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. 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. Our results indicate the importance of the peroxisomal enzymes, transketolase (dihydroxyacetone synthase, Das1), and transaldolase (Tal2), in the xylose alcoholic fermentation of O. polymorpha.

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.

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.

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.

Saccharomyces cerevisiae CL4 was evaluated as a potential probiotic strain based on stress tolerance, safety-related properties, microbial interactions, and effects in rats. The strain grew at 37 °C and showed 71.0 % survival at pH 2.5 and 83.2 % survival under simulated gastric conditions. No hemolytic activity was detected. CL4 did not inhibit Candida albicans, but altered the phenotype of Pseudomonas aeruginosa, an effect also reproduced by cell-free culture fluid. In rats with metabolic syndrome, CL4 administration increased superoxide dismutase activity and affected other antioxidant defense parameters. Keywords: Saccharomyces cerevisiae, probiotic yeast, antioxidant defense, tolerance assay, microbial interactions.

A novel positive selection approach for Ogataea polymorpha based on CAT8 overexpression and metformin-mediated growth inhibition was investigated. Overexpression of CAT8 conferred a growth advantage under selective conditions with glycerol as the carbon source, demonstrating the potential of this system for selection of recombinant O. polymorpha strains. Keywords: Ogataea polymorpha, CAT8, metformin, positive selection, metabolic engineering.