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.
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