**5. Glucose repression in thermotolerant yeast** *K. marxianus*

Glucose repression is a general phenomenon in organisms including yeasts, by which glucose prevents the assimilation of other sugars [122, 123]. This process will disturb the fermentation of mixed sugars like hydrolysate of cellulosic biomass. As mentioned in the previous sections, *K. marxianus* is a well-known budding yeast, which has potential for production of bioethanol, hydrolytic enzymes, food biomass, and food additives [29, 31, 124]. *K. marxianus* DMKU 3-1042 is a thermotolerant yeast from Thailand and efficiently produces ethanol at high temperatures [31]. Although the strain can utilize various sugars including xylose [32, 35, 125], it has an intrinsic system of glucose repression like other microbes. In this section, we describe glucose repression in thermotolerant yeast, *K. marxianus*, and in conventional yeast, *S. cerevisiae*.

#### **5.1. Mechanism of glucose repression in** *S. cerevisiae*

Glucose repression in *S. cerevisiae* has been well studied. Mig1 and Hxk2 play as the main regulator of glucose repression in this species [126]. The former is a C2 H2 zinc finger protein [127], and the latter is a bi-functional protein acting as a hexokinase and transcriptional regulator, which is localized in both the cytoplasm and the nucleus [128, 129]. Hxk2 activity in glucose repression mechanism is influenced by the concentration of glucose. Under high concentrations of glucose, Hxk2 in the cytoplasm moves to the nucleus and, as a complex with dephosphorylated Mig1, Cyc8, and Tup1 [126], represses the transcription of several genes including respiratory and gluconeogenic genes. As a result of Hxk2 binding to Mig1, serine 311 in Mig1 is dephosphorylated, resulting in maintenance of repressive conditions [130]. On the other hand, in the presence of a low concentration or absence of glucose, Hxk2 and Mig1 remain in the cytoplasm, where neither Mig1 nor Hxk2 can repress Mig1-regulated genes [126]. In this situation, Hxk2 does not interact with Mig1 but still interacts with Snf1. No interaction between Hxk2 and Mig1 facilitates phosphorylation of serine 311 in Mig1 by the Snf1 kinase. Snf1 is phosphorylated by Sak1 and forms a complex with Snf4 and Gal8 to become activated. The Snf1 complex inhibits formation of a complex of Mig1-Hxk2-Cyc8-Tup1. In this situation, since Mig1 is also phosphorylated or inactive and absent in the nucleus, Mig1 regulated genes are de-repressed [130].

#### **5.2. Mechanism of glucose repression in** *K. marxianus*

**Figure 4.** Generation and utilization of NADPH in budding yeast. A major source of cellular-reduced NADPH is thought to be produced via the oxidative branch of the pentose phosphate pathway. Oxidation of isocitrate, malate, and acetaldehyde generates NADPH. NADPH is consumed during the synthesis of amino acids and lipids. The reducing power of NADPH is also used to regenerate a variety of antioxidants and antioxidant enzymes, which protect the cell from ROS and engage in deoxyribonucleotide triphosphate (dNTP) synthesis. Abbreviations: G6P, glucose-6-phosphate;

**Figure 3.** Difference of metabolism under the 30X condition from that under the 30D condition in *K. marxianus* DMKU

3-1042 (see more detail in Ref. [35]).

132 Fuel Ethanol Production from Sugarcane

6PGL, 6-phosphogluconolactone; 6PG, 6-phosphogluconate; Ru5P, ribulose-5-phosphate.

*K. marxianus* DMKU 3-1042 exhibits almost no glucose repression on sucrose assimilation unlike *S. cerevisiae* [33]. To acquire glucose repression-defective strains in *K. marxianus*, some researchers performed spontaneous isolation on 2-deoxyglucose (2-DOG) plates or random insertion of *kan*MX4 [131, 132]. According to the characteristics of sugar consumption abilities, cell growth and ethanol accumulation along with cultivation time, only one of 33 isolates of 2-DOG-resistant mutants showed enhanced utilization of xylose in the presence of glucose. Further analysis revealed that this isolate had a single nucleotide mutation to cause amino acid substitution (G270S) in *RAG5* encoding hexokinase and exhibited very low activity of the enzyme [132]. Another technique for obtaining glucose repression-defective strains showed one group of 2-DOG-resitant mutants with intragenical insertion of *Kan*MX4. This group also exhibits enhanced utilization of xylose in the presence of glucose, presumably due to a defect in the glucose-repression mechanism [131].

On the other hand, Zhou et al. focused on the function of Mig1 in *K. marxianus* and showed that the *MIG1* mutation increased hydrolysis of lactose [133] and production of inulinase [134]. Nevertheless, information on the function of Rag5 as a transcriptional regulator is hardly available, and thus construction of the complete disrupted mutation of *RAG5* and its analysis become a challenge. Thus, disrupted mutants of genes for Mig1 and Rag5 were constructed, and their characteristics were compared with those of the corresponding mutants of *S. cerevisiae*. *MIG1* and *RAG5* mutants exhibited more resistance to 2-DOG in YP plates containing sucrose. *RAG5* and *HXK2* mutants showed more resistant to 2-DOG than the corresponding *MIG1* mutants [135].

Several attractive characteristics of *MIG1* and *RAG5* mutants of *K. marxianus* DMKU 3-1042 were uncovered. *MIG1* mutants consumed almost two times faster xylose and accumulated glycerol and xylitol much more than those of the parental strain and the *RAG5* mutant in the liquid media YPX (containing 20 g/L of xylose) and YPDX (containing 20 g/L of glucose and 20 g/L of xylose) at 30°C. The accumulation of glycerol and xylitol may be due to accumulation of NADH. *RAG5* mutants exhibited very slow utilization of glucose in the liquid media of both YPD (containing 20 g/L of glucose) and YPDS (containing 20 g/L of glucose and 20 g/L of sucrose). However, with this mutant, high amounts of fructose (about 11.9 g/L in YPDS at 30°C for 96 h) were accumulated. *MIG1* and *HXK2* mutants of *S. cerevisiae* also accumulated high amounts of fructose in the same medium, but after 12 h, fructose was consumed.

**6. Thermotolerant and ethanologenic yeasts in Vietnam**

**Strains Enzyme activitiesa Gene expression levels**

**Glucohexokinase (U/mg)**

**Inulinase (U/ mg DCW)**

a

The data are from Ref. [135].

in YPD liquid medium.

**6.1. Characteristics of thermotolerant and ethanologenic yeasts**

tion from cheap and available raw materials in the region has been studied.

In Vietnam, ethanol is a compound in many different products from fermentation technology including alcoholic drinks and biofuel. In the national strategy with a vision to 2025 designed by the government, the technology of biofuel production in Vietnam using the various raw material resources that are abundantly available, e.g., pineapple, cassava, sugarcane, etc., will reach the advanced worldwide level. For the scheme on the development of Vietnam's alcoholic beverages with a vision to 2025, the Mekong Delta is one of the top national areas for the improvement of such products. In addition, nowadays due to global warming, the exploration of thermotolerant yeasts for ethanol fermentation at high temperature also falls in the potential priorities in Vietnam.

**Table 3.** Comparison of enzyme activities and gene expression levels in *MIG1-* and *RAG5-*disrupted mutants of *K. marxianus*

**Hexokinase (U/mg)**

DMKU 3-1042 127.38 1.107 0.662 0.087 0.136 0.916 *MIG1 mutant* 160.1 1.466 0.774 0.696 0.141 0.266 *RAG5 mutant* 9838.16 0.007 0.005 1.927 1.495 0.051 *RAG1 mutant* 4229.23 0.203 0.027 1.234 0.606 0.091

*INU1***/***ACT1 GLK1***/***ACT1 RAG1***/***ACT1*

http://dx.doi.org/10.5772/intechopen.79144

135

Potential of Thermotolerant Ethanologenic Yeasts Isolated from ASEAN Countries…

Recent research studies under international programs, such as the Asian Core Program (2008–2012) and the Core-to-Core Program (2014–2018), have addressed the exploration of useful thermotolerant ethanologenic yeasts isolated from Vietnam and their applications for fermentation technology at high temperature. The diversity of yeast isolates with high capacities and stability for the controlled processing of alcoholic winemaking and ethanol produc-

A total of 712 yeast isolates were purified from many different kinds of raw material sources in the Mekong Delta, Vietnam, such as ripe fruits, flowers of fruit-tree, cocoa, fermented products, alcoholic fermentation starters, sugarcane, molasses, sawdust, agricultural by-products, and soil samples. All of these yeast isolates could grow well at 37°C and about 80, 45 and 10% of these yeasts could grow at 40, 43 and 45°C, respectively. More than 80% of yeasts were able to grow in a medium containing 9% (v/v) of ethanol, this number decreased to about 40% of yeasts growing in a medium supplemented with 12% (v/v) of ethanol. For conservation, all pure yeast isolates have been stored at −20 and −80°C in stock culture of glycerol freezing broth. A bank collection of genetically diverse yeasts with thermotolerant ethanologenic capacity at high temperatures was developed and systemized. The full data of morphological,

The fructose accumulation in *RAG5* mutants is probably due to the inability of this mutant to uptake fructose or the lack of kinase activity. To further analyze this phenomenon, Enzyme activities<sup>a</sup> and gene expression levels of inulinase and kinase in *MIG1*- and *RAG5*-disrupted mutants and the parental strain were measured (**Table 3**) [135]. *RAG5* mutants showed very high activities of inulinase, about 77 times higher than those of the parental strain, but almost no activities of hexokinase and glucokinase that are encoded by *RAG5* and *GLK1*, respectively. The inulinase activity in *RAG5* mutant was consistent with the gene expression level of *INU1*, being about 22 times higher than that of the parental strain. However, the expression level of *GLK1* in this mutant was higher, which was inconsistent with glucokinase activity. It is thus likely that there is a post-transcriptional regulation for glucokinase. *MIG1* mutants showed no significant increase in inulinase activity, but *INU1* transcriptional expression was eight times higher than that of the parental strain. This inconsistence may also be due to post-transcriptional regulation for inulinase. These results suggest that Mig1 and Rag5 are related to the glucose repression mechanism in *K. marxianus* and share some functions with Mig1 and Hxk2, respectively, in *S. cerevisiae*.

In conclusion, Mig1 and Rag5 in *K. marxianus* share some functions with Mig1 and Hxk2, respectively, in *S. cerevisiae*. Mig1 and Rag5 in *K. marxianus* may form a complex similar to that consisting of Mig1 and Hxk2 in *S. cerevisiae.*


**Table 3.** Comparison of enzyme activities and gene expression levels in *MIG1-* and *RAG5-*disrupted mutants of *K. marxianus* in YPD liquid medium.
