Metabolic Engineering Of The Glycerol Pathway In Saccharomyces Cerevisiae

With the growing consumption of fossil energy and as a non-renewable energy, ethanolwhich is an alternative to fossil fuels and a renewable and clean energy, causes the attention ofmore people and has brought a profound impact on its social significance. Glycerol is themain byproduct in Saccharomyces cerevisiae producing ethanol under anaerobic conditions.Glycerol consumes about4~10%of carbon source, if this part of carbon source redirectstowards producing ethanol, it will bring enormous social and economic benefits. In thepresent study, we aimed to combine the glycerol synthesis pathway, glycerol decompositionpathway with cofactor regulation by genetic modification strategies in S.cerevisiae. Ourultimate goal was improving the ethanol productivity by decreasing the production of glyceroland cofactor engineering with little effect on the physiological properties of S.cerevisiae.(1) Glycerol was converted to dihydroxyacetone phosphate by the dihydroxyacetonedecomposition pathway which involves a glycerol dehydrogenase encoded by GCY1or YPR1,and a dihydroxyacetone kinase encoded by DAK1or DAK2. GCY1and DAK1wereoverexpressed in Saccharomyces cerevisiae resulted in the recombinant haploid strainsS.cerevisiae GDA1, GDA2, and then the two haploid strains were hybridized to obtain therecombinant polyploid strain S.cerevisiae GDA. The haploid strains S.cerevisiae GDA1andGDA2showed a3.19%,2.82%(relative to the amount of the substrate consumed) increase inethanol production and a19.24%,18.63%(relative to the amount of the substrate consumed)decrease in glycerol production, respectively. In addition, the recombinant polyploid strainS.cerevisiae GDA exhibited a3.93%increase in ethanol production and a20.54%decrease inglycerol production. However, the concentration of the intracellular NADH for therecombinant polyploid strain S.cerevisiae GDA was77.2μmol L-1, which was higher than thatof the wide type S.cerevisiae (40.8μmol L-1).(2) The critical enzyme of the glycerol synthesis pathway is NAD+-dependentglycerol-3-phosphate dehydrogenase encoded by GPD2. And NADH is converted to NADPHby NADH kinase encoded by POS5in the presence of ATP. A disruption cassettegpd2Δ::PGK1PT-POS5-HyBR was constructed and it was transformed into S.cerevisiae toresult in the recombinant strain S.cerevisiae PA, so GPD2was deleted and POS5wasoverexpressed simultaneously in S.cerevisiae. S.cerevisiae PA exhibited a7.47%increase inethanol production and a35.27%decrease in glycerol production. At the same time, theconcentration of intracellular NADH was lower.(3) Based on (1) and (2), the disruption cassette gpd2Δ::PGK1PT-POS5-HyBR wastransformed into the recombinant haploid strains S.cerevisiae GDA1and GDA2and then thetwo constructed haploid recombinant strains were hybridized to get the recombinant strainS.cerevisiae PGDA, which combined the glycerol synthesis pathway, glycerol decompositionpathway with the cofactor regulation. S.cerevisiae PGDA exhibited a8.38%increase inethanol production and a48.96%decrease in glycerol production. Moreover, theconcentration of the intracellular NADH is a little more than that of the wide typeS.cerevisiae. (4) The effusion of the glycerol is regulated by aquaglyceroporin encoded by FPS1.FPS1was deleted in S.cerevisiae to result in the recombinant strain S.cerevisiae FA, but thegrowth rate was inhibited, which exhibited a4.08%increase in ethanol production and a20.98%decrease in glycerol production. Thus, the recombinant plasmid pYX212-POS5-Kanrwas introduced into S.cerevisiae FA to obtain the recombinant strain S.cerevisiae PFA and itsgrowth rate was higher than that of S.cerevisiae FA. S.cerevisiae PFA showed a5.79%increase in ethanol production and a29.46%decrease in glycerol production. What’s more,the lower concentration of intracellular NADH was regulated effectively.