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This research presents a method for the positive selection of mutants with improved xylose and L-arabinose fermentation in the thermotolerant, naturally xylose-utilizing yeast Ogataea polymorpha which is based on isolation of the mutants growing on L-arabinose as sole carbon and energy source. Whole-genome sequencing of the most efficient xylose-fermenting strain, A107, revealed mutations in the API1 and IRA1 genes, which are homologous to bacterial arabinose-5-phosphate isomerase and the Ras-GTPase activating domain in Saccharomyces cerevisiae , respectively. Disruption of the IRA1 gene increased ethanol production during the fermentation of xylose and L-arabinose in O. polymorpha at 45 °C. Overexpression of the API1 gene specifically enhanced L-arabinose fermentation without affecting xylose fermentation. The most productive mutant strain accumulated 20.91 g/L of ethanol in a xylose-containing medium at 45 °C, exceeding the ethanol accumulation level of the wild-type strain (0.40 g/L) by over 50 times. This strain holds potential for application in simultaneous saccharification and fermentation (SSF) processes.

Background Xylose transport is one of the bottlenecks in the conversion of lignocellulosic biomass to ethanol. Xylose consumption by the wild-type strains of xylose-utilizing yeasts occurs once glucose is depleted resulting in a long fermentation process and overall slow and incomplete conversion of sugars liberated from lignocellulosic hydrolysates. Therefore, the engineering of endogenous transporters for the facilitation of glucose-xylose co-consumption is an important prerequisite for efficient ethanol production from lignocellulosic hydrolysates. Results In this study, several engineering approaches formerly used for the low-affinity glucose transporters in Saccharomyces cerevisiae , were successfully applied for earlier identified transporter Hxt1 in Ogataea polymorpha to improve xylose consumption (engineering involved asparagine substitution to alanine at position 358 and replacement of N-terminal lysine residues predicted to be the target of ubiquitination for arginine residues). Moreover, the modified versions of S. cerevisiae Hxt7 and Gal2 transporters also led to improved xylose fermentation when expressed in O. polymorpha . Conclusions The O. polymorpha strains with modified Hxt1 were characterized by simultaneous utilization of both glucose and xylose, in contrast to the wild-type and parental strain with elevated ethanol production from xylose. When the engineered Hxt1 transporter was introduced into constructed earlier advanced ethanol producer form xylose, the resulting strain showed further increase in ethanol accumulation during xylose fermentation. The overexpression of heterologous S. cerevisiae Gal2 had a less profound positive effects on sugars uptake rate, while overexpression of Hxt7 revealed the least impact on sugars consumption.

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 defectsdisplayed 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, thepex3Δ mutant of the non-methyl-otrophic 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.

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.

Thermotolerant yeasts comprise a phylogenetically and ecologically diverse set of species with abundant growth around 45°C.Ogataea polymorpha and K. marxianus exhibit rapid growth, stress resilience, and broad substrate utilization, and grow maximally above 50°C. The exact reasons for such high thermotolerance are not known.Heat tolerance involves heat shock protein expression, membrane adaptation, redox homeostasis, and regulatory rewiring.High-temperature bioprocessing benefits from lower contamination, faster kinetics, and integration with enzymatic hydrolysis of biopolymers.Full exploitation of thermotolerant yeasts is hindered by inefficient pentose utilization, oxygen requirements under industrial conditions, and biosafety concerns for non-Generally Recognized as Safe species, such as Pichia kudiavzevii. Most known yeasts are mesophilic organisms, with optimal growth at 28–30°C. However, a few species, notably Ogataea polymorpha and Kluyveromyces marxianus, can grow at temperatures around 50°C. Their thermotolerance is of both fundamental and applied interest, offering advantages for high-temperature bioprocesses. Notably, cultivation at elevated temperatures facilitates simultaneous saccharification and fermentation (SSF) of starchy and lignocellulosic substrates, reduces sterility requirements, lowers distillation costs, and enhances the rates of biochemical reactions. These features make thermotolerant yeasts attractive platforms for producing biofuels, proteins, and other valuable compounds. Despite their potential, the underlying mechanisms of thermotolerance in these species remain underexplored. Herein, we systematically review the molecular, cellular, and metabolic mechanisms underlying yeast thermotolerance and discuss their implications for high-temperature industrial biotechnology. Most known yeasts are mesophilic organisms, with optimal growth at 28–30°C. However, a few species, notably Ogataea polymorpha and Kluyveromyces marxianus, can grow at temperatures around 50°C. Their thermotolerance is of both fundamental and applied interest, offering advantages for high-temperature bioprocesses. Notably, cultivation at elevated temperatures facilitates simultaneous saccharification and fermentation (SSF) of starchy and lignocellulosic substrates, reduces sterility requirements, lowers distillation costs, and enhances the rates of biochemical reactions. These features make thermotolerant yeasts attractive platforms for producing biofuels, proteins, and other valuable compounds. Despite their potential, the underlying mechanisms of thermotolerance in these species remain underexplored. Herein, we systematically review the molecular, cellular, and metabolic mechanisms underlying yeast thermotolerance and discuss their implications for high-temperature industrial biotechnology.

Successful conversion of cellulosic biomass into biofuels requires organisms capable of efficiently utilizing xylose as well as cellodextrins and glucose. Ogataea (Hansenula) polymorpha is the natural xylose-metabolizing organism and is one of the most thermotolerant yeasts known, with a maximum growth temperature above 50°C. Cellobiose-fermenting strains, derivatives of an improved ethanol producer from xylose O. polymorpha BEP/cat8∆, were constructed in this work by the introduction of heterologous genes encoding cellodextrin transporters (CDTs) and intracellular enzymes (β-glucosidase or cellobiose phosphorylase) that hydrolyze cellobiose. For this purpose, the genes gh1-1 of β-glucosidase, CDT-1m and CDT-2m of cellodextrin transporters from Neurospora crassa and the CBP gene coding for cellobiose phosphorylase from Saccharophagus degradans, were successfully expressed in O. polymorpha. Through metabolic engineering and mutagenesis, strains BEP/cat8∆/gh1-1/CDT-1m and BEP/cat8∆/CBP-1/CDT-2mAM were developed, showing improved parameters for high-temperature alcoholic fermentation of cellobiose. The study highlights the need for further optimization to enhance ethanol yields and elucidate cellobiose metabolism intricacies in O. polymorpha yeast. This is the first report of the successful development of stable methylotrophic thermotolerant strains of O. polymorpha capable of coutilizing cellobiose, glucose, and xylose under high-temperature alcoholic fermentation conditions at 45°C. Strains of xylose-fermenting yeast Ogataea polymorpha have been constructed capable of efficient cellobiose utilization and fermentation at elevated temperature (45°C).

Abstract The production of second-generation (2 G) bioethanol, a key sector in industrial biotechnology, addresses the demand for sustainable energy by utilizing lignocellulosic biomass. Efficient fermentation of all sugars from lignocellulose hydrolysis is essential to enhance ethanol titers, improve biomass-to-biofuel yields, and lower costs. This review compares the potential of recombinant yeast strains for 2 G bioethanol production, focusing on their ability to metabolize diverse sugars, particularly xylose. Saccharomyces cerevisiae, engineered for enhanced pentose and hexose utilization, is compared with the nonconventional yeasts Scheffersomyces stipitis, Kluyveromyces marxianus, and Ogataea polymorpha. Key factors include sugar assimilation pathways, cofermentation with glucose, oxygen requirements, tolerance to hydrolysate inhibitors, and process temperature. Saccharomyces cerevisiae shows high ethanol tolerance but requires genetic modification for xylose use. Scheffersomyces stipitis ferments xylose naturally but lacks robustness. Kluyveromyces marxianus offers thermotolerance and a broad substrate range with lower ethanol yields, while O. polymorpha enables high-temperature fermentation but yields modest ethanol from xylose. The comparative analysis clarifies each yeast’s advantages and limitations, supporting the development of more efficient 2 G bioethanol production strategies. Strain selection must balance ethanol yield, stress tolerance, and temperature adaptability to meet industrial requirements for cost-effective lignocellulosic bioethanol production. This review explores advances in engineering yeast for cellulosic ethanol production, comparing Saccharomyces and nonconventional species in sugar metabolism, fermentation efficiency, and tolerance to lignocellulosic hydrolysate inhibitors.

Abstract The global transition to renewable energy sources requires efficient microbial platforms capable of fermenting carbon sources present in lignocellulosic biomass. Conventional yeasts like Saccharomyces cerevisiae face critical limitations, particularly in pentose sugar utilization and inhibitor resistance. This review focuses on two emerging nonconventional yeasts, Candida famata and Ogataea polymorpha, which exhibit native or engineered capacities to overcome these bottlenecks. We present a comparative analysis of their stress tolerance, metabolic versatility, and recent advances in genetic engineering, adaptive laboratory evolution, and heterologous expression systems. Their ability to grow on a wide range of sugars, tolerate fermentation inhibitors, and operate under industrial conditions underscores their potential as microbial platforms for sustainable bioprocessing. Key challenges and future directions are discussed to guide further development. This review highlights Candida famata and Ogataea polymorpha as promising nonconventional yeasts with enhanced lignocellulosic fermentation capabilities, stress tolerance, and engineering potential for sustainable bioenergy production.

ABSTRACT Glucose is a preferred carbon source for most living organisms. The metabolism and regulation of glucose utilization are well studied mostly for Saccharomyces cerevisiae. Xylose is the main pentose sugar released from the lignocellulosic biomass, which has a high potential as a renewable feedstock for bioethanol production. The thermotolerant yeast Ogataea (Hansenula) polymorpha, in contrast to S. cerevisiae, is able to metabolize and ferment not only glucose but also xylose. However, in non-conventional yeasts, the regulation of glucose and xylose metabolism remains poorly understood. In this study, we characterize the role of transcriptional factors Mig1, Mig2, Tup1 and Hap4 in the natural xylose-fermenting yeast O. polymorpha. The deletion of MIG1 had no significant influence on ethanol production either from xylose or glucose, however the deletion of both MIG1 and MIG2 reduced the amount of ethanol produced from these sugars. The deletion of HAP4-A and TUP1 genes resulted in increased ethanol production from xylose. Inversely, the overexpression of HAP4-A and TUP1 genes reduced ethanol production during xylose alcoholic fermentation. Thus, HAP4-A and TUP1 are involved in repression of xylose metabolism and fermentation in yeast O. polymorpha and their deletion could be a viable strategy to improve ethanol production from this pentose. The investigation of the role of transcriptional factors in xylose and glucose metabolism and fermentation in thermotolerant yeast Ogataea polymorpha.

Glutathione is the most abundant cellular thiol and the low molecular weight peptide present in cells. The methylotrophic yeast Ogataea (Hansenula) polymorpha is considered as a promising cell factory for the synthesis of glutathione. In this study, a competitive O. polymorpha glutathione producer was constructed by overexpression of the GSH2 gene, encoding γ-glutamylcysteine synthetase, the first enzyme involved in glutathione biosynthesis, and the MET4 gene coding for central regulator of sulfur metabolism. Overexpression of MET4 gene in the background of overexpressed GSH2 gene resulted in 5-fold increased glutathione production during shake flask cultivation as compared to the wild-type strain, reaching 2167 mg L-1. During bioreactor cultivation, glutathione accumulation by obtained recombinant strain was 5-fold increased relative to that by the parental strain with overexpressed only GSH2 gene, on the first 25 h of batch cultivation in mineral medium. Obtained results suggest involvement of Met4 transcriptional activator in regulation of GSH synthesis in the methylotrophic yeast O. polymorpha.