*1.1.1. Yeast diversity and metabolism*

Yeast are unicellular fungi, being the simplest eukaryotes. Present in a great number of environments, yeast can be found not only in decomposing fruit, trees, and soils but also in commensal relationships with higher eukaryotes, humans included, and even saltwater. The high diversity of species, almost 1500 species have been described [6], is closely related to this wide distribution. Some of these yeast are adapted to extreme environments, such as high salt concentrations [7], low pH [8], or extremely cold temperatures [9, 10]. The genus *Saccharomy‐ ces*, particularly *Saccharomyces cerevisiae*, is strongly associated with the production of ferment‐ ed products for human consumption, namely, bread, wine, and beer [2]. After several millennia of close coexistence, through phenotypic selection, these species evolved to produce goods with organoleptic properties pleasant to humans. However, given the high degree of diversity found in nature, it is expected to find yeast with new and more interesting characteristics for the industry in new and unexplored niches [11, 12].

Yeast, as other heterotrophic organisms, have the anabolism coupled with catabolism. In one hand, the oxidation of organic molecules, as sugars, yields adenosine 5-triphosphate (ATP) that, in turn, is used as an energy resource for the cell. On the other hand, such organic molecules can also be used as building blocks or to generate intermediary compounds for the synthesis of other compounds, some of which with high commercial value.

The high diversity of environments where yeast can be found is closely related to the variety of carbon sources that can be used. Hexoses such as glucose, fructose, galactose, or mannose are the most common substrates, but some species can use pentoses like xylose or arabinose. Several industrial relevant species can metabolize disaccharides as maltose, lactose, or sucrose, and some, as *Saccharomyces diastaticus*, can even metabolize dextrins (glucose polymers) [13, 14]. Nevertheless, glucose and fructose, to a lesser extent, are the preferred substrates.

**1. Introduction**

108 Food Production and Industry

as water, tea, and coffee [5].

**1.1. Ethanol and lactic acid fermentation**

the industry in new and unexplored niches [11, 12].

*1.1.1. Yeast diversity and metabolism*

Alcoholic beverages have been consumed by mankind since ancient times. These products of fermented sugar-rich goods, namely, cereals, roots, and fruits, are present worldwide since the oldest records [1, 2]. In fact, several of mankind's milestones, such as the dawn of agricul‐ ture, are closely linked with the production of some type of alcoholic beverages. Similar processes of fermentation emerged independently in many civilizations across the globe. Interestingly, the main players of the whole process are relatively few, mostly yeast from the *Saccharomyces* genus and lactic acid bacteria (LAB) [3, 4]. Nowadays, such microorganisms have a significant role in several industrial relevant processes, including the production of beer, wine, cheese, and bread. Importantly, the popularity of fermented beverages, namely, beer and wine, is such that their worldwide consumption is second only to nonalcoholic drinks

This chapter aims to contribute to a comprehensible analysis of the role of yeast and LAB on the production of fermented beverages from South America. The microbiological diversity associated with the fermentation of a wide diversity of raw materials, from sugarcane to

Yeast are unicellular fungi, being the simplest eukaryotes. Present in a great number of environments, yeast can be found not only in decomposing fruit, trees, and soils but also in commensal relationships with higher eukaryotes, humans included, and even saltwater. The high diversity of species, almost 1500 species have been described [6], is closely related to this wide distribution. Some of these yeast are adapted to extreme environments, such as high salt concentrations [7], low pH [8], or extremely cold temperatures [9, 10]. The genus *Saccharomy‐ ces*, particularly *Saccharomyces cerevisiae*, is strongly associated with the production of ferment‐ ed products for human consumption, namely, bread, wine, and beer [2]. After several millennia of close coexistence, through phenotypic selection, these species evolved to produce goods with organoleptic properties pleasant to humans. However, given the high degree of diversity found in nature, it is expected to find yeast with new and more interesting characteristics for

Yeast, as other heterotrophic organisms, have the anabolism coupled with catabolism. In one hand, the oxidation of organic molecules, as sugars, yields adenosine 5-triphosphate (ATP) that, in turn, is used as an energy resource for the cell. On the other hand, such organic molecules can also be used as building blocks or to generate intermediary compounds for the

The high diversity of environments where yeast can be found is closely related to the variety of carbon sources that can be used. Hexoses such as glucose, fructose, galactose, or mannose are the most common substrates, but some species can use pentoses like xylose or arabinose.

synthesis of other compounds, some of which with high commercial value.

cassava, as well as new potential biotechnological applications will be addressed.

In order to use glucose as carbon source, first and foremost, yeast have to sense the presence of this sugar in the surrounding environment and then express the adequate proteins to transport it across the plasma membrane [15, 16]. Whenever glucose is sensed in the medium, changes in the cell proteome will occur. Several processes contribute to the overall change in enzymes levels, including alteration of mRNA translation rates, mRNA stability, or protein synthesis and/or degradation. However, the major response is the extensive upregulation of a large number of genes required for the metabolism of glucose, such as genes encoding glycolytic pathway enzymes, leading to the adaptation to the fermentative metabolism. Moreover, in genes encoding for proteins involved in the metabolism of alternative substrates, gluconeogenic and respiratory pathways are repressed strongly by glucose (for reviews, see [17, 18]). In *S. cerevisiae*, a glucose concentration as little as 15 mM is enough to induce such changes [19].

*S. cerevisiae* presents an extensive family of hexose transporters, including more than 20 members: (i) 18 genes encoding transporters (*HXT*1-*HXT*17, *GAL*2) and (ii) at least two genes encoding sensors (*SNF*3, *RGT*2). Some studies suggest that Gpr1p and Hxk2p may sense glucose levels [17, 20]. The transporters can be divided in two classes regarding glucose affinity: (1) low affinity for glucose and high transport capacity, the most important proteins are Hxt1p and Hxt3p, and (2) high affinity and low transport capacity, the key proteins being Hxt2p, Hxt4p, and Hxt7p.

Following uptake by the hexose transporters, glucose enters the glycolytic pathway in order to be metabolized to pyruvate (Figure 1, steps from glucose to pyruvate), whereby the production of energy in the form of ATP is coupled to the generation of intermediates and reducing power in the form of NADH for biosynthetic pathways [21, 22]. The phosphorylation of glucose to glucose-6-phosphate, requiring ATP, is the initial step of glycolysis, by the action of the hexokinases (Hxk1/2p) and the glucokinase (Glk1p), which are linked to high-affinity glucose uptake. The glucose-6-phosphate is then isomerized to fructose-6-phosphate by the phosphoglucose isomerase, encoded by *PGI1* gene. The next step, done by the phosphofruc‐ tokinase (Pfk1/2p), also requires energy. The fructose-6-phosphate molecule is converted into fructose 1,6-biphosphate through the transfer of inorganic phosphate from ATP. In turn, yeast aldolase (fructose 1,6-bisphosphate aldolase—Fba1p) is responsible for the reversible cleavage of fructose 1,6-bisphosphate to glyceraldehyde 3-phosphate and dihydroxyacetone phosphate.

These two resulting compounds can be interconverted, in a reversible way, by the action of the triosephosphate isomerase (Tpi1p). Glyceraldehyde 3-phosphate is further metabolized to ultimately yield pyruvate, while some of the dihydroxyacetone phosphate follows gluconeo‐ genesis. This step is fundamental for the osmotic and redox homoeostasis, as the dihydrox‐ yacetone can be converted to glycerol yielding NAD+ . Glyceraldehyde 3-phosphate is first oxidized by NAD+ and then phosphorylated under the catalysis of the 3-phosphate dehydro‐ genase (Tdh1/2/3p). The resulting 1,3-diphosphoglycerate is, in turn, converted to 3-phospho‐ glycerate by the action of phosphoglycerate kinase (Pgk1p), yielding 1 molecule of ATP. The enzyme phosphoglycerate mutase (Pgm1p) promotes the relocation of the phosphate group from C3 to C2, allowing the dehydration by the enolase (Eno1/2 p), resulting in the phosphoe‐ nolpyruvate. Then the pyruvate kinase (Pyk1p) converts this highly energetic molecule to pyruvate, yielding a second molecule of ATP.

**Figure 1.** Glycolysis and alcoholic fermentation steps on *S. cerevisiae* (adapted from [23]).

The pyruvate molecule can be further processed through different metabolic alternatives, the respiratory or the fermentative pathways (Figure 2). The selection of one of the route depends greatly on the expression or repression of some genes, which in turn are tightly regulated on the environmental conditions [24]. The genus to which the yeast belongs also plays a role in the prevalence of one route over the other.

The fermentative pathway is particularly relevant to industry, as several important commod‐ ities are produced through this process (characteristic of particular organisms). In *S. cerevi‐* *siae*, the first step is the decarboxylation of pyruvate to yield acetaldehyde and carbon dioxide (CO2), through the action of the pyruvate decarboxylase (Pdc1/5/6p). The acetaldehyde can be further reduced to form ethanol by the enzyme alcohol dehydrogenase (Adh1p), allowing the reoxidation of NADH to NAD+ . Besides the direct products of fermentation, ethanol and CO2, several other by-products are generated during the process, including cell biomass, glycerol, and some organic acids. Overall, the ethanol fermentation is a redox-neutral process since the reduced coenzyme NADH produced during glycolysis, in the oxidation of glyceraldehyde 3 phosphate, is latter reoxidized in the reduction of acetaldehyde to ethanol [25]. Nevertheless, given that biomass is a product of fermentation, and it is in a more oxidized state than glucose, an excess of reducing equivalents may be generated. As mentioned above, glycerol production plays an important role in the redox balance restoration. The glycolytic intermediate dihy‐ droxyacetone is reduced to glycerol 3-phosphate, oxidizing NADH to NAD+ , in a reaction catalyzed by the NAD+ -dependent glycerol 3-phosphate dehydrogenase (Gpd1/2p). Glycerol 3-phosphate is then dephosphorylated to glycerol due to the action of glycerol 3-phosphatase (Gpp1/2p) [5, 26, 27]. The presence of glycerol may contribute to the organoleptic properties in the final product of fermentation, such as wine.

enzyme phosphoglycerate mutase (Pgm1p) promotes the relocation of the phosphate group from C3 to C2, allowing the dehydration by the enolase (Eno1/2 p), resulting in the phosphoe‐ nolpyruvate. Then the pyruvate kinase (Pyk1p) converts this highly energetic molecule to

pyruvate, yielding a second molecule of ATP.

110 Food Production and Industry

**Figure 1.** Glycolysis and alcoholic fermentation steps on *S. cerevisiae* (adapted from [23]).

the prevalence of one route over the other.

The pyruvate molecule can be further processed through different metabolic alternatives, the respiratory or the fermentative pathways (Figure 2). The selection of one of the route depends greatly on the expression or repression of some genes, which in turn are tightly regulated on the environmental conditions [24]. The genus to which the yeast belongs also plays a role in

The fermentative pathway is particularly relevant to industry, as several important commod‐ ities are produced through this process (characteristic of particular organisms). In *S. cerevi‐*

**Figure 2.** Pyruvate metabolic fates. The pyruvate yielded during glycolysis can be converted into two intermediates of TCA cycle: acetyl-CoA, by the pyruvate dehydrogenase complex (PDH), and/or oxaloacetate, by pyruvate carboxylas‐ es (Pyc1/2p). These molecules can be transported into the mitochondria by the pyruvate carriers (Mpc1p or Mpc2p) and the oxaloacetate carrier (Oac1p), respectively. Another alternative is the decarboxylation to acetaldehyde, by the pyruvate decarboxylase (Pdc1/5/6p), which ultimately can yield ethanol. Adh1p—alcohol dehydrogenase; Ald5p—ace‐ taldehyde dehydrogenase; Acs1/2p—acetyl-CoA synthases; Yat1/2p—carnitine acetyltransferases (adapted from [22]).

Although most microorganisms ferment in the absence of oxygen, this is not always the case. Even if oxygen is available, high concentrations of sugars present in the environment will lead yeast to choose fermentation over respiration. This inhibition of aerobic metabolism if glucose is available, both in the presence or absence of oxygen, is denominated the Crabtree effect [28]. *S. cerevisiae* is known as Crabtree positive since it will produce ethanol aerobically if the glucose available is higher than 15 mM [19]. The availability of high sugar concentrations in the surrounding environment stimulates glycolysis, which in turn leads to the production of increasing amounts of ATP, through substrate-level phosphorylation. At the same time, the availability of additional ATP will reduce the respiration and ATP synthesis, through oxidative phosphorylation, leading to a decrease in oxygen consumption. On the other hand, Crabtreenegative yeast do not present a glucose inhibition of aerobic respiration, so these microorgan‐ isms resort to this more efficient form of energy metabolism, producing biomass via tricarboxylic acid (TCA) cycle. Nevertheless, these species are able to ferment, but mainly in anaerobic conditions. Importantly, Crabtree is not exclusive to yeast, as it has been detected in many mammalian tumor cells [29–31].

During aerobic respiration (Figure 3), acetyl-CoA is produced by the decarboxylation of the glycolytic pyruvate, by the action of the pyruvate dehydrogenase complex. Then acetyl-CoA will enter the tricarboxylic acid (TCA) cycle, where it will be used to generate reducing equivalents, NADH and FADH2. These molecules will fuel the oxidative phosphorylation, through the highly conserved electron transport chain. Besides the production of reducing coenzymes, the TCA cycle provides intermediates to several other biochemical pathways, including the synthesis of amino acids and nucleotides (for reviews, see [22, 32]).

**Figure 3.** Aerobic respiration in *S. cerevisiae* (adapted from [33]).
