**4. Complete genome sequence of thermotolerant yeast** *K. marxianus* **DMKU 3-1042 and transcriptomic analysis**

High-temperature fermentation technology with thermotolerant microbes has been expected to reduce the cost of bioconversion of biomass to fuels or chemicals. *K. marxianus* was included in GRAS (FDA) and QPS (EU) lists of safe microorganisms for use in foods [83, 84]. The capacity of *K. marxianus* to utilize a wide variety of sugars reflects its potential for biotechnological applications [29, 84], which has been indicated by many studies with diverse substrates such as whey permeate, crop plants, and lignocellulosic biomass [32, 33, 78, 85, 86]. *K. marxianus* is also distinguished by its thermotolerance [36, 87] and the highest growth rate in eukaryotes [88]. In recent years, interest also increased in several new applications such as production of biomolecules [89, 90], biocatalysts [91, 92], and heterologous protein expression [93, 94].

were induced by xylose. The initial xylose catabolism after its uptake in *K. marxianus* is accomplished by three reactions catalyzed by enzymes, xylose reductase (*XYL1*), xylitol dehydrogenase (*XYL2*), and xylulokinase (*XKS1*), which are involved in the conversion of xylose to xylulose-5-phosphate as an intermediate in the pentose phosphate pathway (PPP). Genes for utilization of various other sugars and alcohol dehydrogenases were also found

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*K. marxianus* showed a high level of phenotypic variation. Recently, the single nucleotide polymorphisms (SNIPs) in 14 strains of *K. marxianus* were analyzed [100]. On the basis of SNIP analysis and flow cytometry, it was found that the isolates included haploid, diploid, and triploid strains. All isolates from dairy environments were diploid or triploid, whereas

A major potential future application of *K. marxianus* may be ethanol production from lignocellulosic biomass, which is an anaerobic or oxygen-limited process where both glucose and xylose may be present. Detailed transcription start site sequencing (TSS Seq) to explore the response of *K. marxianus* DMKU 3-1042 was reported for four different conditions: shaking condition in rich medium at 30°C (30D) or 45°C (45D), static condition in rich medium at 30°C (30DS), and shaking condition in xylose-containing rich medium at 30°C

Under the 30DS condition, there were 159 and 154 significantly upregulated and downregulated genes, respectively. In brief, *K. marxianus* may increase the turnover of RNAs and proteins in addition to suppression of transporters that depend on mitochondrial respiratory activity. Most genes for several oxygen-dependent biosynthetic pathways (**Figure 1**), such as those for heme, sterols, unsaturated fatty acids, pyrimidine, and deoxyribonucleotides [114],

Under the 45D condition, there were 199 and 508 significantly upregulated and downregulated genes, respectively. *K. marxianus* seems to drastically change metabolic pathways under the 45D condition, that is, the enhancement of PPP and the attenuation of TCA cycle after the fumarate-producing step (**Figure 2**). Several genes for homologous recombination and nonhomologous end joining, which function in the repair of DNA-double stranded breaks, were also upregulated. As expected, heat shock proteins and chaperones, such as Hsp26, Hsp60, Hsp78, Hsp82, Ssa3, and Cpr6, are crucial for survival at high temperatures. The thermotolerance of *K. marxianus* is likely achieved by systematic mechanisms consisting of various strategies. The yeast prevents reactive oxygen species (ROS) generation by minimizing mitochondrial activity and mainly acquires ATP from glycolysis rather than from TCA cycle at

most isolates (6 out 7 isolates) from nondairy environments were haploid.

are crucial for the cellular metabolism under the static condition.

[35, 112, 113].

(30X) [35].

high temperatures.

**4.2. Ploidy variation in** *K. marxianus*

**4.3. Transcriptomic analysis**

Genomic and transcriptomic studies have started to shed light on *K. marxianus*, and a growing number of genome sequences of *K. marxianus* strains are now available. Those include KCTC 17555 [34], DMB1 [95], CCT 7735 [96], NBRC1777 [97], DMKU 3-1042 [35], B0399 [98], UFS-Y2791 [99], and other nine strains: L01, L02, L03, L04, L05, CBS397, NBRC0272, NBRC0288, and NBRC0617 [100].

#### **4.1. Genomic information and comparative genomics**

The genome sequence of *K. marxianus* DMKU 3-1042 as one of the most efficient thermotolerant strains was determined, and the complete genome sequence of 11.0 Mb including all centromeric regions and boundary regions containing up to one to several sequence repeats (GGTGTACGGATTTGATTAGTTATGT) of telomeres was obtained [35]. The genome was composed of eight chromosomes in total, including mitochondrial DNA. Annotation of the genome of DMKU 3-1042 revealed a total of 4952 genes. UniProt and KAAS assignments led to the assignment of homologous genes of about 86.4% of predicted genes and KEGG Orthology numbers of 50.5% respectively.

A total of 202 tRNAs and 8 rDNAs were identified. According to the optical mapping experiment, 140 rDNA copies were observed on chromosome 5 instead of 6 rDNA copies found in the genome sequence in the database. The rDNA copy number and the thermotolerance were expected to positively relate. However, there was no such correlation among 10 *K. marxianus* strains, which exhibited different growth at different temperatures, and at least 31 copies of rDNA are sufficient to support its thermotolerance [35].

The yeast shares 1552 genes with other hemiascomycetous yeasts, including *K. lactis, Ashbya gossypii, Candida glabrata, S. cerevisiae, Ogataea parapolymorpha, Debaryomyces hansenii, S. stipitis, Clavispora lusitaniae, Yarrowia lipolytica,* and *Schizosaccharomyces pombe* [101–105]. *K. marxianus* was found to be phylogenetically closest to *K. lactis*. There are 193 genes specific to *K. marxianus*, which may be responsible for its species-specific characteristics [35]. The 422 genes shared between *K. marxianus* and *K. lactis* may be related to their genus-specific characteristics, such as production of β-galactosidase [106], assimilation of a wide variety of inexpensive substrates [84], efficient productivity of heterologous proteins [107–109], and synthesis of a killer toxin against certain ascomycetous yeasts [110, 111].

The two attractive traits of *K. marxianus* for fermentation applications were the thermotolerance and pentose assimilation capability. The thermotolerant ability was also found in *O. parapolymorpha*, and 30 genes were found to be shared between the two thermotolerant yeasts, including genes for three siderophore-iron transporters and three vacuolar proteins. For pentose assimilation capability, there are 27 putative genes for sugar transporters in the *K. marxianus* genome, and some of them (*KLMA\_60073, KLMA\_70145 and KLMA\_80101*) were induced by xylose. The initial xylose catabolism after its uptake in *K. marxianus* is accomplished by three reactions catalyzed by enzymes, xylose reductase (*XYL1*), xylitol dehydrogenase (*XYL2*), and xylulokinase (*XKS1*), which are involved in the conversion of xylose to xylulose-5-phosphate as an intermediate in the pentose phosphate pathway (PPP). Genes for utilization of various other sugars and alcohol dehydrogenases were also found [35, 112, 113].

#### **4.2. Ploidy variation in** *K. marxianus*

in eukaryotes [88]. In recent years, interest also increased in several new applications such as production of biomolecules [89, 90], biocatalysts [91, 92], and heterologous protein expression

Genomic and transcriptomic studies have started to shed light on *K. marxianus*, and a growing number of genome sequences of *K. marxianus* strains are now available. Those include KCTC 17555 [34], DMB1 [95], CCT 7735 [96], NBRC1777 [97], DMKU 3-1042 [35], B0399 [98], UFS-Y2791 [99], and other nine strains: L01, L02, L03, L04, L05, CBS397, NBRC0272, NBRC0288,

The genome sequence of *K. marxianus* DMKU 3-1042 as one of the most efficient thermotolerant strains was determined, and the complete genome sequence of 11.0 Mb including all centromeric regions and boundary regions containing up to one to several sequence repeats (GGTGTACGGATTTGATTAGTTATGT) of telomeres was obtained [35]. The genome was composed of eight chromosomes in total, including mitochondrial DNA. Annotation of the genome of DMKU 3-1042 revealed a total of 4952 genes. UniProt and KAAS assignments led to the assignment of homologous genes of about 86.4% of predicted genes and KEGG

A total of 202 tRNAs and 8 rDNAs were identified. According to the optical mapping experiment, 140 rDNA copies were observed on chromosome 5 instead of 6 rDNA copies found in the genome sequence in the database. The rDNA copy number and the thermotolerance were expected to positively relate. However, there was no such correlation among 10 *K. marxianus* strains, which exhibited different growth at different temperatures, and at least 31 copies of

The yeast shares 1552 genes with other hemiascomycetous yeasts, including *K. lactis, Ashbya gossypii, Candida glabrata, S. cerevisiae, Ogataea parapolymorpha, Debaryomyces hansenii, S. stipitis, Clavispora lusitaniae, Yarrowia lipolytica,* and *Schizosaccharomyces pombe* [101–105]. *K. marxianus* was found to be phylogenetically closest to *K. lactis*. There are 193 genes specific to *K. marxianus*, which may be responsible for its species-specific characteristics [35]. The 422 genes shared between *K. marxianus* and *K. lactis* may be related to their genus-specific characteristics, such as production of β-galactosidase [106], assimilation of a wide variety of inexpensive substrates [84], efficient productivity of heterologous proteins [107–109], and synthesis of a killer toxin

The two attractive traits of *K. marxianus* for fermentation applications were the thermotolerance and pentose assimilation capability. The thermotolerant ability was also found in *O. parapolymorpha*, and 30 genes were found to be shared between the two thermotolerant yeasts, including genes for three siderophore-iron transporters and three vacuolar proteins. For pentose assimilation capability, there are 27 putative genes for sugar transporters in the *K. marxianus* genome, and some of them (*KLMA\_60073, KLMA\_70145 and KLMA\_80101*)

[93, 94].

and NBRC0617 [100].

128 Fuel Ethanol Production from Sugarcane

**4.1. Genomic information and comparative genomics**

rDNA are sufficient to support its thermotolerance [35].

against certain ascomycetous yeasts [110, 111].

Orthology numbers of 50.5% respectively.

*K. marxianus* showed a high level of phenotypic variation. Recently, the single nucleotide polymorphisms (SNIPs) in 14 strains of *K. marxianus* were analyzed [100]. On the basis of SNIP analysis and flow cytometry, it was found that the isolates included haploid, diploid, and triploid strains. All isolates from dairy environments were diploid or triploid, whereas most isolates (6 out 7 isolates) from nondairy environments were haploid.

#### **4.3. Transcriptomic analysis**

A major potential future application of *K. marxianus* may be ethanol production from lignocellulosic biomass, which is an anaerobic or oxygen-limited process where both glucose and xylose may be present. Detailed transcription start site sequencing (TSS Seq) to explore the response of *K. marxianus* DMKU 3-1042 was reported for four different conditions: shaking condition in rich medium at 30°C (30D) or 45°C (45D), static condition in rich medium at 30°C (30DS), and shaking condition in xylose-containing rich medium at 30°C (30X) [35].

Under the 30DS condition, there were 159 and 154 significantly upregulated and downregulated genes, respectively. In brief, *K. marxianus* may increase the turnover of RNAs and proteins in addition to suppression of transporters that depend on mitochondrial respiratory activity. Most genes for several oxygen-dependent biosynthetic pathways (**Figure 1**), such as those for heme, sterols, unsaturated fatty acids, pyrimidine, and deoxyribonucleotides [114], are crucial for the cellular metabolism under the static condition.

Under the 45D condition, there were 199 and 508 significantly upregulated and downregulated genes, respectively. *K. marxianus* seems to drastically change metabolic pathways under the 45D condition, that is, the enhancement of PPP and the attenuation of TCA cycle after the fumarate-producing step (**Figure 2**). Several genes for homologous recombination and nonhomologous end joining, which function in the repair of DNA-double stranded breaks, were also upregulated. As expected, heat shock proteins and chaperones, such as Hsp26, Hsp60, Hsp78, Hsp82, Ssa3, and Cpr6, are crucial for survival at high temperatures. The thermotolerance of *K. marxianus* is likely achieved by systematic mechanisms consisting of various strategies. The yeast prevents reactive oxygen species (ROS) generation by minimizing mitochondrial activity and mainly acquires ATP from glycolysis rather than from TCA cycle at high temperatures.

**Figure 1.** Oxygen-related metabolism in budding yeast. Oxygen is used for the biosynthesis of unsaturated fatty acids, ergosterol, heme, pyrimidine, and deoxyribonucleotides, as well as during disulfide bond formation and fatty acid oxidation. Oxygen is also the final electron acceptor for the electron transport chain, which oxidizes reduced equivalents of nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2 ) for the synthesis of ATP. However, ROS are produced as a by-product during some of these processes. The ROS can cause damage to DNA, proteins, and lipids.

TSS seq analysis revealed that the oxidative stress-response genes were highly induced under the three conditions tested, indicating that ROS is accumulated in the cytoplasm, mitochondria, and peroxisome under the 30DS and 30X conditions and in the cytoplasm and mitochon-

**Figure 2.** Difference of metabolism under the 45D condition from that under the 30D condition in *K. marxianus* DMKU

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Moreover, *K. marxianus* has been exploited as a cell factory to produce valuable enzymes, showing retention of the activity in a broad temperature range [120]. The 30X condition showed high expression of *INU1* for inulinase, which is useful for the production of recombinant proteins [108, 109, 121]. These useful characteristics may allow simultaneous production of ethanol and valuable proteins, thus generating additional revenue from ethanol production. In conclusion, the transcriptome analyses clarified distinctive metabolic pathways under three different growth conditions, static culture, high temperature, and xylose medium, in comparison to the control condition of a glucose medium under a shaking condition at 30°C. Interestingly, the yeast appears to overcome the issue of ROS, which tend to accumulate under all three conditions. Nicotinamide adenine dinucleotide phosphate (NADPH) synthesis from several

dria under the 45D condition.

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

reactions is the key for cells to cope with ROS (**Figure 4**).

Under the 30X condition, there were 89 and 79 significantly upregulated and downregulated genes, respectively. This condition may stimulate the degradation of lipids in the peroxisome and keep a low level of amino acid synthesis, indicating the possibility that fatty acids could be a subsidiary intracellular carbon source in xylose medium (**Figure 3**). Similarly, Schabort et al. [99] also reported that peroxisomal fatty acid catabolism was dramatically upregulated in a defined xylose mineral medium without fatty acids, along with mechanisms to activate fatty acids and transfer products of β-oxidation to the mitochondria. It is known that *K. marxianus* tends to suffer from cofactor imbalance in xylose medium [115, 116]. Redox balancing mechanisms between the cytoplasm and mitochondria are probably used to resolve the NADH/NADPH imbalance owing to lack of transhydrogenases [117]. In *S. cerevisiae*, five cytosolic-mitochondrial redox shuttles have been proposed [118]. Of these, genes for enzymes related to ethanol-acetaldehyde, citrate-oxoglutarate, and oxaloacetate-malate shuttles were relatively upregulated under the 30X condition, which were different from those found in *S. cerevisiae* and *S. stipitis* [103, 119].

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**Figure 2.** Difference of metabolism under the 45D condition from that under the 30D condition in *K. marxianus* DMKU 3-1042 (see more detail in Ref. [35]).

TSS seq analysis revealed that the oxidative stress-response genes were highly induced under the three conditions tested, indicating that ROS is accumulated in the cytoplasm, mitochondria, and peroxisome under the 30DS and 30X conditions and in the cytoplasm and mitochondria under the 45D condition.

Under the 30X condition, there were 89 and 79 significantly upregulated and downregulated genes, respectively. This condition may stimulate the degradation of lipids in the peroxisome and keep a low level of amino acid synthesis, indicating the possibility that fatty acids could be a subsidiary intracellular carbon source in xylose medium (**Figure 3**). Similarly, Schabort et al. [99] also reported that peroxisomal fatty acid catabolism was dramatically upregulated in a defined xylose mineral medium without fatty acids, along with mechanisms to activate fatty acids and transfer products of β-oxidation to the mitochondria. It is known that *K. marxianus* tends to suffer from cofactor imbalance in xylose medium [115, 116]. Redox balancing mechanisms between the cytoplasm and mitochondria are probably used to resolve the NADH/NADPH imbalance owing to lack of transhydrogenases [117]. In *S. cerevisiae*, five cytosolic-mitochondrial redox shuttles have been proposed [118]. Of these, genes for enzymes related to ethanol-acetaldehyde, citrate-oxoglutarate, and oxaloacetate-malate shuttles were relatively upregulated under the 30X condition, which were different from those found in *S. cerevisiae* and *S. stipitis*

**Figure 1.** Oxygen-related metabolism in budding yeast. Oxygen is used for the biosynthesis of unsaturated fatty acids, ergosterol, heme, pyrimidine, and deoxyribonucleotides, as well as during disulfide bond formation and fatty acid oxidation. Oxygen is also the final electron acceptor for the electron transport chain, which oxidizes reduced

ATP. However, ROS are produced as a by-product during some of these processes. The ROS can cause damage to DNA,

) for the synthesis of

equivalents of nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2

[103, 119].

proteins, and lipids.

130 Fuel Ethanol Production from Sugarcane

Moreover, *K. marxianus* has been exploited as a cell factory to produce valuable enzymes, showing retention of the activity in a broad temperature range [120]. The 30X condition showed high expression of *INU1* for inulinase, which is useful for the production of recombinant proteins [108, 109, 121]. These useful characteristics may allow simultaneous production of ethanol and valuable proteins, thus generating additional revenue from ethanol production.

In conclusion, the transcriptome analyses clarified distinctive metabolic pathways under three different growth conditions, static culture, high temperature, and xylose medium, in comparison to the control condition of a glucose medium under a shaking condition at 30°C. Interestingly, the yeast appears to overcome the issue of ROS, which tend to accumulate under all three conditions. Nicotinamide adenine dinucleotide phosphate (NADPH) synthesis from several reactions is the key for cells to cope with ROS (**Figure 4**).

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

regulator of glucose repression in this species [126]. The former is a C2

tional yeast, *S. cerevisiae*.

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

regulated genes are de-repressed [130].

**5.2. Mechanism of glucose repression in** *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 conven-

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Glucose repression in *S. cerevisiae* has been well studied. Mig1 and Hxk2 play as the main

tein [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-

*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

H2

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zinc finger pro-

**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]).

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