Riboflavin overproduction on lignocellulose hydrolysate by the engineered yeast Candida famata.

Lignocellulose (dry plant biomass) is an abundant cheap inedible residue of agriculture and wood industry with great potential as a feedstock for biotechnological processes. Lignocellulosic substrates can serve as valuable resources in fermentation processes, allowing the production of a wide array of chemicals, fuels, and food additives. The main obstacle for cost-effective conversion of lignocellulosic hydrolysates to target products is poor metabolism of the major pentoses, xylose and L-arabinose, which are the second and third most abundant sugars of lignocellulose after glucose. We study the oversynthesis of riboflavin in the flavinogenic yeast Candida famata and found that all major lignocellulosic sugars, including xylose and L-arabinose, support robust growth and riboflavin synthesis in the available strains of C. famata. To further increase riboflavin production from xylose and lignocellulose hydrolysate, genes XYL1 and XYL2 coding for xylose reductase and xylitol dehydrogenase were overexpressed. The resulting strains exhibited increased riboflavin production in both shake flasks and bioreactors using diluted hydrolysate, reaching 1.5 g L-1.

Efficient production of bacterial antibiotics aminoriboflavin and roseoflavin in eukaryotic microorganisms, yeasts.

BACKGROUND: Actinomycetes Streptomyces davaonensis and Streptomyces cinnabarinus synthesize a promising broad-spectrum antibiotic roseoflavin, with its synthesis starting from flavin mononucleotide and proceeding through an immediate precursor, aminoriboflavin, that also has antibiotic properties. Roseoflavin accumulation by the natural producers is rather low, whereas aminoriboflavin accumulation is negligible. Yeasts have many advantages as biotechnological producers relative to bacteria, however, no recombinant producers of bacterial antibiotics in yeasts are known. RESULTS: Roseoflavin biosynthesis genes have been expressed in riboflavin- or FMN-overproducing yeast strains of Candida famata and Komagataella phaffii. Both these strains accumulated aminoriboflavin, whereas only the latter produced roseoflavin. Aminoriboflavin isolated from the culture liquid of C. famata strain inhibited the growth of Staphylococcus aureus (including MRSA) and Listeria monocytogenes. Maximal accumulation of aminoriboflavin in shake-flasks reached 1.5 mg L- 1 (C. famata), and that of roseoflavin was 5 mg L- 1 (K. phaffii). Accumulation of aminoriboflavin and roseoflavin by K. phaffii recombinant strain in a bioreactor reached 22 and 130 mg L- 1, respectively. For comparison, recombinant strains of the native bacterial producer S. davaonensis accumulated near one-order less of roseoflavin while no recombinant producers of aminoriboflavin was reported at all. CONCLUSIONS: Yeast recombinant producers of bacterial antibiotics aminoriboflavin and roseoflavin were constructed and evaluated.

Metabolic engineering of non-conventional yeasts for construction of the advanced producers of biofuels and high-value chemicals.

Non-conventional yeasts, i.e. yeasts different from Saccharomyces cerevisiae, represent heterogenous group of unicellular fungi consisting of near 1500 species. Some of these species have interesting and sometimes unique properties like ability to grow on methanol, n-alkanes, ferment pentose sugars xylose and l-arabinose, grow at high temperatures (50°Ð¡ and more), overproduce riboflavin (vitamin B2) and others. These unique properties are important for development of basic science; moreover, some of them possess also significant applied interest for elaboration of new biotechnologies. Current paper represents review of the recent own results and of those of other authors in the field of non-conventional yeast study for construction of the advanced producers of biofuels (ethanol, isobutanol) from lignocellulosic sugars glucose and xylose or crude glycerol (Ogataea polymorpha, Magnusiomyces magnusii) and vitamin B2 (riboflavin) from glucose and cheese whey (Candida famata).

The Komagatella phaffii ACG1 gene, encoding ß-1,6-N-acetylglucosaminyltransferase, is involved in the autophagy of cytosolic and peroxisomal proteins.

The methylotrophic yeast Komagataella phaffii is considered one of the most effective producers of recombinant proteins of industrial importance. Effective producers should be characterized by the maximal reduction of degradation of the cytosolic recombinant proteins. The mechanisms of degradation of cytosolic proteins in K. phaffii have not been elucidated; however, data suggest that they are partially degraded in the autophagic pathway. To identify factors that influence this process, a developed system for the selection of recombinant strains of K. phaffii with impaired autophagic degradation of the heterologous model cytosolic protein (yeast ß-galactosidase) was used for insertional tagging of the genes involved in cytosolic proteins degradation. In one of the obtained strains, the insertion cassette disrupted the open reading frame of the gene encoding ß-1,6-N-acetylglucosaminyltransferase. A recombinant strain with deletion of this gene was also obtained. The rate of degradation of the ß-galactosidase enzyme was two times slower in the insertion mutant and 1.5 times slower in the deletion strain as compared to the parental strain with native ß-1,6-N-acetylglucosaminyltransferase. The rate of degradation of native K. phaffii cytosolic and peroxisomal enzymes, formaldehyde dehydrogenase, formate dehydrogenase, and alcohol oxidase, respectively, showed similar trends to that of ß-galactosidase-slower degradation in the deletion and insertional mutants as compared to the wild-type strain, but faster protein degradation relative to the strain completely defective in autophagy. We conclude that K. phaffii gene designated ACG1, encoding ß-1,6-N-acetylglucosaminyltransferase, is involved in autophagy of the cytosolic and peroxisomal proteins.

Construction of the advanced flavin mononucleotide producers in the flavinogenic yeast Candida famata.

Flavin mononucleotide (FMN, riboflavin-5'-phosphate) is flavin coenzyme synthesized in all living organisms from riboflavin (vitamin B2 ) after phosphorylation in the reaction catalyzed by riboflavin kinase. FMN has several applications mostly as yellow colorant in food industry due to 200 times better water solubility as compared to riboflavin. Currently, FMN is produced by chemical phosphorylation of riboflavin, however, final product contains up to 25% of flavin impurities. Microbial overproducers of FMN are known, however, they accumulate this coenzyme in glucose medium. Current work shows that the recombinant strains of the flavinogenic yeast Candida famata with overexpressed FMN1 gene coding for riboflavin kinase in the recently isolated by us advanced riboflavin producers due to overexpression of the structural and regulatory genes of riboflavin synthesis and of the putative exporter of riboflavin from the cell, synthesized elevated amounts of FMN in the media not only with glucose but also in lactose and cheese whey. Activation of FMN accumulation on lactose and cheese whey was especially strong in the strains which expressed the gene of transcription activator SEF1 under control of the lactose-induced LAC4 promoter. The accumulation of this coenzyme by the washed cells of the best recombinant strain achieved 540 mg/L in the cheese whey supplemented only with ammonium sulfate during 48 h in shake flask experiments.

Cheese whey supports high riboflavin synthesis by the engineered strains of the flavinogenic yeast Candida famata.

BACKGROUND: Riboflavin is a precursor of FMN and FAD which act as coenzymes of numerous enzymes. Riboflavin is an important biotechnological commodity with annual market sales exceeding nine billion US dollars. It is used primarily as a component of feed premixes, a food colorant, a component of multivitamin mixtures and medicines. Currently, industrial riboflavin production uses the bacterium, Bacillus subtilis, and the filamentous fungus, Ashbya gossypii, and utilizes glucose and/or oils as carbon substrates. RESULTS: We studied riboflavin biosynthesis in the flavinogenic yeast Candida famata that is a genetically stable riboflavin overproducer. Here it was found that the wild type C. famata is characterized by robust growth on lactose and cheese whey and the engineered strains also overproduce riboflavin on whey. The riboflavin synthesis on whey was close to that obtained on glucose. To further enhance riboflavin production on whey, the gene of the transcription activator SEF1 was expressed under control of the lactose-induced promoter of the native ß-galactosidase gene LAC4. These transformants produced elevated amounts of riboflavin on lactose and especially on whey. The strain with additional overexpression of gene RIB6 involved in conversion of ribulose-5-phosphate to riboflavin precursor had the highest titer of accumulated riboflavin in flasks during cultivation on whey. Activation of riboflavin synthesis was also obtained after overexpression of the GND1 gene that is involved in the synthesis of the riboflavin precursor ribulose-5-phosphate. The best engineered strains accumulated 2.5 g of riboflavin/L on whey supplemented only with (NH4)2SO4 during batch cultivation in bioreactor with high yield (more than 300 mg/g dry cell weight). The use of concentrated whey inhibited growth of wild-type and engineered strains of C. famata, so the mutants tolerant to concentrated whey were isolated. CONCLUSIONS: Our data show that the waste of dairy industry is a promising substrate for riboflavin production by C. famata. Possibilities for using the engineered strains of C. famata to produce high-value commodity (riboflavin) from whey are discussed.

The role of hexose transporter-like sensor hxs1 and transcription activator involved in carbohydrate sensing azf1 in xylose and glucose fermentation in the thermotolerant yeast Ogataea polymorpha.

BACKGROUND: Fuel ethanol from lignocellulose could be important source of renewable energy. However, to make the process feasible, more efficient microbial fermentation of pentose sugars, mainly xylose, should be achieved. The native xylose-fermenting thermotolerant yeast Ogataea polymorpha is a promising organism for further development. Efficacy of xylose alcoholic fermentation by O. polymorpha was significantly improved by metabolic engineering. Still, genes involved in regulation of xylose fermentation are insufficiently studied. RESULTS: We isolated an insertional mutant of O. polymorpha with impaired ethanol production from xylose. The insertion occurred in the gene HXS1 that encodes hexose transporter-like sensor, a close homolog of Saccharomyces cerevisiae sensors Snf3 and Rgt2. The role of this gene in xylose utilization and fermentation was not previously elucidated. We additionally analyzed O. polymorpha strains with the deletion and overexpression of the corresponding gene. Strains with deletion of the HXS1 gene had slower rate of glucose and xylose consumption and produced 4 times less ethanol than the wild-type strain, whereas overexpression of HXS1 led to 10% increase of ethanol production from glucose and more than 2 times increase of ethanol production from xylose. We also constructed strains of O. polymorpha with overexpression of the gene AZF1 homologous to S. cerevisiae AZF1 gene which encodes transcription activator involved in carbohydrate sensing. Such transformants produced 10% more ethanol in glucose medium and 2.4 times more ethanol in xylose medium. Besides, we deleted the AZF1 gene in O. polymorpha. Ethanol accumulation in xylose and glucose media in such deletion strains dropped 1.5 and 1.8 times respectively. Overexpression of the HXS1 and AZF1 genes was also obtained in the advanced ethanol producer from xylose. The corresponding strains were characterized by 20-40% elevated ethanol accumulation in xylose medium. To understand underlying mechanisms of the observed phenotypes, specific enzymatic activities were evaluated in the isolated recombinant strains. CONCLUSIONS: This paper shows the important role of hexose sensor Hxs1 and transcription factor Azf1 in xylose and glucose alcoholic fermentation in the native xylose-fermenting yeast O. polymorpha and suggests potential importance of the corresponding genes for construction of the advanced ethanol producers from the major sugars of lignocellulose.

Recent Advances in Construction of the Efficient Producers of Riboflavin and Flavin Nucleotides (FMN, FAD) in the Yeast Candida famata.

The approaches used by the authors to design the Candida famata strains capable to overproduce riboflavin, flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD) are described. The metabolic engineering approaches include overexpression of SEF1 gene encoding positive regulator of riboflavin biosynthesis, IMH3 (coding for IMP dehydrogenase) orthologs from another species of flavinogenic yeast Debaryomyces hansenii, and the homologous genes RIB1 and RIB7 encoding GTP cyclohydrolase II and riboflavin synthase, the first and the last enzymes of riboflavin biosynthesis pathway, respectively. Overexpression of the above mentioned genes in the genetically stable riboflavin overproducer AF-4 obtained by classical selection resulted in fourfold increase of riboflavin production in shake flask experiments.Overexpression of engineered enzymes phosphoribosyl pyrophosphate synthetase and phosphoribosyl pyrophosphate amidotransferase catalyzing the initial steps of purine nucleotide biosynthesis enhances riboflavin synthesis in the flavinogenic yeast C. famata even more.Recombinant strains of C. famata containing FMN1 gene from D. hansenii encoding riboflavin kinase under control of the strong constitutive TEF1 promoter were constructed. Overexpression of the FMN1 gene in the riboflavin-producing mutant led to the 30-fold increase of the riboflavin kinase activity and 400-fold increase of FMN production in the resulting recombinant strains which reached maximally 318.2 mg/L.FAD overproducing strains of C. famata were also constructed. This was achieved by overexpression of FAD1 gene from D. hansenii in C. famata FMN overproducing strain. The 7- to 15-fold increase in FAD synthetase activity as compared to the wild-type strain and FAD accumulation into cultural medium were observed. The maximal FAD titer 451.5 mg/L was achieved.

Overexpression of Riboflavin Excretase Enhances Riboflavin Production in the Yeast Candida famata.

Many microorganisms are capable of riboflavin oversynthesis and accumulation in a medium, suggesting that they efficiently excrete riboflavin. The mechanisms of riboflavin efflux in microorganisms remain elusive. Candida famata are representatives of a group of so-called flavinogenic yeast species that overproduce riboflavin (vitamin B2) in response to iron limitation. The riboflavin overproducers of this yeast species have been obtained by classical mutagenesis and metabolic engineering. Overproduced riboflavin accumulates in the cultural medium rather than in the cells suggesting existence of the special mechanisms involved in riboflavin excretion. The appropriate protein and gene have not been identified in yeasts till recently. At the same time, the gene BCRP (breast cancer resistance protein) has been identified in mammal mammary glands. Several homologs of the mammal BCRP gene encoding putative riboflavin efflux protein (excretase) were identified in the flavinogenic yeasts Debaryomyces hansenii and C. famata. Here we evaluate the yeast homologs of BCRP with respect to improvement of a riboflavin production by C. famata. The closest homologs from D. hansenii or C. famata were expressed under the control of TEF1 promoter of these yeasts in the wild-type and riboflavin-overproducing strains of C. famata. Resulted transformants overexpressed the corresponding genes (designated as DhRFE and CfRFE) and produced 1.4- to 6-fold more riboflavin as compared to the corresponding parental strains. They also were characterized by overexpression of RIB1 and RIB6 genes which encode the first and the last structural enzymes of riboflavin synthesis and exhibited elevated specific activity of GTP cyclohydrolase II. Thus, overexpression of yeast homolog of mammal gene BCRP may be useful to increase the riboflavin yield in a riboflavin production process using a recombinant overproducing C. famata strain or other flavinogenic microorganisms.

Flavocytochrome b2 of the Methylotrophic Yeast Ogataea polymorpha: Construction of Overproducers, Purification, and Bioanalytical Application.

Flavocytochrome b2 (EC 1.1.2.3; L-lactate cytochrome: c oxidoreductase, FC b2) from the thermotolerant methylotrophic yeast Ogataea polymorpha is a thermostable enzyme-prospective for a highly selective L-lactate analysis in the medicine, nutrition sector, and quality control of commercial products. Here we describe the construction of FC b2 producers by overexpression of the CYB2 gene O. polymorpha, encoding FC b2, under the control of a strong alcohol oxidase promoter in the frame of plasmid for multicopy integration with the next transformation of recipient strain O. polymorpha C-105 (gcr1 catX) impaired in the glucose repression and devoid of catalase activity. The selected recombinant strain O. polymorpha "tr1" (gcr1 catX CYB2), characterized by eightfold increased FC b2 activity compared to the initial strain, was used as a source of the enzyme. For purification of FC b2 a new method of affinity chromatography was developed and purified preparations of the enzyme were used for the construction of the highly selective enzymatic kits and amperometric biosensor for L-lactate analysis in human liquids and foods.

Pentose metabolism and conversion to biofuels and high-value chemicals in yeasts.

Pentose sugars are widespread in nature and two of them, D-xylose and L-arabinose belong to the most abundant sugars being the second and third by abundance sugars in dry plant biomass (lignocellulose) and in general on planet. Therefore, it is not surprising that metabolism and bioconversion of these pentoses attract much attention. Several different pathways of D-xylose and L-arabinose catabolism in bacteria and yeasts are known. There are even more common and really ubiquitous though not so abundant pentoses, D-ribose and 2-deoxy-D-ribose, the constituents of all living cells. Thus, ribose metabolism is example of endogenous metabolism whereas metabolism of other pentoses, including xylose and L-arabinose, represents examples of the metabolism of foreign exogenous compounds which normally are not constituents of yeast cells. As a rule, pentose degradation by the wild-type strains of microorganisms does not lead to accumulation of high amounts of valuable substances; however, productive strains have been obtained by random selection and metabolic engineering. There are numerous reviews on xylose and (less) L-arabinose metabolism and conversion to high value substances; however, they mostly are devoted to bacteria or the yeast Saccharomyces cerevisiae. This review is devoted to reviewing pentose metabolism and bioconversion mostly in non-conventional yeasts, which naturally metabolize xylose. Pentose metabolism in the recombinant strains of S. cerevisiae is also considered for comparison. The available data on ribose, xylose, L-arabinose transport, metabolism, regulation of these processes, interaction with glucose catabolism and construction of the productive strains of high-value chemicals or pentose (ribose) itself are described. In addition, genome studies of the natural xylose metabolizing yeasts and available tools for their molecular research are reviewed. Metabolism of other pentoses (2-deoxyribose, D-arabinose, lyxose) is briefly reviewed.

100 Years Later, What Is New in Glycerol Bioproduction?

Industrial production of glycerol by yeast, which began during WWI in the so-called Neuberg fermentation, was the first example of metabolic engineering. However, this process, based on bisulfite addition to fermentation liquid, has many drawbacks and was replaced by other methods of glycerol production. Osmotolerant yeasts and other microorganisms that do not require addition of bisulfite to steer cellular metabolism towards glycerol synthesis have been discovered or engineered. Because the glycerol market is expected to reach 5 billion US$ by 2024, microbial fermentation may again become a promising way to produce glycerol. This review summarizes some problems and perspectives on the production of glycerol by natural or engineered eukaryotic and prokaryotic microorganisms.

Expression of yeast homolog of the mammal BCRP gene coding for riboflavin efflux protein activates vitamin B2 production in the flavinogenic yeast Candida famata.

Candida famata is a representative of a group of so-called flavinogenic yeast species that overproduce riboflavin (vitamin B2 ) in response to iron limitation. Overproduced riboflavin accumulates in the cultural medium rather than in the cells suggesting existence of the special mechanisms involved in riboflavin excretion. The corresponding protein and gene have not been identified in yeasts. At the same time, the corresponding gene BCRP has been identified in mammal mammary glands. Several homologs of the mammal BCRP gene encoding putative riboflavin efflux protein (excretase) were identified in Debaryomyces hansenii. The closest homolog was expressed under the control of D. hansenii TEF1 promoter in the riboflavin overproducing strain of C. famata. Resulted transformants overexpressed the corresponding gene and produced 1.4- to 1.8-fold more riboflavin as compared with the parental strain. They also were characterized by overexpression of RIB1 and RIB6 genes of riboflavin synthesis and exhibited elevated specific activity of GTP-cyclohydrolase II. Membrane localization of the riboflavin excretase was confirmed by fluorescent microscopy.

Modulation of the Purine Pathway for Riboflavin Production in Flavinogenic Recombinant Strain of the Yeast Candida famata.

Riboflavin (vitamin B2 ) is an indispensable nutrient for humans and animals, since it is the precursor of the essential coenzymes flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), involved in variety of metabolic reactions. Riboflavin is produced on commercial scale and is used for feed and food fortification purposes, and in medicine. Until recently, the mutant strains of the flavinogenic yeast Candida famata were used in industry for riboflavin production. Guanosine triphosphate is the immediate precursor of riboflavin synthesis. Therefore, the activation of metabolic flux toward purine nucleotide biosynthesis is a promising approach to improve riboflavin production. The phosphoribosyl pyrophosphate synthetase and phosphoribosyl pyrophosphate amidotransferase are the rate limiting enzymes in purine biosynthesis. Corresponding genes PRS3 and ADE4 from yeast Debaryomyces hansenii are modified to avoid feedback inhibition and cooverexpressed on the background of a previously constructed riboflavin overproducing strain of C. famata. Constructed strain accumulates twofold more riboflavin when compared to the parental strain.

Multinuclear Yeast Magnusiomyces (Dipodascus, Endomyces) magnusii is a Promising Isobutanol Producer.

Higher alcohol isobutanol is a promising liquid fuel. During alcoholic fermentation, Saccharomyces cerevisiae produces only trace amounts of isobutanol. Screening the collection of nonconventional yeasts show that Magnusiomyces magnusii accumulates 440 mg of isobutanol per L in rich YPD medium. Here, the transformation protocol for M. magnusii is adapted based on the use of the dominant markers conferring resistance to nourseothricin or zeocin; the strong constitutive promoter TEF1 is cloned and a reporter system based on LAC4 gene from Kluyveromyces lactis coding for ß-galactosidase is constructed. In order to increase isobutanol production in M. magnusii, the heterologous gene ILV2 from S. cerevisiae is expressed in M. magnusii under control of the TEF1 promoter. The best stabilized transformants produce 620 mg of isobutanol per L in YPD medium and 760 mg L-1 in the medium with 2-oxoisovalerate. This suggests that M. magnusii is a promising organism for further development of a robust isobutanol producer.

Construction of advanced producers of first- and second-generation ethanol in Saccharomyces cerevisiae and selected species of non-conventional yeasts (Scheffersomyces stipitis, Ogataea polymorpha).

This review summarizes progress in the construction of efficient yeast ethanol producers from glucose/sucrose and lignocellulose. Saccharomyces cerevisiae is the major industrial producer of first-generation ethanol. The different approaches to increase ethanol yield and productivity from glucose in S. cerevisiae are described. Construction of the producers of second-generation ethanol is described for S. cerevisiae, one of the best natural xylose fermenters, Scheffersomyces stipitis and the most thermotolerant yeast known Ogataea polymorpha. Each of these organisms has some advantages and drawbacks. S. cerevisiae is the primary industrial ethanol producer and is the most ethanol tolerant natural yeast known and, however, cannot metabolize xylose. S. stipitis can effectively ferment both glucose and xylose and, however, has low ethanol tolerance and requires oxygen for growth. O. polymorpha grows and ferments at high temperatures and, however, produces very low amounts of ethanol from xylose. Review describes how the mentioned drawbacks could be overcome.

Virulence and antifungal susceptibility of microsatellite genotypes of Candida albicans from superficial and deep locations.

A set of 185 strains of Candida albicans from patients with vulvovaginal candidiasis (VVC) and from non-VVC clinical sources in southwest China was analysed. Strains were subjected to genotyping using CAI microsatellite typing and amplification of an intron-containing region of the 25S rRNA gene. Microsatellite genotypes of strains from non-VVC sources showed high polymorphism, whereas those of VVC were dominated by few, closely similar genotypes. However, among non-VVC strains, two genotypes were particularly prevalent in patients with lung cancer. 25S rDNA genotype A was dominant in VVC sources (86.7%), whereas genotypes A, B, and C were rather evenly distributed among non-VVC sources; known genotypes D and E were not found. In an experimental mouse model, isolates from lung cancer and AIDS patients proved to have higher virulence than VVC strains. Among 156 mice infected with C. albicans, 19 developed non-invasive urothelial carcinoma. No correlation could be established between parameters of virulence, source of infection, and incidence of carcinoma. C. albicans strains from VVC were less susceptible to itraconazole than the strains from non-VVC sources, whereas there was small difference in antifungal susceptibility between different 25S rDNA genotypes of C. albicans tested against amphotericin B, itraconazole, fluconazole, and flucytosine.

Peroxisomes and peroxisomal transketolase and transaldolase enzymes are essential for xylose alcoholic fermentation by the methylotrophic thermotolerant yeast, Ogataea (Hansenula) polymorpha.

BACKGROUND: Ogataea (Hansenula) polymorpha is one of the most thermotolerant xylose-fermenting yeast species reported to date. Several metabolic engineering approaches have been successfully demonstrated to improve high-temperature alcoholic fermentation by O. polymorpha. Further improvement of ethanol production from xylose in O. polymorpha depends on the identification of bottlenecks in the xylose conversion pathway to ethanol. RESULTS: Involvement of peroxisomal enzymes in xylose metabolism has not been described to date. Here, we found that peroxisomal transketolase (known also as dihydroxyacetone synthase) and peroxisomal transaldolase (enzyme with unknown function) in the thermotolerant methylotrophic yeast, Ogataea (Hansenula) polymorpha, are required for xylose alcoholic fermentation, but not for growth on this pentose sugar. Mutants with knockout of DAS1 and TAL2 coding for peroxisomal transketolase and peroxisomal transaldolase, respectively, normally grow on xylose. However, these mutants were found to be unable to support ethanol production. The O. polymorpha mutant with the TAL1 knockout (coding for cytosolic transaldolase) normally grew on glucose and did not grow on xylose; this defect was rescued by overexpression of TAL2. The conditional mutant, pYNR1-TKL1, that expresses the cytosolic transketolase gene under control of the ammonium repressible nitrate reductase promoter did not grow on xylose and grew poorly on glucose media supplemented with ammonium. Overexpression of DAS1 only partially restored the defects displayed by the pYNR1-TKL1 mutant. The mutants defective in peroxisome biogenesis, pex3Δ and pex6Δ, showed normal growth on xylose, but were unable to ferment this sugar. Moreover, the pex3Δ mutant of the non-methylotrophic yeast, Scheffersomyces (Pichia) stipitis, normally grows on and ferments xylose. Separate overexpression or co-overexpression of DAS1 and TAL2 in the wild-type strain increased ethanol synthesis from xylose 2 to 4 times with no effect on the alcoholic fermentation of glucose. Overexpression of TKL1 and TAL1 also elevated ethanol production from xylose. Finally, co-overexpression of DAS1 and TAL2 in the best previously isolated O. polymorpha xylose to ethanol producer led to increase in ethanol accumulation up to 16.5 g/L at 45 °C; or 30-40 times more ethanol than is produced by the wild-type strain. CONCLUSIONS: Our results indicate the importance of the peroxisomal enzymes, transketolase (dihydroxyacetone synthase, Das1), and transaldolase (Tal2), in the xylose alcoholic fermentation of O. polymorpha.

Autophagy-related gene ATG13 is involved in control of xylose alcoholic fermentation in the thermotolerant methylotrophic yeast Ogataea polymorpha.

Lignocellulosic biomass belongs to main sustainable renewable sources for global energy supply. One of the main challenges in the conversion of saccharified lignocellulosic biomass into bioethanol is the utilization of xylose, since lignocellulosic feedstocks contain a significant amount of this pentose. The non-conventional thermotolerant yeast Ogataea polymorpha naturally ferments xylose to ethanol at elevated temperatures (45°C). Studying the molecular mechanisms of regulation of xylose metabolism is a promising way toward increased xylose conversion to ethanol. Insertional mutagenesis was applied to yeast O. polymorpha to identify genes involved in regulation of xylose fermentation. An insertional mutant selected as 3-bromopyruvate resistant strain possessed 50% increase in ethanol production as compared to the parental strain. Increase in ethanol production was caused by disruption of an autophagy-related gene ATG13. Involvement of Atg13 in regulation of xylose fermentation was confirmed by deletion of that gene. The atg13Δ strain also produced an elevated amount of ethanol from xylose. Insertion in ATG13 gene did not disrupt HORMA domain and did not lead to defects in autophagy whereas knock out of this gene impaired autophagy process. We suggest that Atg13 plays two different functions and its role in regulation of xylose fermentation differs from that in autophagy.