Systematic Investigation On Adaptive Evolution Of Saccharomyces Cerevisiae To Repeated-batch Vacuum Fermentation

In the ethanol production using cellulose hydrolyses, volatile substance in the fermentation broth can have toxicity to yeast cells, and consequently cause negative effects to the productivity. Repeated-batch vacuum fermentation (RBVF) can effectively eliminate the inhibitory effects of the volatile products on yeast cells, and can improve the ethanol productivity. However, yeast cells have to encounter the stresses from RBVF, which can induce changes in their cell morphology and cell behaviors, and can potentially affect the ethanol productivity. Therefore, obtain a new vacuum-adapted strain and understand the relationship between adaptive evolution and fermentation behaviors will be of great importance to RBVF process. In this thesis, the complex adaptive evolution and the mechanism of how the S.cerevisiae responses to RBVF were explored systematically in view of proteome, metabolome and lipidome. Furthermore, a new strain that can better adapt to vacuum fermentation and achieve higher ethanol productivity was obtained. Finally the correlation between adaptive evolution and fermentation behavior was investigated by integrating omics analysis to the differential mechanisms of parental and vacuum-adapted yeast to RBVF.RBVF was performed under 50mba vacuum pressure, with 30 cycles for a total period of 420 hours. The glucose concentration in broth was maintained between 20-100g/L by feeding suitable YPD medium at each cycle. The viability of S.cerevisiae and their fermentative rate started to decrease quickly after the 13th cycle. Unexpectedly, at the 30th cycle the fermentative rate recovered to the level at the 18th cycle, which suggested the yeast was adapted to vacuum fermentation. The adapted-vacuum yeast obtained by recovery of aerobically recultivation from the 13th cycle, exhibites better property in RBVF. Compared with the parental yeast, adapted-vacuum yeast has six times higher ethanol productivity with a higher vialbility at 98%.The mechanisms how S.cerevisiae adapts to the RBVF were explored in view of proteome using proteomic, metabolome and lipidome, respectively. (1) Protomics level analysis shows that the proteins, which involved in carbohydrate metabolisms and associated with heat stress and oxidative response, were upregulated; while the essential gene proteins were expressed differentially. The variations of proteins suggest that vacuum fermentation can induce redistribution of the metabolic fluxes, affect cell redox homeostasis and modify the cell morphology and membrane structure. (2) Studies on metabolomic level elucidate that the adapted evolution of S.cerevisiae to RBVF includes three phases. In the first phase, cells sensed and responded the stresses, then produce protective substances chemicals such as treholase and glycerol. In the second phase, the decrease of those protective chemicals in vivo meant the completion of adaptive process. During the third phase, both the yeast viability and the glucose consumption gradually decreased. Additionally, glycolysis and TCA cycle intermediates were enhanced, whereas glycerol biosynthesis was depressed by vacuum. The decreases of most amino acids might be related to the increases in intermediates of glycolysis and TCA cycle as vacuum fermentation progressed. These findings provide new insights into the underlying mechanisms in adaptive evolution of yeast under vacuum condition. (3) Lipidomic analysis demonstrates that, RBVF can cause eburicol accumulation, suggesting that vacuum can activate the branch of the ergosterol biosynthesis pathway which contributes to the weakened viability and poor ethanol productivity. Furthermore, the synthesis of PC via CDP–choline, and the turnover of PC were enhanced. PIs and PCs with short carbon chain were increased for cell membrane components.Comparison of the protomic, metabolite and lipidomic levels between parental and vacuum-adapted yeast reveals that, the adapted evolution is performed by regulation of cell membrane components on curvature and the ratios of ergosterol and lipid. As a result of the adapted evolution in vacuum-adapted yeast, the glycosis and PPP pathway were suppressed; the TCA cycle was activated and subsequentially compensated the loss of NADPH induced by PPP pathway. Furthmore, ATP futile cycle, which regulates the synthesis and the degradation of treholase and glycerol, can offset the unbalance caused by RBVF. Finally, anaplerotic reaction from oxaloacetate to pyruvate catalysed by PCK can supply pyruvate which contributed to the higher ethanol productivity in adapted-vacuum yeast.