, Faculty of Science and Letters, Istanbul Technical University, Maslak, Istanbul, Turkey

2 Istanbul Technical University, Dr. Orhan Öcalgiray Molecular Biology, Biotechnology and Genetics Research Center (ITU-MOBGAM), Maslak, Istanbul, Turkey

† These authors have equal contribution to this work

#### **References**


[17] Shi D, Gao Y, Sun L, Chen M. Superabsorbent poly(acrylamide co itaconic acid) hydrogel microspheres : Preparation, characterization and absorbency. Polymer Science Series A. 2014;**56**:275-282

[3] Bailey JE, Sburlati A, Hatzimanikatis V, Lee K, Renner WA, Tsai PS. Inverse metabolic engineering: A strategy for directed genetic engineering of useful phenotypes. Biotechnology

[4] Bro C, Nielsen J. Impact of "ome" analyses on inverse metabolic engineering. Metabolic

[5] Winkler JD, Kao KC. Recent advances in the evolutionary engineering of industrial bio-

[6] Chubukov V, Mukhopadhyay A, Petzold CJ, Keasling JD, Martín HC. Synthetic and systems biology for microbial production of commodity chemicals. NPJ Systems Biology

[7] Biddy MJ, Scarlata C, Kinchin C. Chemicals from Biomass: A Market Assessment of Bioproducts with Near-Term Potential. No. NREL/TP-5100-65509. Golden, CO: National

[8] Adkins J, Pugh S, McKenna R, Nielsen DR. Engineering microbial chemical factories to

[9] Cherubini F. The biorefinery concept : Using biomass instead of oil for producing energy

[10] Bozell JJ, Peterson GR. Technology development for the production of biobased products from biorefinery carbohydrates — the US Department of Energy's "Top 10" revis-

[11] Patel M, Crank M, Dornburg V, Hermann B, Roes L, Husing B, Overbeek L, Terragni F, Recchia E. Medium and long-term opportunities and risks of the biotechnological production of bulk chemicals from renewable resources: The Potential of White

[12] Beauprez JJ, De Mey M, Soetaert WK. Microbial succinic acid production : Natural versus metabolic engineered producers. Process Biochemistry. 2010;**45**:1103-1114

[13] Ahn JH, Jang Y, Lee SY. Production of succinic acid by metabolically engineered micro-

[14] Raab AM, Gebhardt G, Bolotina N, Weuster-Botz D, Lang C. Metabolic engineering of *Saccharomyces cerevisiae* for the biotechnological production of succinic acid. Metabolic

[15] Agren R, Otero JM, Nielsen J. Genome-scale modeling enables metabolic engineering of *Saccharomyces cerevisiae* for succinic acid production. Journal of Industrial Microbiology

[16] Ito Y, Hirasawa T, Shimizu H. Metabolic engineering of *Saccharomyces cerevisiae* to improve succinic acid production based on metabolic profiling. Bioscience Biotechnology

produce renewable "biomonomers." Frontiers in Microbiology. 2012;**3**:1-12

and chemicals. Energy Conversation and Management. 2010;**51**:1412-1421

Biotechnology. Utrecht, Netherlands: The BREW Project; 2006

organisms. Current Opinion in Biotechnology. 2016;**42**:54-66

and Bioengineering. 1996;**52**:109-121

catalysts. Genomics. 2014;**104**:406-411

Renewable Energy Laboratory (NREL); 2016

ited. Green Chemistry. 2010;**12**:539-554

Engineering. 2010;**12**:518-525

and Biotechnology. 2013;**40**:735-747

and Biochemistry. 2014;**78**:151-159

Engineering. 2004;**6**:204-211

80 Old Yeasts - New Questions

and Applications. 2016;**2**:16009


[43] Küçükgöze G, Alkım C, Yılmaz Ü, Kısakesen Hİ, Gündüz S, Akman S, Çakar ZP. Evolutionary engineering and transcriptomic analysis of nickel-resistant *Saccharomyces cerevisiae*. FEMS Yeast Research. 2013;**13**:731-734

[30] Ishida N, Saitoh S, Ohnishi T, Tokuhiro K, Nagamori E, Kitamoto K, Takahashi H. Metabolic engineering of *Saccharomyces cerevisiae* for efficient production of pure L-(+) lactic acid. In: McMillan JD, Adney WS, Mielenz JR, Klasson KT, editors. Twenty-Seventh Symposium on Biotechnology for Fuels and Chemicals. Totowa, New Jersey: Humana

[31] Baek S, Kwon EY, Kim YH, Hahn J. Metabolic engineering and adaptive evolution for efficient production of D-lactic acid in *Saccharomyces cerevisiae*. Applied Microbiology

[32] Rossi G, Sauer M, Porro D, Branduardi P. Effect of HXT1 and HXT7 hexose transporter overexpression on wild-type and lactic acid producing *Saccharomyces cerevisiae* cells.

[33] Song J, Park J, Kang CD, Cho H, Yang D, Lee S, Myung K. Introduction of a bacterial acetyl-CoA synthesis pathway improves lactic acid production in *Saccharomyces cerevi-*

[34] Pacheco A, Sa-Pessoa J, Bessa D, Goncalves MJ, Paiva S, Casal M, Queiros O. Lactic acid production in *Saccharomyces cerevisiae* is modulated by expression of the monocarboxyl-

[35] Sugiyama M, Akase S, Nakanishi R, Kaneko Y, Harashima S. Overexpression of ESBP6 improves lactic acid resistance and production in *Saccharomyces cerevisiae*. Journal of

[37] Dragosits M, Mattanovich D. Adaptive laboratory evolution—Principles and applica-

[38] David F, Nielsen J, Siewers V. Flux control at the malonyl-CoA node through hierarchical dynamic pathway regulation in *Saccharomyces cerevisiae*. ACS Synthetic Biology.

[39] Alkım C, Turanlı-Yıldız B, Çakar ZP. Evolutionary engineering of yeast. In: Mapelli V, editor. Yeast Metabolic Engineering: Methods and Protocols. New York: Springer; 2014.

[40] Cakar ZP, Seker UOS, Tamerler C, Sonderegger M, Sauer U. Evolutionary engineering of multiple-stress resistant *Saccharomyces cerevisiae*. FEMS Yeast Research. 2005;**5**:569-578

[41] Cakar ZP, Alkim C, Turanli B, Tokman N, Akman S, Sarikaya M, Tamerler C, Benbadis L, François JM. Isolation of cobalt hyper-resistant mutants of *Saccharomyces cerevisiae* by *in vivo* evolutionary engineering approach. Journal of Biotechnology. 2009;**143**:130-138

[42] Alkim C, Benbadis L, Yilmaz U, Cakar ZP, Francois JM. Mechanisms other than activation of the iron regulon account for the hyper-resistance to cobalt of a *Saccharomyces cerevisiae* strain obtained by evolutionary engineering. Metallomics. 2013;**5**:1043-1060

ate transporters Jen1 and Ady2. FEMS Yeast Research. 2012;**12**:375-381

[36] Skyta B. Techniques in Applied Microbiology. Prague: Elsevier; 1995. p. 122

tions for biotechnology. Microbial Cell Factories. 2013;**12**:64

Press; 2006. pp. 795-807

82 Old Yeasts - New Questions

2016;**5**:224-233

pp. 169-183

and Biotechnology. 2016;**100**:2737-2748

*siae*. Metabolic Engineering. 2015;**35**:38-45

Bioscience and Bioengineering. 2016;**122**:415-420

Microbial Cell Factories. 2010;**9**:15


biosynthetic pathway in *Saccharomyces cerevisiae* coupled to β-ionone production. Journal of Biotechnology. 2014;**192**:383-392


[68] Ignea C, Ioannou E, Georgantea P, Trikka FA, Athanasakoglou A, Loupassaki S, Roussis V, Makris AM, Kampranis SC. Production of the forskolin precursor 11 β-hydroxy-manoyl oxide in yeast using surrogate enzymatic activities. Microbial Cell Factories. 2016;**15**:46

biosynthetic pathway in *Saccharomyces cerevisiae* coupled to β-ionone production. Journal

[56] Romagnoli G, Knijnenburg TA, Liti G, Louis EJ, Pronk JT, Daran J. Deletion of the *Saccharomyces cerevisiae* ARO8 gene, encoding an aromatic amino acid transaminase,

[57] Shen L, Nishimura Y, Matsuda F, Ishii J, Kondo A. Overexpressing enzymes of the Ehrlich pathway and deleting genes of the competing pathway in *Saccharomyces cerevisiae* for increasing 2-phenylethanol production from glucose. Journal of Bioscience and

[58] Orumets K, Kevvai K, Nisamedtinov I, Tamm T, Paalme T. *YAP1* over-expression in *Saccharomyces cerevisiae* enhances glutathione accumulation at its biosynthesis and sub-

[59] Hara KY, Kiriyama K, Inagaki A. Improvement of glutathione production by metabolic engineering the sulfate assimilation pathway of *Saccharomyces cerevisiae*. Applied

[60] Hara KY, Aoki N, Kobayashi J, Kiriyama K. Improvement of oxidized glutathione fermentation by thiol redox metabolism engineering in *Saccharomyces cerevisiae*. Applied

[61] Anett P, Steiger MG, Caterina H, Lang C, Mattanovich D, Sauer M. Enhanced glutathione production by evolutionary engineering of *Saccharomyces cerevisiae* strains.

[62] Yin H, Ma Y, Deng Y, Xu Z, Liu J, Zhao J, Dong J. Genome shuffling of *Saccharomyces cerevisiae* for enhanced glutathione yield and relative gene expression analysis using fluorescent quantitation reverse transcription polymerase chain reaction. Journal of Micro-

[63] Ye VM, Bhatia SK. Metabolic engineering for the production of clinically important molecules: Omega-3 fatty acids, artemisinin, and taxol. Biotechnology Journal. 2012;**7**:20-33

[64] Keasling JD. Manufacturing molecules through metabolic engineering. Science. 2010;**330**:

[65] Pscheidt B, Glieder A. Yeast cell factories for fine chemical and API production. Microbial

[66] Engels B, Dahm P, Jennewein S. Metabolic engineering of taxadiene biosynthesis in yeast as a first step towards Taxol (Paclitaxel) production. Metabolic Engineering. 2008;

[67] Ding MZ, Yan HF, Li LF, Zhai F, Shang LQ, Yin Z, Yuan YJ. Biosynthesis of taxadiene in *Saccharomyces cerevisiae*: Selection of geranylgeranyl diphosphate synthase directed by a

computer-aided docking strategy. PLoS One. 2014;**9**:10 e109348

enhances phenylethanol production from glucose. Yeast. 2015;**32**:29-45

strate availability levels. Biotechnology Journal. 2012;**7**:566-568

Microbiology and Biotechnology. 2012;**94**:1313-1319

Microbiology and Biotechnology. 2015;**99**:9771-9778

Biotechnology Journal. 2015;**10**:1719-1726

biological Methods. 2016;**127**:188-192

1355-1358

**10**:201-206

Cell Factories. 2008;**7**:25

of Biotechnology. 2014;**192**:383-392

84 Old Yeasts - New Questions

Bioengineering. 2016;**122**:34-39


**Provisional chapter**

#### **Non-Conventional Yeasts in Fermentation Processes: Potentialities and Limitations Potentialities and Limitations**

**Non-Conventional Yeasts in Fermentation Processes:** 

DOI: 10.5772/intechopen.70404

Dorota Kręgiel, Ewelina Pawlikowska and Hubert Antolak Hubert Antolak Additional information is available at the end of the chapter

Dorota Kręgiel, Ewelina Pawlikowska and

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.70404

#### **Abstract**

[81] Hawkins KM, Smolke CD. Production of benzylisoquinoline alkaloids in *Saccharomyces* 

[82] Galanie S, Smolke CD. Optimization of yeast-based production of medicinal protober-

[83] Fossati E, Ekins A, Narcross L, Zhu Y, Falgueyret J, Beaudoin GAW, Facchini PJ, Martin VJJ. Reconstitution of a 10-gene pathway for synthesis of the plant alkaloid dihydrosan-

[84] Li Y, Smolke CD. Engineering biosynthesis of the anticancer alkaloid noscapine in yeast.

[85] Fossati E, Narcross L, Ekins A, Falgueyret J, Vincent J. Synthesis of morphinan alkaloids

[86] Galanie S, Thodey K, Trenchard IJ, Filsinger Interrante M, Smolke CD. Complete biosyn-

guinarine in *Saccharomyces cerevisiae*. Nature Communications. 2014;**5**:1-11

*cerevisiae*. Nature Chemical Biology. 2008;**4**:564-573

berine alkaloids. Microbial Cell Factories. 2015;**14**:144

in *Saccharomyces cerevisiae*. PLoS One. 2015;**10**:1-15

thesis of opioids in yeast. Science. 2015;**349**:1095-1100

Nature Communications. 2016;**7**:1-14

86 Old Yeasts - New Questions

Traditionally the term 'yeast' means *Saccharomyces cerevisiae* and its close relatives. This yeast is used in traditional fermentation processes, mainly for ethanol formation, baking, winemaking and beer production. The classical carbon substrates for typical yeast processes are glucose or sucrose, however, the successful expansion of industrial biotechnology drives research toward the utilization of alternative carbon sources. New technologies require very specific challenges and differ from those found in conventional fermentation processes. Most microbial habitats, especially in modern biotechnological processes, do not provide culture media rich in mono- and disaccharides. They include fermentation environments with various compositions of carbon and energy sources as well as the presence of various cytotoxic compounds which inhibit the growth of industrial yeasts. About 1500 various yeast species have been identified nowadays. Microbiologists and biotechnologists have named all non-*S. cerevisiae* yeasts as 'non-conventional' yeasts. Their features present a potential that can be used for non-conventional processes. Non-*Saccharomyces* strains provide alternative metabolic routes for substrate utilization and product formation. The diversity of these yeasts includes many species possessing useful, and sometimes uncommon, metabolic features potentially interesting for biotechnology. The selected strains of non-conventional yeasts could be used as pure or mixed cultures for improving industrial fermentations.

**Keywords:** non-*Saccharomyces*, yeasts, fermentation, stress resistance

#### **1. Introduction**

Yeasts belong to the most studied microorganisms. More than 1500 species of yeast have been described so far [1]. Many of them have been used in various fermentation processes [2].

Taxonomic analysis of the microflora in active spontaneous fermentations revealed variety of yeasts, but still the predominant genera is *Saccharomyces* [3, 4]. This yeast has become the model organism for research studies and valuable results for numerous eukaryotic cells have been obtained [5]. *S. cerevisiae* was also the first species whose genome was sequenced [6]. The ability of *S. cerevisiae* to conduct metabolic processes under both aerobic and anaerobic conditions, and ethanol production meant that this species has been used for many years as starter cultures for production of bread and numerous fermented beverages [7]. This yeast has also been used in the biofuel industry and for the production of heterologous proteins, human insulin, hepatitis and human papillomavirus vaccines [8].

However, new technological processes, for example, production of second-generation bioethanol, are different from those encountered in conventional fermentation processes. These new technologies pose special challenges. They include fermentations in various environments, with wide spectrum of carbon and energy sources, as well as with significant content of numerous cytotoxic compounds that may inhibit the growth of industrial microorganisms [9, 10]. Strong pressure to improve the economic viability of bioethanol production from waste plant materials makes strains of *S. cerevisiae* rather ineffective in fermentation processes with lignocellulosic hydrolysates. This fact stimulates research to use other non-*Saccharomyces* strains that exhibit broad spectrum of assimilated carbon compounds and higher resistance to cytotoxic compounds.

#### **2. Ethanol production**

Ethanol production on the industrial scale has been carried out in the conventional manner using mesophilic strains of *Saccharomyces* spp. The commonly used carbon sources are molasses, beet juice, beet sugar, corn or potato starch. However, these raw materials are expensive and their availability is usually dependent on seasonal productivity. Additionally, the use of plant food such as corn and potatoes in biofuel production is morally and socially controversial. Therefore, diversified actions have been taken to convert a variety of agricultural and forestry wastes, rich in lignocellulosic sugars, into biofuels (**Table 1**).


**Table 1.** Ethanol yields from selected waste biomass [11].

**Figure 1.** Flow diagram of biofuel production from plant biomass. (1) Pretreatment, (2) fermentation and (3) separation and purification.

According to the Directives 2008/98/EC and Regulation (EU) No 1357/2014, by-products generated can be used directly, without further processing, but wastes may be subjected to recovery, disposed of or liquidated. Proper management of waste biomass is an important issue for environmental protection. However, the use of production residues not only minimizes the negative impact on the environment, but it is also possible to get additional economic benefits [12]. Organic waste from the agroindustry and forestry, according to their physicochemical properties, can be used for the production of bioethanol, butanol, acetone and new chemical building blocks for advanced materials [13, 14] (**Figure 1**).

#### **3. Starch and lignocellulosic biomass**

Taxonomic analysis of the microflora in active spontaneous fermentations revealed variety of yeasts, but still the predominant genera is *Saccharomyces* [3, 4]. This yeast has become the model organism for research studies and valuable results for numerous eukaryotic cells have been obtained [5]. *S. cerevisiae* was also the first species whose genome was sequenced [6]. The ability of *S. cerevisiae* to conduct metabolic processes under both aerobic and anaerobic conditions, and ethanol production meant that this species has been used for many years as starter cultures for production of bread and numerous fermented beverages [7]. This yeast has also been used in the biofuel industry and for the production of heterologous proteins, human

However, new technological processes, for example, production of second-generation bioethanol, are different from those encountered in conventional fermentation processes. These new technologies pose special challenges. They include fermentations in various environments, with wide spectrum of carbon and energy sources, as well as with significant content of numerous cytotoxic compounds that may inhibit the growth of industrial microorganisms [9, 10]. Strong pressure to improve the economic viability of bioethanol production from waste plant materials makes strains of *S. cerevisiae* rather ineffective in fermentation processes with lignocellulosic hydrolysates. This fact stimulates research to use other non-*Saccharomyces* strains that exhibit broad spectrum of assimilated carbon compounds and higher resistance

Ethanol production on the industrial scale has been carried out in the conventional manner using mesophilic strains of *Saccharomyces* spp. The commonly used carbon sources are molasses, beet juice, beet sugar, corn or potato starch. However, these raw materials are expensive and their availability is usually dependent on seasonal productivity. Additionally, the use of plant food such as corn and potatoes in biofuel production is morally and socially controversial. Therefore, diversified actions have been taken to convert a variety of agricultural and

forestry wastes, rich in lignocellulosic sugars, into biofuels (**Table 1**).

**Biomass Ethanol yield (litres per dry metric ton)**

insulin, hepatitis and human papillomavirus vaccines [8].

to cytotoxic compounds.

88 Old Yeasts - New Questions

**2. Ethanol production**

Hardwood 350 Softwood 420 Corn stover 275–300 Wheat straw 250–300 Sugarcane bagasse 314 Municipal solid waste 170–486

**Table 1.** Ethanol yields from selected waste biomass [11].

Starch is the carbohydrate accumulated in plants, made up of long chains of glucose units joined by α-l,4 linkages and joined at branch points by α-1,6 bonds. Many microorganisms, including *S. cerevisiae*, are not able to degrade starch since they do not produce starch decomposing enzymes such as α-amylase, β-amylase, pullulanase, isoamylase and glucoamylase. To simplify the fermentation process by eliminating the separate saccharification step, numerous genetically engineered *S. cerevisiae* strains capable of secreting glucoamylase or α-amylase were constructed. However, starch decomposition abilities presented by these yeast strains are usually unsatisfactory because of the limited amounts of secreted amylolytic enzymes [15].

Lignocellulose is the most abundant renewable biomass on earth. It is composed mainly of cellulose, hemicellulose and lignin. Both the cellulose and hemicellulose fractions are polymers of sugars and thereby a potential source of fermentable carbon sources. Hence the interest in research on procedures for chemical degradation of the lignocellulosic structure and for maximization of its decomposition into glucose, xylose and phenolic compounds. The resulting carbon substances can then be assimilated by yeast, which considerably increases the efficiency of biodegradation [16, 17].

Different pretreatment technologies published in public literature are described in terms of the involved mechanisms, advantages versus disadvantages, and economic calculation. Pretreatment technologies for lignocellulosic biomass include biological, mechanical or chemical methods, and their various combinations in particular. It is not possible to define the best pretreatment method because it depends on the type of lignocellulosic biomass and desired products. The acidic (H2 SO4 ) or alkali (NaOH) hydrolysis, oxidation techniques (H2 O2 ), heat and enzymatic (cellulases, cellobiase and xylanase) treatments are the most frequently used for this purpose [18, 19]. However, each of these methods leads to release of various decomposition products. When fermentable sugars are produced, special attention must be paid to the formation of fermentation inhibitors. Especially the formation of phenolic compounds from lignin degradation, as well as the formation of furfural and 5-(hydroxymethyl)-2-furfural from sugar degradation should be limited by keeping the process parameters: temperature and time as low and as short as possible. Therefore, the choice of the appropriate pretreatment method of plant biomass hydrolysis is the crucial step for effectiveness of fermentation processes [20].

#### **4.** *S. cerevisiae* **or non-conventional yeasts?**

All yeasts are capable of assimilating glucose, almost all such as fructose and mannose, while galactose can also be assimilated by many species. Among the disaccharides, sucrose is the most commonly used. However, in ethanol production, second generation classical yeast *Saccharomyces* spp. are not useful because they are not able to ferment pentoses, exhibit low tolerance to alcohols, acids and solvents. Additionally, they are characterized by high sensitivity to pH changes and cytotoxic compounds: furfural, 5-(hydroxymethyl)-2-furfural and other organic compounds produced during hydrolysis. Limitations of *S. cerevisiae* make the course of new industrial fermentation processes very difficult.

The fuel ethanol production from lignocellulosic materials requires co-fermentation of both hexoses and pentoses, mainly d-xylose and l-arabinose. *S. cerevisiae* cannot utilize pentoses because of the lack of specific metabolic pathways and transport systems. Genomic resources from a variety of microorganisms as well as biological systems combined with mutagenesis have been used to engineer yeast with pentose fermentation abilities [21]. By expressing heterologous d-xylose or l-arabinose pathways, *S. cerevisiae* could obtain the metabolic capacity but this efficiency still needs to be improved [22, 23].

The main strategies for constructing d-xylose-utilizing *S. cerevisiae* include two paths. The first one is XR-XDH pathway, containing d-xylose reductase (XR) and xylitol dehydrogenase (XDH), and converts d-xylose to xylulose. Due to the cofactor imbalance in this pathway, the accumulation of byproduct is the main problem, which needs to be solved. Another one is XI pathway, which only needs to introduce one d-xylose isomerase (XI) that directly converts d-xylose to xylulose. However, the activity of XI still needs to be increased. The xylulose from both pathways could be phosphorylated to xylulose-5-P by endogenous xylulokinase. Subsequently xylulose-5-P can be further entered into the endogenous pentose phosphate pathway (PPP) to produce ethanol [23].

There are also two main l-arabinose metabolic pathways which are both candidates for constructing l-arabinose-metabolic yeasts. l-Arabinose could be converted to d-xylulose-5-phosphate that then enters into PPP. This pathway needs five important enzymes, including aldose reductase, l-arabinitol-4-dehydrogenase, l-xylulose reductase, d-xylulose reductase and xylulokinase. In addition, this pathway contained two reduction reactions which utilize NADPH, two oxidation reactions which generate NADH, and a kinase reaction [23]. Therefore, the construction of stable *S. cerevisiae* strains able to ferment xylose and/or arabinose is not easy. The co-utilization of d-xylose and l-arabinose was obtained in engineered *S. cerevisiae* strain with a high ethanol yield 0.43 g/g of total sugar [24]. Also selected strains of other yeast belonging to *Pichia stipitis* were shown to ferment hydrolysates with ethanol yields of 0.45 g/g of sugar, so commercialization seems feasible for some applications [25].

for this purpose [18, 19]. However, each of these methods leads to release of various decomposition products. When fermentable sugars are produced, special attention must be paid to the formation of fermentation inhibitors. Especially the formation of phenolic compounds from lignin degradation, as well as the formation of furfural and 5-(hydroxymethyl)-2-furfural from sugar degradation should be limited by keeping the process parameters: temperature and time as low and as short as possible. Therefore, the choice of the appropriate pretreatment method of plant biomass hydrolysis is the crucial step for effectiveness of fermentation

All yeasts are capable of assimilating glucose, almost all such as fructose and mannose, while galactose can also be assimilated by many species. Among the disaccharides, sucrose is the most commonly used. However, in ethanol production, second generation classical yeast *Saccharomyces* spp. are not useful because they are not able to ferment pentoses, exhibit low tolerance to alcohols, acids and solvents. Additionally, they are characterized by high sensitivity to pH changes and cytotoxic compounds: furfural, 5-(hydroxymethyl)-2-furfural and other organic compounds produced during hydrolysis. Limitations of *S. cerevisiae* make the

The fuel ethanol production from lignocellulosic materials requires co-fermentation of both hexoses and pentoses, mainly d-xylose and l-arabinose. *S. cerevisiae* cannot utilize pentoses because of the lack of specific metabolic pathways and transport systems. Genomic resources from a variety of microorganisms as well as biological systems combined with mutagenesis have been used to engineer yeast with pentose fermentation abilities [21]. By expressing heterologous d-xylose or l-arabinose pathways, *S. cerevisiae* could obtain the metabolic capacity

The main strategies for constructing d-xylose-utilizing *S. cerevisiae* include two paths. The first one is XR-XDH pathway, containing d-xylose reductase (XR) and xylitol dehydrogenase (XDH), and converts d-xylose to xylulose. Due to the cofactor imbalance in this pathway, the accumulation of byproduct is the main problem, which needs to be solved. Another one is XI pathway, which only needs to introduce one d-xylose isomerase (XI) that directly converts d-xylose to xylulose. However, the activity of XI still needs to be increased. The xylulose from both pathways could be phosphorylated to xylulose-5-P by endogenous xylulokinase. Subsequently xylulose-5-P can be further entered into the endogenous pentose phosphate

There are also two main l-arabinose metabolic pathways which are both candidates for constructing l-arabinose-metabolic yeasts. l-Arabinose could be converted to d-xylulose-5-phosphate that then enters into PPP. This pathway needs five important enzymes, including aldose reductase, l-arabinitol-4-dehydrogenase, l-xylulose reductase, d-xylulose reductase and xylulokinase. In addition, this pathway contained two reduction reactions which utilize NADPH, two oxidation reactions which generate NADH, and a kinase reaction [23].

processes [20].

90 Old Yeasts - New Questions

**4.** *S. cerevisiae* **or non-conventional yeasts?**

course of new industrial fermentation processes very difficult.

but this efficiency still needs to be improved [22, 23].

pathway (PPP) to produce ethanol [23].

An additional problem for the simultaneous consumption of pentoses and hexoses is the inhibition of pentose uptake by d-glucose. Researchers have engineered xylose metabolism in *S. cerevisiae* by over-expressing genes for aldose (xylose) reductase, xylitol dehydrogenase and moderate levels of xylulokinase-enabled xylose assimilation and fermentation. The results obtained by Subtil and Boles suggested that co-fermentation of pentoses in the presence of d-glucose can significantly be improved by the overexpression of pentose transporters, especially if they are not inhibited by d-glucose [26]. However, a balanced proportion of NAD(P) and NAD(P)H must be maintained to avoid xylitol production. It was noted that respiration is critical for growth on xylose by both native and recombinant xylose-fermenting yeasts. Reducing the respiration capacity of xylose-metabolizing yeasts increases ethanol production. In studies conducted by Jeffries and Jin, *S. cerevisiae* was engineered for d-xylose utilization through the heterologous expression of genes for aldose reductase, xylitol dehydrogenase and d-xylulokinase and produced only limited amounts of ethanol in xylose medium. It was observed that levels for glycolytic, fermentative and pentose phosphate enzymes did not influence significantly on glucose or xylose under aeration or oxygen limitation. However, expression of genes encoding the tricarboxylic acid cycle and respiration enzymes increased significantly when cells were cultivated on xylose, and the genes for respiration were even more elevated under oxygen limitation. These results suggest that recombinant *S. cerevisiae* does not recognize xylose as a fermentable carbon source. However, the petite respiration-deficient engineered strain produced more ethanol and accumulated less xylitol from xylose [25, 27].

The results obtained by Wang et al. for co-utilization of d-glucose, d-xylose and l-arabinose in engineered *S. cerevisiae* showed that the pentose metabolic capacity is prominently lower than that of d-glucose due to d-glucose-inhibition effect. To alleviate the phenomenon, the pentose metabolic flux can be improved and a pentose specific transporter without inhibition by d-glucose might also be needed [23].

The progress in fermentation of pentose sugars has gone on slow pace as there are few microorganisms known, which are capable of pentose metabolism. While numerous metabolic engineering strategies have been developed in laboratory yeast strains, only a few approaches have been realized in industrial strains. Ethanol yields of more than 0.4 g of ethanol/g of sugar have been achieved with several xylose-fermenting industrial strains with the heterologous xylose utilization pathway consisting of xylose reductase and xylitol dehydrogenase, which demonstrates the potential of pentose fermentation in lignocellulosic ethanol production [28]. In the future, desired perspective is to find organisms that would be able to ferment high density hydrolysates without purification. The genetic and metabolic engineering routes also should be continued. Also a direct or a sequential fermentation system using mixed populations of yeasts needs to be worked out [29].

The interest of microbiologists has been also directed to the use of yeasts belonging to other genera than genus *Saccharomyces* or *Schizosaccharomyces*, commonly called 'non-conventional' yeasts. Due to the information collected on abilities of some of these yeasts, as well as their applications in many fields, their 'unconventional' status may change in the future. Some of the 'non-conventional' yeasts of today will be the 'conventional' yeasts of tomorrow [30]. The similar thesis was given by Sibirny and Scheffers [31]. They highlighted that, since an increasing number of non-conventional yeasts and increasing importance in both fundamental and applied sciences, the term 'non-conventional' is gradually losing significance and usefulness.

There is an enormous biodiversity of non-conventional yeasts. Currently 1500 species have been described although this is only thought to be 1% of yeast that may exist on Earth. These yeasts are phylogenetically diverse and thus may probably harbor industrially relevant traits to augment the currently used *S. cerevisiae*. In addition, due to the carbon substrates utilization range, as well as a poor stress tolerance drawback, there is need to search for novel traits in other yeasts. Therefore, biodiversity is an alternative approach to genetically improved yeasts [32]. Due to the progress in identification and characteristic of a new species found in nature, it is possible to increase the diversity and number of yeasts used in industrial purposes. It is indisputable that the exploration for new species will lead to additional novel technologies, including fermentation of pentoses to ethanol.

A lot of genera different than *Saccharomyces* may also be interesting for their use in specific technological applications. In fact, some species have already attracted researchers in the last years on different aspects: *Kluyveromyces lactis* as a possible utilizer of the residual whey in dairy industries; some methylotrophic yeasts for the production of heterologous proteins; *Yarrowia lipolytica* for its ability to grow on particular substrates and its high protein excretion capacity. As it was mentioned above, transport of carbohydrates into cells is the very important step in yeast metabolism, except in those cases in which di- or trisaccharides are hydrolyzed outside the cell. Transport of monosaccharides such as glucose, fructose or mannose in *S. cerevisiae* is a facilitated diffusion process; however, the situation may be different in other yeasts. For example, in *K. lactis* glucose transport appears to proceed by facilitated diffusion. In *Candida utilis*, the popular 'fodder yeast', glucose appears to be transported by a proton symport when the organism is grown at low glucose concentration [33].

The non-conventional yeasts may overcome many problems related with narrow spectrum of carbon sources assimilation presented by conventional *S. cerevisiae* [15]. Some non-conventional yeasts show many uncommon, metabolic features potentially interesting to biotechnology. Non-conventional yeasts represent the vast majority of genera and species so far described. Several yeast species are diverged by evolution from *S. cerevisiae* and possess several unique genes and growth characteristics to withstand different stress conditions [34]. These exceptional strains are able to utilize various sources of carbon such as starch, cellulose, raffinose, arabinose, xylose and sugar alcohols (xylitol, sorbitol, mannitol, etc.) [8, 35].

At least 22 yeast strains have been shown to produce some ethanol from d-xylose. However, only six strains such as *Brettanomyces naardenensis*, *C. shehatae*, *C. tenuis*, *Pachysolen tannophilus*, *P. segobiensis* and *P. stipitis* are able to produce significant amounts of ethanol, and of these, only three: *C. shehatae*, *P. tannophilus* and *P. stipitis* have been studied extensively [36, 37].

The production systems exploiting some non-*Saccharomyces* yeasts have one important advantage—they are not pathogenic organisms received the 'generally recognized as safe' (GRAS) designation from the Food and Drug Administration (FDA) [38–40].

The interest of microbiologists has been also directed to the use of yeasts belonging to other genera than genus *Saccharomyces* or *Schizosaccharomyces*, commonly called 'non-conventional' yeasts. Due to the information collected on abilities of some of these yeasts, as well as their applications in many fields, their 'unconventional' status may change in the future. Some of the 'non-conventional' yeasts of today will be the 'conventional' yeasts of tomorrow [30]. The similar thesis was given by Sibirny and Scheffers [31]. They highlighted that, since an increasing number of non-conventional yeasts and increasing importance in both fundamental and applied sciences, the term 'non-conventional' is gradually losing significance and usefulness. There is an enormous biodiversity of non-conventional yeasts. Currently 1500 species have been described although this is only thought to be 1% of yeast that may exist on Earth. These yeasts are phylogenetically diverse and thus may probably harbor industrially relevant traits to augment the currently used *S. cerevisiae*. In addition, due to the carbon substrates utilization range, as well as a poor stress tolerance drawback, there is need to search for novel traits in other yeasts. Therefore, biodiversity is an alternative approach to genetically improved yeasts [32]. Due to the progress in identification and characteristic of a new species found in nature, it is possible to increase the diversity and number of yeasts used in industrial purposes. It is indisputable that the exploration for new species will lead to additional novel technologies,

A lot of genera different than *Saccharomyces* may also be interesting for their use in specific technological applications. In fact, some species have already attracted researchers in the last years on different aspects: *Kluyveromyces lactis* as a possible utilizer of the residual whey in dairy industries; some methylotrophic yeasts for the production of heterologous proteins; *Yarrowia lipolytica* for its ability to grow on particular substrates and its high protein excretion capacity. As it was mentioned above, transport of carbohydrates into cells is the very important step in yeast metabolism, except in those cases in which di- or trisaccharides are hydrolyzed outside the cell. Transport of monosaccharides such as glucose, fructose or mannose in *S. cerevisiae* is a facilitated diffusion process; however, the situation may be different in other yeasts. For example, in *K. lactis* glucose transport appears to proceed by facilitated diffusion. In *Candida utilis*, the popular 'fodder yeast', glucose appears to be transported by a proton

The non-conventional yeasts may overcome many problems related with narrow spectrum of carbon sources assimilation presented by conventional *S. cerevisiae* [15]. Some non-conventional yeasts show many uncommon, metabolic features potentially interesting to biotechnology. Non-conventional yeasts represent the vast majority of genera and species so far described. Several yeast species are diverged by evolution from *S. cerevisiae* and possess several unique genes and growth characteristics to withstand different stress conditions [34]. These exceptional strains are able to utilize various sources of carbon such as starch, cellulose,

raffinose, arabinose, xylose and sugar alcohols (xylitol, sorbitol, mannitol, etc.) [8, 35].

*C. shehatae*, *P. tannophilus* and *P. stipitis* have been studied extensively [36, 37].

At least 22 yeast strains have been shown to produce some ethanol from d-xylose. However, only six strains such as *Brettanomyces naardenensis*, *C. shehatae*, *C. tenuis*, *Pachysolen tannophilus*, *P. segobiensis* and *P. stipitis* are able to produce significant amounts of ethanol, and of these, only three:

symport when the organism is grown at low glucose concentration [33].

including fermentation of pentoses to ethanol.

92 Old Yeasts - New Questions

The non-conventional yeast systems may have several beneficial traits like ethanol tolerance, thermotolerance, inhibitor tolerance, genetic diversity, etc. However, not all non-conventional yeasts possess these important characteristics. Currently, studies on non-conventional yeasts concern limited number of species like *Hansenula polymorpha*, *K. lactis*, *P. pastoris* and *Y. lipolytica* [21, 40]. However, more non-conventional yeasts are worth the special attention. For example, amylases from non-conventional yeasts were found to have the ability to hydrolyze starch. These interesting enzymes are α-amylase and glucoamylase from *Debaryomyces* (*Schwanniomyces*) *castelli*, glucoamylase from *Saccharomycopsis fibuligera* and *C. antarctica*, α-amylase from *Cryptococcus* sp., *C. lusitaniae*, *C. famata*, amylopullulanase from *Clavispora lusitaniae* and pullulanase from *Aureobasidium pullulans*. There is a number of reviews on bacterial and fungal amylases and their applications. They clearly indicate that α-amylases and pullulanase from yeasts are one of the most popular and important forms of industrial amylases. Non-conventional yeasts were studied as both free and immobilized cells for production of amylolytic enzymes [41–44] (**Photo 1**).

Fermentation trials with immobilized conventional *S. cerevisiae* and non-conventional cells *D. occidentalis* showed that both tested yeasts are able to adapt to the specific conditions inside carrier materials. Nevertheless, the mechanical endurance of alginate carriers, commonly used in yeast immobilization, shows better applications in industrial fermentation especially with non-conventional yeasts. In the case of fermentative yeast, *S. cerevisiae* alginate beads may be destroyed, as a result of intense CO2 formation [44]. Furthermore, *Debaryomyces* spp. ability to tolerate and decompose both phenols and polyphenols at concentrations that are highly toxic to bacteria and other yeast species, demonstrated that these yeasts may be an attractive system for biofuel production from renewable starch sources [15]. It is worth to note that methylotrophic yeasts belonging to *Hansenula*, *Candida*, *Pichia* and *Torulopsis* genera are able to metabolize monocarbonic compounds like methanol and formaldehyde [45]. As

**Photo 1.** Amylolytic non-conventional yeast *D. occidentalis* immobilized in alginate (author: Dorota Kregiel).

a result, the use of non-conventional yeasts allows for the utilization of various waste plant biomass, and—which is worth emphasizing—to receive the post-fermentation yeast biomass rich in protein and amino acids [39, 46, 47].

Non-conventional yeasts are large but not fully known, diverse group of microorganisms. Yeast species other than *Saccharomyces* spp., in addition to the previously mentioned ability to use the complex substrates, exhibit features particularly important in the industrial processes—thermotolerance and tolerance to the presence of chemical inhibitors. The majority of non-conventional yeasts have been isolated and characterized as microflora of spoiled food and beverages [48, 49]. It can be assumed that some non-conventional yeasts have developed specific mechanisms to survive in various natural environmental conditions. Therefore, it is believed that most species of non-conventional yeasts have acquired specific mechanisms that are not included in the classical model yeast *S. cerevisiae* [49–51]. It is worth to explore the molecular basis of their tolerance to numerous environmental stress such as increased osmotic pressure, high concentrations of ethanol, high temperature or the presence of toxic compounds.

The eukaryotic microorganism most studied for its tolerance is still *S. cerevisiae*. However, this yeast is rather sensitive and not able to adapt to 'non-regular' conditions. For example, some species like *D. hansenii* or *Hortaea werneckii* have been isolated from natural hypersaline environments. Therefore, these non-conventional yeasts are more suitable model organisms to study halotolerance in eukaryotes than *S. cerevisiae* [52]. It should be also noted that non-conventional yeasts show a high growth rate in fermentation processes, and are capable of producing important enzymes. For example, *D. occidentalis* is capable of secretion of α-amylase and glucoamylase, and *K. marxianus* of producing intracellular lactase, intra and extracellular pectinase and intra and extracellular inulinase [42, 44, 53–55]. These examples show the important potential of non-conventional yeasts that can be utilized in use of various waste materials.

#### **5. Osmotolerance**

Yeast cells are exposed to osmotic stress during industrial fermentations. The processes carried out in media with significant levels of saccharides above 300 g/L, need osmotolerant yeasts in particular [56–58]. Accordingly, there is a growing interest among microbiologists and technicians to obtain yeast strains that are able to grow in environments with high concentrations of salts or saccharides. The molecular mechanisms for responsibility of osmotolerant *S. cerevisiae* strains have been described extensively in available literature [59, 60]. *S. cerevisiae* remains the model organism to study the molecular basis of important physiological features, but researchers have isolated and identified non-conventional osmotolerant yeasts belonging to *Zygosaccharomyces rouxii* [48, 61].

*Z. rouxii* is known for its high tolerance to osmotic stress, which is thought to be caused by sets of specific genes. Important differences were found for salt tolerance and assimilation of glycerol in comparison to *S. cerevisiae*. *Zygosaccharomyces* strains show a higher resistance to salts, higher glycerol production and are able to assimilate glycerol. Under conditions of osmotic stress, the glycerol production in *Z. rouxii* strains may be much lower than in *S. cerevisiae*, which suggests the presence of a system that efficiently retains glycerol inside *Z. rouxii* cells [48, 61, 62].

*D. hansenii* is also one of the most halotolerant species. This yeast was isolated from saline environments sea water, concentrated brines and salty food. It can grow in media containing as high as 4 M NaCl, while the growth of *S. cerevisiae* is limited in media with more than 1.7 M NaCl [63].

The adaptation of yeast cells to osmotic stress is a complex mechanism that combines network regulatory genes and signaling pathways that may vary depending on the species and osmotic agent in the surrounding environment [64]. Generally, the behavior of *Z. rouxii* cells resembles the activity of *S. cerevisiae* in the transport of Na+ ions from yeast cell, while halotolerant yeast *D. hansenii* accumulates sodium ions inside its cells.

The results obtained by Gonzalez-Hernandez et al. confirmed that *D. hansenii* grows better in the presence of moderate concentrations (0.6 M) of NaCl and KCl than in the absence or at higher salt concentration. Therefore *D. hansenii* can be considered moderate halophile yeast [65]. This ability is associated with the accumulation of high concentrations of K+ or Na+ . For this reason *D. hansenii* has been called a 'sodium-includer' [66, 67]. The mechanism of the adaptation is probably an intrinsic resistance to the toxic effects of cations, not observed in other yeasts, particularly *S. cerevisiae* [65]. The problems, how yeasts regulate the intracellular ion concentration, and how ions are tolerated by enzymes promoting survival, remains controversial [67, 68]. In *D. hansenii*, the vacuolar concentration of Na+ was described to be equal to the one of cytoplasm, while in *S. cerevisiae* the differences between these concentrations were described [67].

*Z. rouxii* integrates general and osmoticum-specific adaptive responses under sugar and salts stresses, including regulation of Na+ and K+ -fluxes across the plasma membrane, modulation of cell wall properties, compatible osmolyte production and accumulation and stress signaling pathways [69, 70]. According to Leandro et al., *Z. rouxii* is capable of growing in osmolarity of 3 M NaCl and glucose concentrations of 90%, due to the presence of unique transporters in plasma membrane which is higher than *S. cerevisiae* [62]. Dakal et al. described internal reactions that occur in yeast cells under different osmotic agents. They suggested that sugars and polyols modify the osmotic pressure, while salts induce changes in both osmotic pressure and ionic homeostasis [70].

According to Pribylova et al., the less osmotolerant yeasts strain possesses a more rigid cell wall than the more osmotolerant ones. They suggested that the differences in the osmotolerance are related to resistance to the lysing enzymes—lyticase and zymolyase, cell-wall polymer content and cell wall micromorphology [69].

Availability of genome sequence of osmotolerant and halotolerant strains may open up new perspectives in this direction [71].

#### **6. Thermotolerance**

a result, the use of non-conventional yeasts allows for the utilization of various waste plant biomass, and—which is worth emphasizing—to receive the post-fermentation yeast biomass

Non-conventional yeasts are large but not fully known, diverse group of microorganisms. Yeast species other than *Saccharomyces* spp., in addition to the previously mentioned ability to use the complex substrates, exhibit features particularly important in the industrial processes—thermotolerance and tolerance to the presence of chemical inhibitors. The majority of non-conventional yeasts have been isolated and characterized as microflora of spoiled food and beverages [48, 49]. It can be assumed that some non-conventional yeasts have developed specific mechanisms to survive in various natural environmental conditions. Therefore, it is believed that most species of non-conventional yeasts have acquired specific mechanisms that are not included in the classical model yeast *S. cerevisiae* [49–51]. It is worth to explore the molecular basis of their tolerance to numerous environmental stress such as increased osmotic pressure, high concentrations of ethanol, high temperature or the presence of toxic compounds. The eukaryotic microorganism most studied for its tolerance is still *S. cerevisiae*. However, this yeast is rather sensitive and not able to adapt to 'non-regular' conditions. For example, some species like *D. hansenii* or *Hortaea werneckii* have been isolated from natural hypersaline environments. Therefore, these non-conventional yeasts are more suitable model organisms to study halotolerance in eukaryotes than *S. cerevisiae* [52]. It should be also noted that non-conventional yeasts show a high growth rate in fermentation processes, and are capable of producing important enzymes. For example, *D. occidentalis* is capable of secretion of α-amylase and glucoamylase, and *K. marxianus* of producing intracellular lactase, intra and extracellular pectinase and intra and extracellular inulinase [42, 44, 53–55]. These examples show the important potential of non-conventional yeasts that can be utilized in use of various

Yeast cells are exposed to osmotic stress during industrial fermentations. The processes carried out in media with significant levels of saccharides above 300 g/L, need osmotolerant yeasts in particular [56–58]. Accordingly, there is a growing interest among microbiologists and technicians to obtain yeast strains that are able to grow in environments with high concentrations of salts or saccharides. The molecular mechanisms for responsibility of osmotolerant *S. cerevisiae* strains have been described extensively in available literature [59, 60]. *S. cerevisiae* remains the model organism to study the molecular basis of important physiological features, but researchers have isolated and identified non-conventional osmotolerant

*Z. rouxii* is known for its high tolerance to osmotic stress, which is thought to be caused by sets of specific genes. Important differences were found for salt tolerance and assimilation of glycerol in comparison to *S. cerevisiae*. *Zygosaccharomyces* strains show a higher resistance to salts, higher glycerol production and are able to assimilate glycerol. Under conditions of osmotic stress, the glycerol production in *Z. rouxii* strains may be much lower

rich in protein and amino acids [39, 46, 47].

94 Old Yeasts - New Questions

waste materials.

**5. Osmotolerance**

yeasts belonging to *Zygosaccharomyces rouxii* [48, 61].

Thermotolerance of yeast cells is a highly desirable feature for fermentation processes. Efficient process for bioethanol production from lignocellulosic substrates requires relatively high temperatures (~50°C) for conducting the enzymatic hydrolysis of biomass before fermentation [72]. Moreover, fermentations carried out at high temperatures significantly reduce the costs of cooling, as well as the risk of microbial contamination [73]. A limited temperature tolerance in yeast *S. cerevisiae*, with the optimal range of 25–37°C, increases the overall cost of ethanol production [74, 75]. Therefore, in order to achieve efficient fermentation at high temperatures, thermotolerant microorganisms may be used. These strains are not only able to survive, but also to produce ethanol efficiently [75, 76]. Non-conventional strains of *K. marxianus* show ability to ferment carbon sources at the temperature of 45°C. Thermotolerance, a broad enzymatic activity and fermentation ability in high concentration of saccharides makes the yeast *K. marxianus* a good material to conduct various fermentation processes [77]. Also other non-conventional yeast species-like *Ogataea polymorpha* (syn. *H. polymorpha*) have been found to ferment xylose at 45°C [78].

Yeast thermotolerance is the result of many factors, including trehalose, heat shock proteins, ATPase, the ubiquitin-proteasome pathway, gene expression responses and heat-induced antioxidant defenses [79]. Some processes may be specific to basal thermotolerance, others may be induced during acquired thermotolerance, and many may be involved in both. High temperatures are known to affect membrane-linked processes due to alterations in membrane fluidity and permeability. Enzymes are also sensitive to higher temperatures. Heat-induced protein denaturation can lead to imbalance in metabolic pathways or to complete enzyme inactivation. These changes lead to the production of active oxygen species and, consequently, heat-induced oxidative stress [80].

The best-characterized aspect of acquired thermotolerance is the production of heat shock proteins (HSPs) consisting of a helix-turn-helix class DNA binding domain, a leucine zipper domain required for trimerization, and a carboxy-terminal transcriptional activation domain. In *S. cerevisiae*, heat shock factor (HSF) is encoded by a single, essential gene, *HSF1*. It was documented that Hsf1p protein from *S. cerevisiae* and HSF from yeast *K. lactis* both contain a unique transcriptional activation domain amino-terminal to the DNA binding domain. Hsf1p appears to be primarily responsible for production of protein chaperones during heat shock [81]. At higher temperatures, organisms induce massive transcription and translation of HSPs. These proteins are proposed to act as molecular chaperones to protect cellular proteins against irreversible heat-induced denaturation and to facilitate refolding of heat-damaged proteins. Genetic evidence established that the Hsp100 family proteins are essential for the acquisition of thermotolerance [82].

The major role for the pathway in heat shock response is mediated by expression of genes required for the synthesis and degradation of the disaccharide trehalose. Originally thought to function as a storage carbohydrate, trehalose accumulates to extremely high levels in stationary phase cells. Logarithmic-phase cells have very low levels of trehalose, which are rapidly increased upon stress exposure. This acts as cytoprotectant, blocking thermally induced protein aggregation. Importantly, trehalose-stabilized proteins are maintained in a partially folded state, ready for reactivation by protein chaperones. Accordingly, the continued presence of trehalose inhibits protein refolding. Stress recovery therefore requires reduction of cellular trehalose levels. Trehalose can thus be considered a chemical chaperone for protein folding with properties remarkably similar to the chaperone Hsp104p – the ability to stabilize unfolded proteins and prevent aggregation [83].

It was documented that temperature affects both growth and ethanol tolerance. Decreasing temperature decreases membrane fluidity; increasing temperature increases membrane fluidity. Yeasts are able to adapt to low temperatures by increasing the proportion of cis-unsaturated fatty-acyl groups in lipids forming cell membranes. Physical principles suggest that fluidity would decrease as the ratio of saturated to unsaturated fatty acids increases because desaturation introduces a bend in the fatty acid chain. However, the majority of fatty acids in the membranes of *S. cerevisiae* are unsaturated, so other factors may be more important. It was found that the unsaturation level of *S. cerevisiae* cellular fatty acids increases at both sublethal or supraoptimal temperatures. On the other side, it was noted that the high content of unsaturated fatty acids is rather result from activation of oxygen-consuming desaturase activity. Membrane fluidity is also affected by the ratios of cell lipids and proteins. These vary with the yeast strain and the conditions under which it is cultivated [37, 84, 85].

(~50°C) for conducting the enzymatic hydrolysis of biomass before fermentation [72]. Moreover, fermentations carried out at high temperatures significantly reduce the costs of cooling, as well as the risk of microbial contamination [73]. A limited temperature tolerance in yeast *S. cerevisiae*, with the optimal range of 25–37°C, increases the overall cost of ethanol production [74, 75]. Therefore, in order to achieve efficient fermentation at high temperatures, thermotolerant microorganisms may be used. These strains are not only able to survive, but also to produce ethanol efficiently [75, 76]. Non-conventional strains of *K. marxianus* show ability to ferment carbon sources at the temperature of 45°C. Thermotolerance, a broad enzymatic activity and fermentation ability in high concentration of saccharides makes the yeast *K. marxianus* a good material to conduct various fermentation processes [77]. Also other non-conventional yeast species-like *Ogataea polymorpha* (syn. *H. polymor-*

Yeast thermotolerance is the result of many factors, including trehalose, heat shock proteins, ATPase, the ubiquitin-proteasome pathway, gene expression responses and heat-induced antioxidant defenses [79]. Some processes may be specific to basal thermotolerance, others may be induced during acquired thermotolerance, and many may be involved in both. High temperatures are known to affect membrane-linked processes due to alterations in membrane fluidity and permeability. Enzymes are also sensitive to higher temperatures. Heat-induced protein denaturation can lead to imbalance in metabolic pathways or to complete enzyme inactivation. These changes lead to the production of active oxygen species and, consequently,

The best-characterized aspect of acquired thermotolerance is the production of heat shock proteins (HSPs) consisting of a helix-turn-helix class DNA binding domain, a leucine zipper domain required for trimerization, and a carboxy-terminal transcriptional activation domain. In *S. cerevisiae*, heat shock factor (HSF) is encoded by a single, essential gene, *HSF1*. It was documented that Hsf1p protein from *S. cerevisiae* and HSF from yeast *K. lactis* both contain a unique transcriptional activation domain amino-terminal to the DNA binding domain. Hsf1p appears to be primarily responsible for production of protein chaperones during heat shock [81]. At higher temperatures, organisms induce massive transcription and translation of HSPs. These proteins are proposed to act as molecular chaperones to protect cellular proteins against irreversible heat-induced denaturation and to facilitate refolding of heat-damaged proteins. Genetic evidence established that the Hsp100 family proteins are essential for the

The major role for the pathway in heat shock response is mediated by expression of genes required for the synthesis and degradation of the disaccharide trehalose. Originally thought to function as a storage carbohydrate, trehalose accumulates to extremely high levels in stationary phase cells. Logarithmic-phase cells have very low levels of trehalose, which are rapidly increased upon stress exposure. This acts as cytoprotectant, blocking thermally induced protein aggregation. Importantly, trehalose-stabilized proteins are maintained in a partially folded state, ready for reactivation by protein chaperones. Accordingly, the continued presence of trehalose inhibits protein refolding. Stress recovery therefore requires reduction of cellular trehalose levels. Trehalose can thus be considered a chemical chaperone for protein folding with properties remarkably similar to the chaperone Hsp104p – the ability to stabilize

*pha*) have been found to ferment xylose at 45°C [78].

heat-induced oxidative stress [80].

96 Old Yeasts - New Questions

acquisition of thermotolerance [82].

unfolded proteins and prevent aggregation [83].

Ethanol also affects membrane fluidity, but through different mechanisms. The presence of alcohols, results in the decrease of the temperature required for maximal activation of heatshock genes, and the concentration of alcohol needed decreases with alcohol chain length. Ethanol is thought to alter membrane organization and permeability by entering the hydrophobic interior and increasing the polarity of this region [37].

The plasma membrane proton pump (H+ -ATPase) of yeast couples ATP hydrolysis to proton extrusion, thereby providing the means for solute uptake by secondary transporters and for regulating cytoplasmic pH. By pumping protons out of the cytoplasm, the H+ -ATPase acidifies the external medium, and makes the cytoplasm relatively alkaline. *S. cerevisiae* possesses two isoforms of this enzyme Pma1 and Pma2. They are 89% identical at the protein level, but they exhibit different activation, kinetic and regulatory properties, which may suggest their different functions. The specific activity of Pma1 increases with growth temperature. However, the increase in activity following stress is not attributable to synthesis of new protein, but rather to activation of the existing enzyme. Additionally, in *S. cerevisiae*, protein Hsp30 is a stress-inducible regulator of ATPase activity. Hsp30 is induced by heat shock, ethanol exposure, severe osmostress, weak organic acid exposure and glucose limitation. Hsp30 induction downregulates stimulation of H+ -ATPase caused by stress. There were also extensive studies of ATPase activity in non-conventional yeast *P. stipitis*. The enzyme from this yeast attained its highest activity at 35°C. It is unclear whether ATPase activity in *P. stipitis* involves one protein or two, as in the case of *S. cerevisiae*. Plasma membrane ATPase activity is essential for basal heat resistance. Moreover, thermotolerance is enhanced by prior exposure to stress. Prestressed cells are able to protect the proton gradient longer than cells that have not adapted to heat [86].

High-temperature stress causes multiple changes in the cell that ultimately affect protein structures and function, leading to inhibition of cell growth or cell death. The denatured or aggregated proteins in live cells may be degraded via the ubiquitin proteasome pathway (UPP). It is the one of main defense strategies to ensure survival in stress conditions [87]. This is ATP-dependent process, and timely destruction is vital for controlled cell division, as well as proteins unable to fold properly within the endoplasmic reticulum. The UPP is carried out by three classes of enzymes. A 'ubiquitin activating enzyme' (E1) forms of a thio-ester bond with ubiquitin that is a highly conserved 76-amino acid protein. The next reaction allows binding of ubiquitin to a 'ubiquitin conjugating enzyme' (E2), followed by the formation of the isopeptide bond between C-terminus of ubiquitin and the lysine rest by 'ubiquitin ligase' (E3) action. The UPP selectively eliminates abnormally folded or damaged proteins that have arisen by missense or nonsense mutations, biosynthetic errors, or damage by oxygen radicals or by denaturation, especially at high temperatures [88].

The mechanisms of yeast thermotolerance are largely controlled through the activation and regulation of specific stress-related genes involved in the synthesis of specific compounds that protect the organism from high-temperature stress. Elucidation of the function of these genes and/or proteins will give insight into the various mechanisms underlying yeast response to high-temperature stress, providing useful information to improve bioethanol production at higher temperatures.

Genetic data indicate that different genes contribute to heat tolerance at different stages of the plant life cycle and that different genes may be essential for basal and acquired thermotolerance [82]. Studies conducted by Gibney et al. have shown that gene deletions may also lead to higher thermosensitivity. Functional analysis of some identified genes confirmed that metabolism, cellular signaling and chromatin regulation play key role in controlling of yeast thermotolerance. However, the molecular mechanism of these actions remains still imprecise. They suggest that survival after heat shock depends on a small number of genes that function in assessing the metabolic health of the cell and/or regulate its growth in a changing environment [89]. To understand the mechanism of thermoadaptation, Shui et al. performed proteomic analysis for both parental and evolved strains of *S. cerevisiae*. They showed that some proteins were differentially regulated at heat-stress conditions in the parental and evolved strains. Additionally, the proteomic response of the industrial strains adapted to stress conditions was substantially different in comparison to the response of laboratory yeast to unexpected heat stress [90].

#### **7. Fermentation activity and ethanol tolerance**

Oxygen is one of key factors in regulation of fermentation in yeast. According to the role of oxygen in their metabolism, yeasts can be classified as: (a) obligatory aerobic, with only respiratory metabolism; (b) facultative fermentative or respiro-fermentative, displaying both respiratory and fermentative metabolism and (c) obligatory fermentative [91]. Although the majority of yeast species described so far is able to ferment sugars into ethanol and carbon dioxide, most of the respire-fermentative yeasts do not grow well under strictly anaerobic conditions [92].

Van Dijken and Scheffers explained the central role of two redox couples NAD<sup>+</sup> /NADH and NADP+ /NADPH in the metabolism of sugars by yeasts. NADH is preferentially used in dissimilatory metabolism, whereas NADPH is generally required for assimilatory reactions. In *S. cerevisiae*, *C. utilis* and probably in the yeasts in general, NADH and NADPH cannot be interconverted owing to the absence of a transhydrogenase activity [93].

Barnett [94] described 678 yeast species, and around 60% are considered to be fermentative on the basis of taxonomic tests such as gas production (Durham tubes) in laboratory conditions. However, this number is even higher since, under certain conditions, some of those species considered as non-fermentative are also able to ferment glucose. The ability to ferment glucose under oxygen limitation turns out to be a common feature of the different yeast species, but the capability of growth under anaerobic conditions is not widespread among these microorganisms. In fact, only very few yeast species are capable of fast growth under those conditions and *S. cerevisiae* stands out as the yeast generally acknowledged as a facultative anaerobe. Anaerobic growth is associated with a low energy yield compared with that observed under complete oxidative processes [94].

binding of ubiquitin to a 'ubiquitin conjugating enzyme' (E2), followed by the formation of the isopeptide bond between C-terminus of ubiquitin and the lysine rest by 'ubiquitin ligase' (E3) action. The UPP selectively eliminates abnormally folded or damaged proteins that have arisen by missense or nonsense mutations, biosynthetic errors, or damage by oxygen radicals

The mechanisms of yeast thermotolerance are largely controlled through the activation and regulation of specific stress-related genes involved in the synthesis of specific compounds that protect the organism from high-temperature stress. Elucidation of the function of these genes and/or proteins will give insight into the various mechanisms underlying yeast response to high-temperature stress, providing useful information to improve bioethanol production at higher temperatures. Genetic data indicate that different genes contribute to heat tolerance at different stages of the plant life cycle and that different genes may be essential for basal and acquired thermotolerance [82]. Studies conducted by Gibney et al. have shown that gene deletions may also lead to higher thermosensitivity. Functional analysis of some identified genes confirmed that metabolism, cellular signaling and chromatin regulation play key role in controlling of yeast thermotolerance. However, the molecular mechanism of these actions remains still imprecise. They suggest that survival after heat shock depends on a small number of genes that function in assessing the metabolic health of the cell and/or regulate its growth in a changing environment [89]. To understand the mechanism of thermoadaptation, Shui et al. performed proteomic analysis for both parental and evolved strains of *S. cerevisiae*. They showed that some proteins were differentially regulated at heat-stress conditions in the parental and evolved strains. Additionally, the proteomic response of the industrial strains adapted to stress conditions was substantially different in comparison to the response of laboratory yeast to unexpected heat stress [90].

Oxygen is one of key factors in regulation of fermentation in yeast. According to the role of oxygen in their metabolism, yeasts can be classified as: (a) obligatory aerobic, with only respiratory metabolism; (b) facultative fermentative or respiro-fermentative, displaying both respiratory and fermentative metabolism and (c) obligatory fermentative [91]. Although the majority of yeast species described so far is able to ferment sugars into ethanol and carbon dioxide, most of the respire-fermentative yeasts do not grow well under strictly anaerobic

/NADPH in the metabolism of sugars by yeasts. NADH is preferentially used in dissimilatory metabolism, whereas NADPH is generally required for assimilatory reactions. In *S. cerevisiae*, *C. utilis* and probably in the yeasts in general, NADH and NADPH cannot be

Barnett [94] described 678 yeast species, and around 60% are considered to be fermentative on the basis of taxonomic tests such as gas production (Durham tubes) in laboratory conditions. However, this number is even higher since, under certain conditions, some of those

/NADH and

Van Dijken and Scheffers explained the central role of two redox couples NAD<sup>+</sup>

interconverted owing to the absence of a transhydrogenase activity [93].

or by denaturation, especially at high temperatures [88].

98 Old Yeasts - New Questions

**7. Fermentation activity and ethanol tolerance**

conditions [92].

NADP+

The Pasteur and the Crabtree effects are the examples of special competition between respiration and fermentation of glucose [93]. The Pasteur effect refers to an activation of anaerobic glycolysis in order to meet cellular ATP demands owing to the lower efficiency of ATP production by fermentation compared with respiration. The Crabtree effect is currently defined as the occurrence of alcoholic fermentation under aerobic conditions. These two regulatory effects are very important in industrial fermentation [95, 96].

*S. cerevisiae* utilizes glucose by fermentative pathway (Crabtree positive) and some nonconventional yeasts like *K. lactis*, *P. pastoris* and *Y. lipolytica* are predominantly oxidative (Crabtree negative). However, among non-conventional yeasts are also Crabtree-positive ones. *S. cerevisiae* shows tolerance and good adaptation to high concentrations of ethanol. It was found that *S. cerevisiae* cells grown in the presence of ethanol appear to increase the amount of monounsaturated fatty acids in cellular lipids [97]. However, several nonconventional yeasts such as *Dekkera bruxellensis*, *P. kudriavzevii*, *Torulaspora delbrueckii* or *Wickerhamomyces anomalus* show quite good fermentation abilities and similar levels of ethanol tolerance in comparison to *S. cerevisiae* [98–103]. Especially Crabtree-positive *D. bruxellensis* strains are able to remain viable in fermentation media containing up to 16% ethanol. It has been shown that the yield of ethanol formation by *D. bruxellensis* in batch culture under anaerobic conditions is comparable with conventional yeasts. Additionally, *D. bruxellensis* shows the ability to 'compete' with conventional yeasts in industrial conditions, presumably due to the predominance of *S. cerevisiae* in the assimilation of nitrates [101, 102].

Several attempts were initiated to increase ability of yeast fermentation or to convert Crabtreenegative yeasts into Crabtree-positive for improving ethanol fermentation efficiency. Schifferdecker et al. created a metabolically engineered strain *D. bruxellensis* by increasing its fermentation capability. The gene encoding for alcohol dehydrogenase was overexpressed under the control of highly active *TEF1* promoter. As result, the improved strain produced 1.4–1.7 times more ethanol than the parental yeast [104]. Other unconventional strain of *K. lactis* was constructed as a mutant in the single gene encoding for a mitochondrial alternative internal dehydrogenase. This strain showed unaffected rate of exogenous NADH oxidation, but this mutation shifted the metabolism from respiration to fermentation. As a consequence, the mutant of *K. lactis* showed the increased rate of ethanol production [105].

Cost-effective fermentation depends on, among other factors, rapid and high yielding conversion of carbohydrates to ethanol, which in itself depends on improvements in the survival and performance of yeast cells under industrial conditions. Conventional *S. cerevisiae* is responsible for industrial alcoholic fermentation. On the other hand, most non-conventional yeasts that do not show such regulatory effect, which does not allow for efficient ethanol production in industrial conditions. Therefore, in traditional fermentation processes (beer production and winemaking), the non-*Saccharomyces* yeasts, initially present in fermentation medium at high numbers (ranging from 103 to 105 cells/ml), grow only during the early stages (up to 4–5% v/v of ethanol) and they are soon overtaken by strongly fermentative *S. cerevisiae* strains that complete the fermentation process [92].

Ethanol is well known as an inhibitor of microbial growth. Large concentrations of ethanol can be toxic to yeasts. Ethanol in low amounts inhibits cell division, decreases cell volume and specific growth rate, while high ethanol concentrations reduce cell vitality and increase cell death [106]. Ethanol also influences cell metabolism and macromolecular biosynthesis. The main results of these changes are production of heat shock-like proteins, low rate of RNA and protein accumulation, numerous petite mutants, denaturation of intracellular proteins and reduction of glycolytic enzymes activity. The response of yeasts to ethanol stress is complex, involving various aspects of cell sensing, signal transduction, transcriptional control, protein-targeting, accumulation of protectants and increased activity of repair functions. The efficiency of these processes in a given yeast strain determines its worth in industrial processes [107].

#### **8. Furan and acetate tolerance**

The use of hydrolysates obtained from plant biomass for the production of second generation bioethanol may be very problematic. In the pre-treatment processes, a lot of by-products toxic to the yeast cells may be formed [10]. The composition and concentration of inhibitory compounds is variable and depends on the type of lignocellulosic raw material and the method of its pretreatment [108]. Generally, after the pre-treatment and enzymatic hydrolysis of the hemicellulose fraction, hexoses: d-glucose, d-galactose, d-mannose and d-rhamnose and pentoses: d-xylose and l-arabinose are obtained [109, 110]. However, under high temperature and pressure, hexoses and pentoses may be degraded to 5-(hydroxymethyl)-2-furfural and furfural. The harmful effects of these compounds, even at low concentration, have been confirmed. RNA, DNA, proteins and membranes of yeast cells are particularly sensitive [111, 112].

The removal of toxic compounds from the fermentation medium is usually very expensive. Therefore, in order to improve the fermentation processes, the use of furan-tolerant yeast strains is more practical. Scientists recognize the molecular basis of cell tolerance to furan and its derivatives for model yeast *S. cerevisiae*. It has been found that *SIZ1* gene encoding the ligase E3, can bring the significant increase in tolerance to furfural. Some non-conventional yeast species, namely *W. anomalus*, *P. kudriavzevii*, *C. stellata*, *C. ethanolica*, *P. fermentans* and *Z. bailii*, show good tolerance to furfural and its derivatives. For example, the resistance of *P. kudriavzevii* to hydroxymethyl reaches up to 7 g/L [103].

The tolerance to weak acids is essential in the second generation bioethanol production. During the pretreatment of the lignocellulosic feedstock, released hemicellulose acetyl groups form acetic acid in the concentration of 5–10 g/L [113, 114]. It is known that weak acids exhibit cytotoxic effects. These compounds are transported through the cell membrane into the yeast cells by passive diffusion in non-dissociated form. In the yeast cells they are subject to dissociation, and protons are accumulated in the cytoplasm, causing acidification of cytosol [115–118]. In this case the cell metabolism slows down significantly by inhibiting glycolytic enzymes and NADH dehydrogenase [119–122]. Low intracellular pH inhibits the growth of yeasts, the adaptive phase increases and consequently, the efficiency of ethanol production decreases [123, 124]. Therefore, the use of yeast strains resistant to weak acids is essential for industrial production of bioethanol. Non-conventional yeast *Z. bailii* has been described as the most resistant to acetic acid. This yeast can grow at the concentration as high as 24 g/L, while conventional *S. cerevisiae* shows sensitivity at 9 g/L of acetate [125].

#### **9. Mixed populations and biocontrol**

in industrial conditions. Therefore, in traditional fermentation processes (beer production and winemaking), the non-*Saccharomyces* yeasts, initially present in fermentation medium at

4–5% v/v of ethanol) and they are soon overtaken by strongly fermentative *S. cerevisiae* strains

Ethanol is well known as an inhibitor of microbial growth. Large concentrations of ethanol can be toxic to yeasts. Ethanol in low amounts inhibits cell division, decreases cell volume and specific growth rate, while high ethanol concentrations reduce cell vitality and increase cell death [106]. Ethanol also influences cell metabolism and macromolecular biosynthesis. The main results of these changes are production of heat shock-like proteins, low rate of RNA and protein accumulation, numerous petite mutants, denaturation of intracellular proteins and reduction of glycolytic enzymes activity. The response of yeasts to ethanol stress is complex, involving various aspects of cell sensing, signal transduction, transcriptional control, protein-targeting, accumulation of protectants and increased activity of repair functions. The efficiency of these processes in a given yeast strain determines its worth in industrial pro-

The use of hydrolysates obtained from plant biomass for the production of second generation bioethanol may be very problematic. In the pre-treatment processes, a lot of by-products toxic to the yeast cells may be formed [10]. The composition and concentration of inhibitory compounds is variable and depends on the type of lignocellulosic raw material and the method of its pretreatment [108]. Generally, after the pre-treatment and enzymatic hydrolysis of the hemicellulose fraction, hexoses: d-glucose, d-galactose, d-mannose and d-rhamnose and pentoses: d-xylose and l-arabinose are obtained [109, 110]. However, under high temperature and pressure, hexoses and pentoses may be degraded to 5-(hydroxymethyl)-2-furfural and furfural. The harmful effects of these compounds, even at low concentration, have been confirmed. RNA, DNA, proteins and membranes of yeast cells are particularly sensitive [111, 112]. The removal of toxic compounds from the fermentation medium is usually very expensive. Therefore, in order to improve the fermentation processes, the use of furan-tolerant yeast strains is more practical. Scientists recognize the molecular basis of cell tolerance to furan and its derivatives for model yeast *S. cerevisiae*. It has been found that *SIZ1* gene encoding the ligase E3, can bring the significant increase in tolerance to furfural. Some non-conventional yeast species, namely *W. anomalus*, *P. kudriavzevii*, *C. stellata*, *C. ethanolica*, *P. fermentans* and *Z. bailii*, show good tolerance to furfural and its derivatives. For example, the resistance of *P.* 

The tolerance to weak acids is essential in the second generation bioethanol production. During the pretreatment of the lignocellulosic feedstock, released hemicellulose acetyl groups form acetic acid in the concentration of 5–10 g/L [113, 114]. It is known that weak acids exhibit cytotoxic effects. These compounds are transported through the cell membrane into the yeast

cells/ml), grow only during the early stages (up to

to 105

high numbers (ranging from 103

100 Old Yeasts - New Questions

**8. Furan and acetate tolerance**

*kudriavzevii* to hydroxymethyl reaches up to 7 g/L [103].

cesses [107].

that complete the fermentation process [92].

*S. cerevisiae* are able to produce high concentrations of ethanol reaching approximately 20% (v/v) but in conventional media. This yeast shows high fermentation rates, whereas they are unexpectedly less tolerant to high concentrations of ethanol and other toxic compounds. That is the reason why several ethanol-tolerant yeasts are used in industrial fermentations.

The profusion of selected starter cultures has allowed the more widespread use of inoculated fermentations, with consequent improvements to the control of the fermentation process, and the use of new biotechnological processes. Mixed fermentations using controlled inoculation of *S. cerevisiae* starter cultures and non-*Saccharomyces* yeasts represent a feasible way toward improving the complexity and enhancing the particular and specific characteristics of fermentation products [126–128].

Mixed cultures with different yeasts also provide an advantage in bioethanol production. In starchy media, using raw unhydrolysed starch in a single-step fermentation, ethanol production by a co-culture of *S. diastaticus* and *S. cerevisiae* was 24.8 g/L. This was 48% higher than the yield obtained with the monoculture of *S. diastaticus* (16.8 g/L). In another coculture fermentation with *Endomycopsis capsularis* and *S. cerevisiae*, maximum ethanol yield was 16.0 g/L, higher than *E. capsularis* the yield with the monoculture [129].

In second-generation ethanol production, xylose and arabinose are the significant fraction of lignocellulosic biomass. Therefore, their utilization is essential for a feasible bioethanol production process. The selection of yeast strains for the fermentation of pentoses has a large effect on ethanol yield [130]. The naturally xylose-fermenting non-conventional yeasts such as *C. shehatae* and *P. stipitis* have been widely studied because of their ability to ferment xylose into ethanol [131]. *P. stipitis* is considered as a promising strain because it can ferment a wide range of sugars, including cellobiose. *Candida* species have been shown to ferment d-xylose to ethanol as the major product. Strain improvement by mutation is one of the best methods to increase the ethanol yield, and in this case, two strains capable of producing significantly higher ethanol yields than the parental strains were obtained [132].

The influence of non-*Saccharomyces* yeasts on fermentation processes was studied and their biotechnological potential was evaluated. The industrial yeast market, which was historically focused on *S. cerevisiae*, now offers *S. cerevisiae*/non-*Saccharomyces* multi-starters. However, the development of these mixed populations requires knowledge about possible interactions between yeast strains. Considering the use of mixed populations, the special attention should be paid not only to the selection of the proper assimilation-competent strains, their inoculation, culture media, but also to the interactions between these yeast monocultures. The interesting results were obtained by Yamaoka et al. [128]. This research was carried out to investigate the influence of non-*Saccharomyces* yeast, *K. lactis*, on metabolite formation and the ethanol tolerance of *S. cerevisiae* in mixed cultures in synthetic minimal medium containing 20% glucose. It was noted that co-cultivation of *K. lactis* seems to prompt *S. cerevisiae* to be ethanol tolerant by forming protective metabolites such as glycerol.

In turn, studies on mixed cultures *S. cerevisiae*/*T. delbrueckii* showed that physical contact between yeast cells induced rapid death of *T. delbrueckii*. This phenomenon was previously described as a cell-cell contact mechanism. However, when these yeast cultures were physically separated from each other, the sensitive strain of *Torulaspora* sp. kept its viability [133].

The mixed yeast populations have been explored not only for improvement of ethanol yield but also as biological control—an alternative to the use of synthetic chemicals for prevention of microbial spoilage. The possibility of using the selected antagonistic yeasts against undesirable spoilage microorganisms is the subject of interest for both scientists and technologists. The presence of undesired microflora may lead to significant reduction in the efficiency of biotechnological processes. Non-conventional yeasts, characterized by antagonistic activity against spoilage microflora include genera *Pichia*, *Candida*, *Aureobasidium*, *Metschnikowia* and *Debaryomyces*. The interactions between microorganisms have been described in numerous scientific studies [126, 127, 134–136]. Industrial yeast strains, due to the high reproductive potential and rich enzymatic equipment, have the ability to colonize fermentation environments rapidly. The presence of microbial contamination not only reduces available nutrients for industrial microorganisms, but also reduces the potential living space. Low nutrient availability is one of the most important mechanisms of competition between yeast strains.

The killer phenomenon was first observed in yeast *S. cerevisiae* in the 1960s of the last century. However, the killer features have also been found in representatives of non-conventional yeasts belonging to genera *Debaryomyces*, *Pichia*, *Kluyveromyces*, *Candida*, *Cryptococcus*, *Ustilago*, *Rhodotorula*, *Williopsis*, *Torulopsis*, *Zygosaccharomyces*, *Hansenula* and *Hanseniaspora*. Killer protein toxins show specific activity spectra dependent on pH level, temperature and aeration conditions. These toxins differ in resistance to proteolytic enzymes, chemicals, pH, and they are mutually antagonistic. The impact of killer yeasts to sensitive yeast cells include killer protein receptors on the cell wall of sensitive cells. The consequences of killer toxin binding to cell wall are physiological changes that lead to death of the sensitive cells. Initially, there is a breakdown of amino acids and proton gradient, leakage of potassium ions from ATP, reduction of metabolite levels and the destruction of the pH gradient. All these processes lead to a gradual death of sensitive yeast cells [4, 137, 138].

It has been found that yeast strains *Metschnikowia pulcherrima* have a great potential to be a leading natural and biological control against a broad spectrum of pathogens [139–141]. *M. pulcherrima* forms pulcherimic acid, which is accumulated in growth medium and forms red pulcherrimin—a chelate complex with Fe(III) ions (**Photo 2**).

focused on *S. cerevisiae*, now offers *S. cerevisiae*/non-*Saccharomyces* multi-starters. However, the development of these mixed populations requires knowledge about possible interactions between yeast strains. Considering the use of mixed populations, the special attention should be paid not only to the selection of the proper assimilation-competent strains, their inoculation, culture media, but also to the interactions between these yeast monocultures. The interesting results were obtained by Yamaoka et al. [128]. This research was carried out to investigate the influence of non-*Saccharomyces* yeast, *K. lactis*, on metabolite formation and the ethanol tolerance of *S. cerevisiae* in mixed cultures in synthetic minimal medium containing 20% glucose. It was noted that co-cultivation of *K. lactis* seems to prompt *S. cerevisiae* to be

In turn, studies on mixed cultures *S. cerevisiae*/*T. delbrueckii* showed that physical contact between yeast cells induced rapid death of *T. delbrueckii*. This phenomenon was previously described as a cell-cell contact mechanism. However, when these yeast cultures were physically separated from each other, the sensitive strain of *Torulaspora* sp. kept its

The mixed yeast populations have been explored not only for improvement of ethanol yield but also as biological control—an alternative to the use of synthetic chemicals for prevention of microbial spoilage. The possibility of using the selected antagonistic yeasts against undesirable spoilage microorganisms is the subject of interest for both scientists and technologists. The presence of undesired microflora may lead to significant reduction in the efficiency of biotechnological processes. Non-conventional yeasts, characterized by antagonistic activity against spoilage microflora include genera *Pichia*, *Candida*, *Aureobasidium*, *Metschnikowia* and *Debaryomyces*. The interactions between microorganisms have been described in numerous scientific studies [126, 127, 134–136]. Industrial yeast strains, due to the high reproductive potential and rich enzymatic equipment, have the ability to colonize fermentation environments rapidly. The presence of microbial contamination not only reduces available nutrients for industrial microorganisms, but also reduces the potential living space. Low nutrient availability is one of the most important mechanisms of competi-

The killer phenomenon was first observed in yeast *S. cerevisiae* in the 1960s of the last century. However, the killer features have also been found in representatives of non-conventional yeasts belonging to genera *Debaryomyces*, *Pichia*, *Kluyveromyces*, *Candida*, *Cryptococcus*, *Ustilago*, *Rhodotorula*, *Williopsis*, *Torulopsis*, *Zygosaccharomyces*, *Hansenula* and *Hanseniaspora*. Killer protein toxins show specific activity spectra dependent on pH level, temperature and aeration conditions. These toxins differ in resistance to proteolytic enzymes, chemicals, pH, and they are mutually antagonistic. The impact of killer yeasts to sensitive yeast cells include killer protein receptors on the cell wall of sensitive cells. The consequences of killer toxin binding to cell wall are physiological changes that lead to death of the sensitive cells. Initially, there is a breakdown of amino acids and proton gradient, leakage of potassium ions from ATP, reduction of metabolite levels and the destruction of the pH gradient. All these processes

It has been found that yeast strains *Metschnikowia pulcherrima* have a great potential to be a leading natural and biological control against a broad spectrum of pathogens [139–141].

ethanol tolerant by forming protective metabolites such as glycerol.

viability [133].

102 Old Yeasts - New Questions

tion between yeast strains.

lead to a gradual death of sensitive yeast cells [4, 137, 138].

It has been shown that the antibacterial and antifungal activity of yeast depends on pulcherrimin formation [139]. Therefore, strains that produce large amounts of pulcherrimin are of great interest to engineers and microbiologists, as biocontrol agents inhibiting growth of pathogenic bacteria, yeasts and molds. This substance may be an alternative to antibiotics and fungicides. Oro et al. evaluated *M. pulcherrima* for the antimicrobial activity against numerous yeast strains belonging to *Pichia*, *Candida*, *Hanseniaspora*, *Kluyveromyces*, *Saccharomycodes*, *Torulaspora*, *Brettanomyces* and *Saccharomyces* genera [141]. *M. pulcherrima* displayed a broad and effective antimicrobial action on undesired wild spoilage yeasts (*Brettanomyces/Dekkera* spp.*, Hanseniaspora* spp., *Pichia* spp.). Interestingly, the antimicrobial activity of *M. pulcherrima* did not have any influence on the growth of *S. cerevisiae*. The oxygen availability strongly influences population dynamics in mixed populations of conventional and non-conventional yeasts. Additionally, in the presence of non-*Saccharomyces* yeasts, species-specific chemical volatile profiles were noted, in particular increases in some higher alcohols and medium chain fatty acids. This data show the potential use of selected *M. pulcherrima* strains in controlled multi-starter fermentations with *S. cerevisiae* starter cultures [142, 143].

**Photo 2.** Pulcherrimin formation by *M. pulcherrima* on YPD agar with Fe(III) ions (author: Ewelina Pawlikowska).

#### **10. Conclusion**

The conventional yeast *S. cerevisiae* is the best known species used in numerous industrial high-tech processes. However, in new technologies, including second generation ethanol production, the use of this yeast may encounter a number of difficulties. Research studies suggest that it is possible and even necessary to use selected non-conventional yeast strains to increase the use of carbon sources as well as to improve the economic effects of ethanol production from plant waste materials. It is worth paying attention to one more aspect—many species of non-conventional yeasts produce unique biocontrol compounds, which can be seen as an additional valuable feature for conducting fermentation processes. These yeasts may find use as monocultures or mixed complementary populations. Although the exploration of existing natural biodiversity of non-conventional yeasts is attractive, the major bottleneck is that industrially applicable traits are not commonly found in nature. However, there are multiples of classical approaches to develop strains with improved phenotypes such as mutagenesis, sexual hybridization, genetic modification, adaptive evolution and other emerging tools. Among them, non-genetic modification, adaptive evolution, is preferable; as the use of strains developed using genetic methods in the food industry remains controversial. In addition, such a traditional phenotype improvement based on random appearance of adaptive mutations based on selective regimes requires no prior knowledge of the genetic background of the strains is under development. This is important, as the current limitation in applications of non-conventional yeasts is that they are less studied and their genetic architectures and pathways are less understood. Therefore, we can conclude that era of research on nonconventional yeasts has just begun.

#### **Author details**

Dorota Kręgiel\*, Ewelina Pawlikowska and Hubert Antolak

\*Address all correspondence to: dorota.kregiel@p.lodz.pl

Institute of Fermentation Technology and Microbiology, Lodz University of Technology, Poland

#### **References**


[4] Steensels J, Verstrepen KJ. Taming wild yeast: Potential of conventional and nonconventional yeasts in industrial fermentations. Annual Review of Microbiology. 2014;**68**:61-80. DOI: 10.1146/annurev-micro-091213-113025

**10. Conclusion**

104 Old Yeasts - New Questions

conventional yeasts has just begun.

Dorota Kręgiel\*, Ewelina Pawlikowska and Hubert Antolak

\*Address all correspondence to: dorota.kregiel@p.lodz.pl

**Author details**

**References**

434449a

crvi.2010.12.016

10.1073/pnas.1317377110

The conventional yeast *S. cerevisiae* is the best known species used in numerous industrial high-tech processes. However, in new technologies, including second generation ethanol production, the use of this yeast may encounter a number of difficulties. Research studies suggest that it is possible and even necessary to use selected non-conventional yeast strains to increase the use of carbon sources as well as to improve the economic effects of ethanol production from plant waste materials. It is worth paying attention to one more aspect—many species of non-conventional yeasts produce unique biocontrol compounds, which can be seen as an additional valuable feature for conducting fermentation processes. These yeasts may find use as monocultures or mixed complementary populations. Although the exploration of existing natural biodiversity of non-conventional yeasts is attractive, the major bottleneck is that industrially applicable traits are not commonly found in nature. However, there are multiples of classical approaches to develop strains with improved phenotypes such as mutagenesis, sexual hybridization, genetic modification, adaptive evolution and other emerging tools. Among them, non-genetic modification, adaptive evolution, is preferable; as the use of strains developed using genetic methods in the food industry remains controversial. In addition, such a traditional phenotype improvement based on random appearance of adaptive mutations based on selective regimes requires no prior knowledge of the genetic background of the strains is under development. This is important, as the current limitation in applications of non-conventional yeasts is that they are less studied and their genetic architectures and pathways are less understood. Therefore, we can conclude that era of research on non-

Institute of Fermentation Technology and Microbiology, Lodz University of Technology, Poland

[1] Boekhout T. Biodiversity – Gut feeling for yeasts. Nature. 2005;**434**:449-451. DOI: 10.1038/

[2] Sicard D, Legras JL. Bread, beer and wine: Yeast domestication in the *Saccharomyces sensu stricto* complex. Comptes Rendus Biologies. 2011;**334**:229-236. DOI: 10.1016/j.

[3] Bokulich NA, Thorngate JH, Richardson PM, Mills DA. Microbial biogeography of wine grapes is conditioned by cultivar, vintage, and climate. Proceedings of the National Academy of Sciences of the United States of America (PNAS). 2014;**111**:139-148. DOI:


[29] Kuhad RC, Gupta R, Pal Khasa Y, Singh A, Zhang YHP. Bioethanol production from pentose sugars: Current status and future prospects. Renewable and Sustainable Energy Reviews. 2011;**15**(9):4950-4962. DOI: 10.1016/j.rser.2011.07.058

[17] Wyman CE, Dale BE, Elander RT, Holtzapple M, Ladisch MR, Lee YY. Coordinated development of leading biomass pretreatment technologies. Bioresource Technology.

[18] Kang J, Kim D, Lee T. Hydrogen production and microbial diversity in sewage sludge fermentation preceded by heat and alkaline treatment. Bioresource Technology.

[19] Michalska K, Miazek K, Krzystek L, Ledakowicz S. Influence of pretreatment with Fenton's reagent on biogas production and methane yield from lignocellulosic biomass.

[20] Harmsen PFH, Huijgen WJJ, Bermúdez López LM, Bakker RRC. Literature Review of Physical and Chemical Pretreatment Processes for Lignocellulosic Biomass. ECN-E-10-013; 2010. Available from: https://www.ecn.nl/docs/library/report/2010/e10013.pdf

[21] Rallis Ch. New tricks for an old-favorite model. Genome Biology. 2009;**10**:315. DOI:

[22] Moysés DN, Reis VCB, Moreira de Almeida JR, Pepe de Moraes LM, Torre FAG. Xylose fermentation by *Saccharomyces cerevisiae*: Challenges and prospects. International Journal

[23] Wang C, Zhao J, Qiu C, Wang S, Shen Y, Du B, Ding Y, Ba X. Coutilization of D-glucose, D-xylose, and L-arabinose in *Saccharomyces cerevisiae* by coexpressing the metabolic pathways and evolutionary engineering. BioMed Research International. 2017, Article

[24] Wisselink HW, Toirkens MJ, Wu O, Pronk JT, van Maris AJA. Novel evolutionary engineering approach for accelerated utilization of glucose, xylose, and arabinose mixtures by engineered *Saccharomyces cerevisiae* strains. Applied Microbiology and Biotechnology.

[25] Jeffries TW, Jin Y-S. Metabolic engineering for improved fermentation of pentoses by yeasts. Applied Microbiology and Biotechnology. 2004;**63**(5):495-509. DOI: 10.1007/s00253-

[26] Subtil T, Boles E. Competition between pentoses and glucose during uptake and catabolism in recombinant *Saccharomyces cerevisiae*. Biotechnology for Biofuels. 2012;**5**:14. Available from: http://www.biotechnologyforbiofuels.com/content/5/1/14 [Accessed:

[27] Jin Y-S, Laplaza JM, Jeffries TW. *Saccharomyces cerevisiae* engineered for xylose metabolism exhibits a respiratory response. Applied and Environmental Microbiology.

[28] Hahn-Hägerdal B, Karhumaa K, Fonseca C, Gorwa-Grauslund MF. Towards industrial pentose-fermenting yeast strains. Applied Microbiology and Biotechnology.

2004;**70**:6816-6825. DOI: 10.1128/AEM.70.11.6816-6825.2004

2007;**74**(5):937-953. DOI: 10.1007/s00253-006-0827-2

of Molecular Sciences. 2016;**17**:207. DOI: 10.3390/ijms17030207

ID 5318232, 8 pages. DOI: 10.1155/2017/5318232

2009;**75**(4):907-914. DOI: 10.1128/AEM.02268-08

Bioresource Technology. 2012;**119**:72-78. DOI: 10.1016/j.biortech.2012.05.105

2005;**96**:1959-1966. DOI: 10.1016/j.biortech.2005.01.010

2012;**109**:239-243. DOI: 10.1016/j.biortech.2012.01.048

[Accessed: February 14, 2017]

10.1186/gb-2009-10-9-315

106 Old Yeasts - New Questions

003-1450-0

May 21, 2017]


[52] Gunde-Cimerman N, Ramos J, Plemenitaš A. Halotolerant and halophilic fungi. Mycological Research. 2009;**113**(11):1231-1241. DOI: 10.1016/j.mycres.2009.09.002

[41] Kregiel D. Immobilization of yeast cells in alginate gels for ethanol production – Potentialities and limitations. Biotechnology and Food Science. 2005;**69**:59-66

[42] Djekrif DS, Gillmann L, Bennamoun L, Ait-Kaki A, Labbani K, Nouadri T, Meraihi Z. Amylolytic yeasts: Producers of α-amylase and pullulanase. International Journal of Life Sciences Scientific Research. 2016;**2**:339-354. Available from: http://ijlssr.com/curren-

[43] Kregiel D.Amylolytic activity of Kluyver-positive *Debaryomyces occidentalis* cells immobilized in foamed alginate gel. Journal of Microbiology, Biotechnology and Food Sciences.

[44] Kregiel D, Berlowska J, Ambroziak W. Growth and metabolic activity of conventional and non-conventional yeasts immobilized in foamed alginate. Enzyme and Microbial

[45] Negruta O, Csutak O, Stoica I, Rusu E, Vassu T. Methylotrophic yeasts: Diversity and methanol metabolism. Romanian Biotechnological Letters. 2010;**5**:5369-5375. Available from: http://www.rombio.eu/rbl4vol15/1%20Negruta.pdf [Accessed: February 14, 2017]

[46] Berlowska J, Dudkiewicz M, Kregiel D, Czyzowska A, Witonska I. Cell lysis induced by membrane-damaging detergent saponins from *Quillaja saponaria*. Enzyme and Microbial

[47] Berlowska J, Dudkiewicz-Kolodziejska M, Pawlikowska E, Pielech-Przybylska K, Balcerek M, Czyzowska A, Kregiel D. Utilisation of post-fermentation yeasts for yeast extract production by autolysis: The effect of yeast strain and saponin *Q. saponaria*.

[48] Martorell P, Stratford M, Steels H, Fernandez-Espinar MT, Querol A. Physiological characterization of spoilage strains of *Zygosaccharomyces bailii* and *Zygosaccharomyces rouxii* isolated from high sugar environments. International Journal of Food Microbiology.

[49] Dujon B. Yeast evolutionary genomics. Nature Reviews Genetics. 2010;**11**:512-524. DOI:

[50] Riley R, Haridas S, Wolfe KH, Lopes MR, Hittinger CT, Göker M, Salamov AA, Wisecaver JH, Long TM, Calvey CH, Aerts AL, Barry KW, Choi C, Clum A, Coughlan AY, Deshpande S, Douglass AP, Hanson SJ, Klenk H-P, LaButti KM, Lapidus A, Lindquist EA, Lipzen AM, Meier-Kolthoff JP, Ohm RA, Otillar RP, Pangilinan JL, Peng Y, Rokas A, Rosa CA, Scheuner C, Sibirny AA, Slot JC, Stielow JB, Sun H, Kurtzman CP, Blackwell M, Grigoriev IV, Jeffries TW. Comparative genomics of biotechnologically important

[51] Masneuf-Pomarede I, Bely M, Marullo P, Albertin W. The genetics of non-conventional wine yeasts: Current knowledge and future challenges. Frontiers in Microbiology.

yeasts. PNAS. 2016;**113**(35):9882-9887. DOI: 10.1073/pnas.1603941113

Technology. 2013;**53**:229-234. DOI: 10.1016/j.enzmictec.2013.05.0102013

Technology. 2015;**75-76**:44-48. DOI: 10.1016/j.enzmictec.2015.04.007

Journal of the Institute of Brewing. 2017. DOI: 10.1002/jib.438

2007;**114**:234-242. DOI: 10.1016/j.ijfoodmicro.2006.09.014

2016;**6**:1563. DOI: 10.3389/fmicb.2015.01563

10.1038/nrg2811

108 Old Yeasts - New Questions

tissue/IJLSSR-1115-10-2015.pdf [Accessed: February 14, 2017]

2016;**5**:311-313. DOI: 10.15414/jmbfs.2016.5.4.311-313


[76] Limtong S, Sringiew C, Yongmanitchai W. Production of fuel ethanol at high temperature from sugar juice by a newly isolated *Kluyveromyces marxianus*. Bioresource Technology. 2007;**98**:3367-3374. DOI: 10.1016/j.biortech.2006.10.044

[64] Hohman S. Osmotic stress signaling and osmoadaptation in yeasts. Microbiology and Molecular Biology Reviews. 2002;**66**(2):300-372. DOI: 10.1128/MMBR.66.2.300-372.2002

[65] Gonzalez-Hernandez JC, Cárdenas-Monroy CA, Peña A. Sodium and potassium transport in the halophilic yeast *Debaryomyces hansenii*. Yeast. 2004;**21**:403-412. DOI: 10.1002/

[66] Prista C, Michán C, Miranda IM, Ramos J. The halotolerant *Debaryomyces hansenii*, the Cinderella of non-conventional yeasts. Yeast Primer. 2016;**33**(10):523-533. DOI: 10.1002/

and K+

Cloning and expression in *Saccharomyces cerevisiae* of DhNHX1. FEMS Yeast Research.

[68] Neves ML, Oliveira RP, Lucas CM. Metabolic flux response to salt-induced stress in the halotolerant yeast *Debaryomyces hansenii*. Microbiology. 1997;**143**:1133-1139. DOI:

[69] Pribylova L, Farkaš V, Slaninová I, de Montigny J, Sychrová H. Differences in osmotolerant and cell-wall properties of two *Zygosaccharomyces rouxii* strains. Folia Microbiologica.

[70] Dakal TC, Solieri L, Giudici P. Adaptive response and tolerance to sugar and salt stress in the food yeast *Zygosaccharomyces rouxii*. International Journal of Food Microbiology.

[71] Kumar S, Randhawa A, Ganesan K, Raghava GPS, Mondal AK. Draft genome sequence of salt-tolerant yeast *Debaryomyces hansenii* var. *hansenii* MTCC 234. Eukaryotic Cell.

[72] Tabka MG, Gimbert I, Monod F, Sigoillot JC. Enzymatic saccharification of wheat straw for bioethanol production by a combined cellulose xylanase and feruloyl esterase treatment. Enzyme and Microbial Technology. 2006;**39**:897-902. DOI: 10.1016/j.

[73] Anderson PJ, McNeil K, Watson K. High-efficiency carbohydrate fermentation to ethanol at temperatures above 40°C by *Kluyveromyces marxianus* var. *marxianus* isolated from sugar mills. Applied and Environmental Microbiology. 1986;**51**:1314-1320. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC239064/ [Accessed: February 14, 2017]

[74] Nonklang S, Abdel-Banat BMA, Cha-aim K, Moonjai N, Hoshida H, Limtong S, Yamada M, Akada R. High-temperature ethanol fermentation and transformation with linear DNA in the thermotolerant yeast *Kluyveromyces marxianus* DMKU3-1042. Applied and

[75] Abdel-Banat BMA, Hoshida H, Ano A, Nonklang S, Akada R. High-temperature fermentation: How can processes for ethanol production at high temperatures become superior to the traditional process using mesophilic yeast? Applied Microbiology and

Environmental Microbiology. 2008;**74**:7514-7521. DOI: 10.1128/AEM.01854-08

Biotechnology. 2010;**85**:861-867. DOI: 10.1007/s00253-009-2248-5

distribution in *Debaryomyces hansenii*.

yea.1108

110 Old Yeasts - New Questions

yea.3177

[67] Montiel V, Ramos J. Intracellular Na+

10.1099/00221287-143-4-1133

enzmictec.2006.01.021

2007;**52**:241-245. DOI: 10.1007/BF02931305

2012;**11**(7):961-962. DOI: 10.1128/EC.00137-12

2007;**7**:102-109. DOI: 10.1111/j.1567-1364.2006.00115.x

2014;**185**:140-157. DOI: 10.1016/j.ijfoodmicro.2014.05.015


[101] Ruyters S, Mukherjee V, Verstrepen KJ, Thevelein JM, Willems KA, Lievens B.Assessing the potential of wild yeasts for bioethanol production. Journal of Industrial Microbiology and Biotechnology. 2015;**42**:39-48. DOI: 10.1007/s10295-014-1544-y

[89] Gibney PA, Lub C, Caudy AA, Hess DC, Botstein D. Yeast metabolic and signaling genes are required for heat-shock survival and have little overlap with the heat-induced genes. PNAS. 2013;**28**:E4393-E4402. DOI: 10.1073/pnas.1318100110/-/DCSupplemental

[90] Shui W, Xiong Y, Xiao W, Qi X, Zhang Y, Lin Y, Guo Y, Zhang Z, Wang Q, Ma Y. Understanding the mechanism of thermotolerance distinct from heat shock response through proteomic analysis of industrial strains of *Saccharomyces cerevisiae*. Molecular &

[91] Jolly NP, Varela C, Pretorius IS. Not your ordinary yeast: Non-*Saccharomyces* yeasts in wine production uncovered. FEMS Yeast Research. 2014;**14**:215-237. DOI:

[92] Albergaria H, Arneborg N. Dominance of *Saccharomyces cerevisiae* in alcoholic fermentation processes: Role of physiological fitness and microbial interactions. Applied Microbiology and Biotechnology. 2016;**100**:2035-2046. DOI: 10.1007/s00253-015-7255-0

[93] van Dijken JP, Scheffers WA. Redox balances in the metabolism of sugars by yeasts. FEMS Microbiology Letters. 1986;**32**:199-224. DOI: 10.1111/j.15746968.1986.tb01194.x

[94] Barnett JA. A history of research on yeast 2: Louis Pasteur and his contemporaries, 1850- 1880. Yeast. 2000;**16**:755-771. DOI: 10.1002/1097-0061(20000615)16:8<755::AID-YEA587>

[95] Rodrigues F, Ludovico P, Leao C.Sugar metabolism in yeasts: An overview of aerobic and anaerobic glucose. In: Peter G, Rosa C, editors. Biodiversity and Ecophysiology of Yeasts. Berlin, Heidelberg: Springer-Verlag; 2006. pp. 101-121. DOI: 10.1007/3-540-30985-3\_6

[96] Hagman A, Säll T, Piškur J. Analysis of the yeast short-term Crabtree effect and its ori-

[97] Henderson CM, Block DE. Examining the role of membrane lipid composition in determining the ethanol tolerance of *Saccharomyces cerevisiae*. Applied Environmental

[98] Galafassi S, Merico A, Pizza F, Hellborg L, Molinari F, Piskur J, Compagno C. *Dekkera*/*Brettanomyces* yeast for ethanol production from renewable sources under oxygen-limited and low-pH conditions. Journal of Industrial Microbiology and

[99] Zha Y, Hossain AH, Tobola F, Sedee N, Havekes M, Punt PJ. *Pichia anomala* 29X: A resistant strain for lignocellulosic biomass hydrolysate fermentation. FEMS Yeast Research.

[100] Mukherjee V, Steensels J, Lievens B, Van de Voorde I, Verplaetse A, Aerts G, Willems KA, Thevelein JM, Verstrepen KJ, Ruyters S. Phenotypic evaluation of natural and industrial *Saccharomyces* yeasts for different traits desirable in industrial bioethanol production. Applied Microbiology and Biotechnology. 2014;**98**:9483-9498. DOI: 10.1007/

gin. The FEBS Journal. 2014;**281**:4805-4814. DOI: 10.1111/febs.13019

Microbiology. 2014;**80**:2966-2972. DOI: 10.1128/AEM.04151-13

Biotechnology. 2011;**38**:1079-1088. DOI: 10.1007/s10295-010-0885-4

2013;**13**:609-617. DOI: 10.1111/1567-1364.12062

s00253-014-6090-z

Cellular Proteomics. 2015;**14**:1885-1897. DOI: 10.1074/mcp.M114.045781

10.1111/1567-1364.12111

3.0.CO;2-4

112 Old Yeasts - New Questions


[125] Lindberg L, Santos AXS, Riezman H, Olsson L, Bettiga M. Lipidomic profiling of *Saccharomyces cerevisiae* and *Zygosaccharomyces bailii* reveals critical changes in lipid composition in response to acetic acid stress. PLoS One. 2013;**8**:e73936. DOI: 10.1371/ journal.pone.0073936

[112] Lin FM, Qiao B, Yuan YJ. Comparative proteomic analysis of tolerance and adaptation of ethanologenic *Saccharomyces cerevisiae* to furfural, a lignocellulosic inhibitory compound. Applied and Environmental Microbiology. 2009;**75**:3765-3776. DOI: 10.1128/

[113] Martinez A, Rodriguez ME, Wells ML, York SW, Preston JF, Ingram LO. Detoxification of dilute acid hydrolysates of lignocellulose with lime. Biotechnology Progress.

[114] Qian M, Tian S, Li X, Zhang J, Pan Y, Yang X. Ethanol production from dilute-acid softwood hydrolysate by co-culture. Applied Biochemistry and Biotechnology.

[115] Villarreal MLM, Prata AMR, Felipe MGA, Silva JBAE. Detoxification procedures of eucalyptus hemicellulose hydrolysate for xylitol production by *Candida guilliermondii*. Enzyme and Microbial Technology. 2006;**40**:17-24. DOI: 10.1016/j.enzmictec.2005.10.032

[116] Chandel AK, Kapoor RK, Singh A, Kuhad RC. Detoxification of sugarcane bagasse hydrolysate improves ethanol production by *Candida shehatea* NCIM 3501. Bioresource

[117] Tian S, Zhou G, Yan F, Yu Y, Yang X. Yeast strains for ethanol production from lignocellulosic hydrolysates during in situ detoxification. Biotechnology Advances. 2009;**27**:656-660.

[118] Mollapour M, Piper PW. Hog1 mitogen-activated protein kinase phosphorylation target the yeast Fps1 aquaglyceroporin for endocytosis, thereby rendering cell resistant to acetic acid. Molecular and Cellular Biology. 2007;**27**:6446-6456. DOI: 10.1128/

[119] Arneborg N, Jespersen L, Jakobsen M. Individual cells of *Saccharomyces cerevisiae* and *Zygosaccharomyces bailii* ehibit different short term intracellular pH responses to acetic

[121] Pampulha ME, Loureiro-Dias MC. Activity of glycolytic enzymes of *Saccharomyces cerevisiae* in the presence of acetic acid. Applied Microbiology and Biotechnology.

[122] Ding J, Huang X, Zhang L, Zhao N, Yang D, Zhang K. Tolerance and stress response to ethanol on the yeast *Saccharomyces cerevisiae*. Applied Microbiology and Biotechnology.

[123] Casey JR, Grinstein S, Orlowski J. Sensors and regulators of intracellular pH. Nature

[124] Cantarella M, Contarella L, Gallifuoco A, Spera A, Alfani F. Effect of inhibitors released during steam-explosion treatment of poplar wood on subsequent enzymatic hydrolysis

and SSF. Biotechnology Progress. 2004;**20**:200-206. DOI: 10.1021/bp0257978

Reviews Molecular Cell Biology. 2010;**11**:50-61. DOI: 10.1038/nrm2820

Nhx1 regulates cellular pH to control vesicle trafficking. Molecular Biology of the Cell.

(K+ )/H+

exchanger

acid. Archives of Microbiology. 2000;**174**:125-128. DOI: 10.1007/s002030000185

[120] Brett CL, Tukaye DN, Mukherjee S, Rao R. The yeast endosomal Na<sup>+</sup>

2005;**16**:1396-1405. DOI: 10.1091/mbc.E04-11-0999

2009;**85**:253-263. DOI: 10.1007/s00253-009-2223-1

1990;**34**:375-380. DOI: 10.1007/BF00170063

Technology. 2007;**98**:1947-1950. DOI: 10.1016/j.biortech.2006.07.047

AEM.02594-08

114 Old Yeasts - New Questions

MCB.02205-06

2001;**17**:287-293. DOI: 10.1021/bp0001720

DOI: 10.1016/j.biotechadv.2009.04.008

2006;**134**:273-283. DOI: 10.1385/ABAB:134:3:273

