Cloning And Expression Condition Optimization Of Gene Related To Saccharomyces Cerevisiae Ethanol Fermentation

In this study, we firstly obtained haploid Saccharomyces cerevisiae strains with higher ethanol production through spore separation. S. cerevisiae diploid ZJD3 series were cultured on McClary medium at 25℃for 7 days, and 71 haploid were resulted. Through mating type testification, 30 MATa. and 18 MATαstrains in the 71 haploid were determined. With fermentation capacity analysis, we screened one MATa and three MATαstrains: zs-ll(a, ZJD3-7), zs-5(a, ZJD3-3), zs-13(a, ZJD3-7) and zs-14(a, ZJD3-11), respectively. The ethanol production was 3% higher than their own parental strains.GapN gene, encoding glyceraldehyde 3-phosphate dehydrogenase, was cloned from Streptococcus mutans. Recombinant plasmids pUG36-gapN and pDB20-gapN were reconstructed on the basis of pUG36 and pDB20. After deleting URA3 gene in zs-11(a, ZJD3-7), we transformed the recombinant plasmids into the URA3-\acking strains, which resulted in an engineered haploid S. cerevisiae strain ZSD pUG36-gapN, with 2.9% higher ethanol production than its parental strain zs-11.Through traditional sporulation and gene engineering, we constructed S. cerevisiae industrial strains with higher ethanol yield. Meanwhile, we primarily discussed the effect of heterologous gapN gene to intercellular metabolic fluxes.