Effects On Ethanol Fermentation By Industral Strain Of Saccharomyces Cerevisiae With GPD Null Mutation

Fuel ethanol, as a clean energy to replace fossil fuels, has become a hot-spot for researchers worldwide. A major problem about ethanol production by anaerobic fermentation of Saccharomyces cerevisiae is that 4 % to 10 % of the carbon source may be converted to glycerol. Strains impaired glycerol synthesis would lead to increase yield of ethanol and efficiency of utilization of the carbon source. The majorst rategy is to interdict or manipulate the metabolism, or to express another gene in S.cerevisiae to redirect the carbon flux. With glucose as the carbon source, glycerol is formed by conversion of dihydroxyacetone phosphate to glycerol-3-phosphate (G-3-P) and subsequent dephosphorylation of G-3-P to yield glycerol. The first reaction is catalyzed by glycerol-3-phosphate dehydrogenase (GPD). There are two isogenes encoding different forms of GPD, GPD1 and GPD2.Firstly, we choose the McClary medium which reached a maximum sporulation rate for Saccharomyces cerevisiae of optimum sporulation medium. Six haploid strains were got after sporulation of Saccharomyces cerevisiae. Take the selected 6 strains and W303-1A as hybridization strains, and make group hybridization, we found two different haploid strains(a type andαtype).Based on homologous recombination, a recombinant plasmid pUC19-GPD1::?Km was constructed and introduced into haploid (a type) by electroporation after linearized. The haploid mutant S.cerevisiae Y(a,△gpd1) was generated and comfirmed by the method of PCR. The recombinant plasmid pPIC-GPD2-bgl-hyg was digested by HindIII and transformed into tne two different haploid strains(a type andαtype)by electroporation, then the haploid mutants S.cerevisiae-Y(a,△gpd2) and S.cerevisiae-Y(α,△gpd2)were generated. Yeast-two hybrid technique used for reference, after hybridization between S.cerevisiae-Y(a,△gpd1) and S.cerevisiae-Y (α) , S.cerevisiae-Y ( a,△gpd2 ) and S.cerevisiae-Y(α,△gpd2), the diploid mutants S.cerevisiae-Y(△gpd1,△gpd1) and S.cerevisiae-Y(△gpd2,△gpd2)were generated and comfirmed by the method of sporulation and PCR.In ethanol fermentation of glucose carbon source, the recombinants S.cerevisiae-Y(a,△gpd1) and S.cerevisiae-Y(α,△gpd2)'s glycerol production decreased by 24.83% and 22.35% than their parent strains. And the recombinants both showed a slight increase in ethanol formation. The result of anaerobic fermentation showed that the rate of growth and glucose consumption of the S.cerevisiae-Y(△gpd1,△gpd1) and S.cerevisiae-Y(△gpd2,△gpd2)recombinants were slower than S.cerevisiae-Y. On the other hand, when compared to the original strain,there were 11.66 % and 20.71 % reduction in glycerol formation for S.cerevisiae-Y(△gpd1,△gpd1) and S.cerevisiae-Y(△gpd2,△gpd2),respectively. Ethanol production increased by 5.9 % for S.cerevisiae-Y(△gpd1,△gpd1) and 13.2 % for S.cerevisiae-Y(△gpd2,△gpd2). Dramatic reduction in acetate and pyruvic acid was also observed in both recombinants compared to the original strains.