No abstract

Current large-scale, anaerobic industrial processes for ethanol production from renewable carbohydrates predominantly rely on the mesophilic yeast Saccharomyces cerevisiae. Use of thermotolerant, facultatively fermentative yeasts such as Kluyveromyces marxianus could confer significant economic benefits. However, in contrast to S. cerevisiae, these yeasts cannot grow in the absence of oxygen. Response of K. marxianus and S. cerevisiae to different oxygen-limitation regimes were analyzed in chemostats. Genome and transcriptome analysis, physiological responses to sterol supplementation and sterol-uptake measurements identified absence of a functional sterol-uptake mechanism as a key factor underlying the oxygen requirement of K. marxianus. Heterologous expression of a squalene-tetrahymanol cyclase enabled oxygen-independent synthesis of the sterol surrogate tetrahymanol in K. marxianus. After a brief adaptation under oxygen-limited conditions, tetrahymanol-expressing K. marxianus strains grew anaerobically on glucose at temperatures of up to 45 °C. These results open up new directions in the development of thermotolerant yeast strains for anaerobic industrial applications.

While thermotolerance is an attractive trait for yeasts used in industrial ethanol production, oxygen requirements of known thermotolerant species are incompatible with process requirements. Analysis of oxygen-sufficient and oxygen-limited chemostat cultures of the facultatively fermentative, thermotolerant species Ogataea parapolymorpha showed its minimum oxygen requirements to be an order of magnitude larger than those reported for the thermotolerant yeast Kluyveromyces marxianus. High oxygen requirements of O. parapolymorpha coincided with a near absence of glycerol, a key NADH/NAD+ redox-cofactor-balancing product in many other yeasts, in oxygen-limited cultures. Genome analysis indicated absence of orthologs of the S. cerevisiae glycerol-3-phosphate-phosphatase genes GPP1 and GPP2. Co-feeding of acetoin, whose conversion to 2,3-butanediol enables reoxidation of cytosolic NADH, supported a 2.5-fold increase of the biomass concentration in oxygen-limited cultures. An O. parapolymorpha strain in which key genes involved in mitochondrial reoxidation of NADH were inactivated did produce glycerol, but transcriptome analysis did not reveal a clear candidate for a responsible phosphatase. Expression of S. cerevisiae GPD2, which encodes NAD+-dependent glycerol-3-phosphate dehydrogenase, and GPP1 supported increased glycerol production by oxygen-limited chemostat cultures of O. parapolymorpha. These results identify dependence on respiration for NADH reoxidation as a key contributor to unexpectedly high oxygen requirements of O. parapolymorpha.

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.