PT  - JOURNAL ARTICLE
AU  - Wijbrand J. C. Dekker
AU  - Hannes Juergens
AU  - Raúl A. Ortiz-Merino
AU  - Christiaan Mooiman
AU  - Remon van den Berg
AU  - Astrid Kaljouw
AU  - Robert Mans
AU  - Jack T. Pronk
TI  - Re-oxidation of cytosolic NADH is a major contributor to the high oxygen requirements of the thermotolerant yeast <em>Ogataea parapolymorpha</em> in oxygen-limited cultures
AID  - 10.1101/2021.04.30.442227
DP  - 2021 Jan 01
TA  - bioRxiv
PG  - 2021.04.30.442227
4099  - http://biorxiv.org/content/early/2021/05/04/2021.04.30.442227.short
4100  - http://biorxiv.org/content/early/2021/05/04/2021.04.30.442227.full
AB  - 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 (ScGPP1, ScGPP2). 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 ScGPD2, encoding NAD+-dependent glycerol-3P dehydrogenase, and ScGPP1 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.Importance Thermotolerant yeasts hold great potential for anaerobic fermentation processes but their application is so far hampered by incompletely understood oxygen requirements. Based on quantitative physiological studies in oxygen-limited chemostat cultures, this study shows that the thermotolerant yeast Ogataea parapolymorpha has a much higher oxygen requirement than other, previously investigated facultatively fermentative yeasts. The large oxygen requirement of O. parapolymorpha was linked to an inability of oxygen-limited cultures to efficiently reoxidize NADH formed in biosynthetic processes by glycerol formation. These results provide a basis for reducing the oxygen requirements of O. parapolymorpha by targeted metabolic engineering. In addition, this study shows that diversity of oxygen requirements should be taken into account in selecting yeast species for application in anaerobic or oxygen-limited industrial processes.