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Abstract:

Thermotolerance is an attractive feature for yeast-based industrial ethanol production. However, incompletely understood oxygen requirements of known thermotolerant yeasts are incompatible with process requirements. To study the magnitude and molecular basis of these oxygen requirements in the facultatively fermentative, thermotolerant yeast Ogataea parapolymorpha , chemostat studies were performed under defined oxygen-sufficient and oxygen-limited cultivation regimes. The minimum oxygen requirements of O. parapolymorpha were found to be at least an order of magnitude larger than those of the thermotolerant yeast Kluyveromyces marxianus . This high oxygen requirement coincided with absence of glycerol formation, which plays a key role in NADH reoxidation in oxygen-limited cultures of other facultatively fermentative yeasts. Co-feeding of acetoin, whose reduction to 2,3-butanediol can reoxidize cytosolic NADH, supported a 2.5-fold higher biomass concentration in oxygen-limited cultures. The apparent inability of O. parapolymorpha to produce glycerol correlated with absence of orthologs of the S. cerevisiae genes encoding glycerol-3P phosphatase (Sc GPP1 , Sc GPP2 ). Glycerol production was observed in aerobic batch cultures of a strain in which genes including key enzymes in mitochondrial reoxidation of NADH were deleted. However, transcriptome analysis did not identify a clear candidate for the responsible phosphatase. Expression of Sc GPD2 , encoding NAD + -dependent glycerol-3P dehydrogenase, and Sc GPP1 in O. parapolymorpha resulted in increased glycerol production in oxygen-limited chemostats, but glycerol production rates remained substantially lower than observed in S. cerevisiae and K. marxianus . These results identify a dependency on aerobic respiration for reoxidation of NADH generated in biosynthesis as a key factor in the unexpectedly high oxygen requirements of O. parapolymorpha .

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