Optimization Of Ethanol Production In Saccharomyces Cerevisiae By Genetic Engineering Of Minimizing Glycerol Biosynthesis

The purpose of this investigation was to increase ethanol production ofSaccharomyces cerevisiae through minimizing glycerol formation by employingmutants defective in GPD1 and GPD2 encoding glycerol-3-phosphatedehydrogenases, and FPS1 encoding a channel protein which mediated glycerolexport. In addition, we also overexpressed GLN1 that encodes the glutaminesynthetase and GLT1 that encodes the glutamate synthase (the GS-GOGAT system) toovercome the redox imbalancing problem in the GPD1 and GPD2 double mutants.GPD1 and GPD2 were deleted using one-step gene replacement method;GLN1 andGLT1 were overexpressed by two-step gene replacement method. HaploidSaccharomyces cerevisiae cells of opposite mating type (i.e., mating type α andmating type a) can mate to produce an a/α diploid. If one haploid carries a dominantmutant allele and the other carries a recessive wild-type allele of the same gene, theresulting heterozygous diploid will express the dominant trait. Under certainconditions, a diploid cell will form a tetrad of four haploid spores. Two of the sporesin the tetrad would express the recessive trait and the others would express thedominant trait. By using these methods, we constructed 30 mutant strains withdifferent genotype.Fermentation experiments were performed under microaerobic conditions withstrains, DC124, L-2, ZAL63, ZAL66, ZAL69, ZAL805, ZAL806, ZAL807, ZAL808,ZA809 and ZAL912. The results showed that L-2 (fps1?:: LEU2), ZAL66(fps1△:: LEU2 gpd1△::URA3), ZAL69 (fps1?:: LEU2 gpd2?::URA3), ZAL64(gpd1△::URA3) and ZAL63 (gpd2△::URA3) had 17%, 23%, 51%, 17% and 46%lower glycerol yield respectively compared to the wild type in microaerobicfermentations. The ethanol formation of ZAL63 and ZAL69 were elevatedsignificantly compared to the wild type, but the ethanol yield of the other strains didnot have significant change compared to the wild type. The strain ZAL806(GLT1-PGK1 GLN1-PGK1) did not have a higher ethanol yield than the wild typestrain does, but the glycerol yield decreased slightly compared to the wild type strain.Strains ZAL809 (fps1?:: LEU2 gpd1?::URA3 gpd2?::URA3 GLT1-PGK1 GLN1-PGK1) and ZAL912 (gpd1?::URA3 gpd2?::URA3 GLT1-PGK1 GLN1-PGK1) didnot produce glycerol but the ethanol yield decreased significantly compared to thewild type strain.In this study plasmid pUC18-RYUR was also made, which could be used forconstruction of yeast multiple deletion strains using the URA3 gene as the soleselectable marker gene.