The Expression Strategy For Glycerol Decomposition Based On Endocellular Redox Equilibrium In Saccharomyces Cerevisiae

As the reserves of petroleum decrease year by year, bioethanol, which is renewable andgreen, has become the focus and hotspot of many countries. Glycerol is a major by-product inindustrial ethanol fermentation. If the carbon flow towards glycerol could be redirectedtowards ethanol, the ethanol yield and utilization ratio of raw materials will be improved. Inthis study, we aimed to develop the most promising genetic modification strategies to increaseethanol yield and decrease glycerol production.Interruption of glycerol production by deleting GPD1and GPD2, encoding glycerol3-phosphate dehydrogenase in the glycerol pathway, were unsuccessful because the growthrate and product formation in such engineered strains were severely lowered. In addtition,glycerol can be covnverted to glycolytic intermediates by glycerol-3-phosphate routeaerobically and by dihydroxyacetone route anaerobically in Saccharomyces cerevisiae. Thedihydroxyacetone pathway involves a glycerol dehydrogenase encoded by GCY1or YPR1,and a dihydroxyacetone kinase encoded by DAK1or DAK2. GCY1and DAK1wereoverexpressed in S.cerevisiae, with rDNA as homologous recombination locus, resulted inrecombinant strain S.cerevisiae GDS1(PPGK-GCY1-DAK1-kan). The growth and compoundyields of parent yeast and recombinant strain GDS1were compared during anaerobic batchgrowth on15%glucose. The yeast expressing GCY1and DAK1produced73.4g ethanol l-1and showed a2.9%increase in ethanol production and24.9%decrease in glycerol productioncompared to the wild type. Additionally, the intracellular NADH concentration forrecombinant strain S.cerevisiae GDS1is0.0273mmol/g-DCW which is higher than that ofthe parent yeast (0.0152mmol/g-DCW).To further increase ethanol yields, Bacillus cereus gapN gene encodingnon-phosphorylating NADP+-dependent glyceraldehyde-3-phosphate dehydrognease,S.cerevisiae POS5gene encoding NADH kinase, Escherichia coli mhpF gene encodingacetylating NAD+-dependent acetaldehyde dehydrogenase and frdA gene encoding theNAD+-dependent fumarate reductase, was introduced into S.cerevisiae respectively. Therecombinant plasmids pYX212-gapN-kan, pYX212-POS5-kan, pYX212-mhpF-kan andpYX212-frdA-kan obtained were introduced into S.cerevisiae CICIMY0086via the lithiumacetate method, resulted in recombinant strains GAS1, POS1, MHS1and FRS1. Comparationof growth and compound yields among wild type and recombinant strains showed thatglycerol production decreased in the recombinant strains GAS1, POS1and MHS1by cofactorregulation. The yeast expressing mhpF gene produced74.3g ethanol L-1, showed4.6%increase in ethanol yield and40%decrease in glycerol yield compared to the wild type, whenacetic acid was added before inoculation.Then mhpF gene was expressed in recombinant strain GDS1(PPGK-GCY1-DAK1), whoseG418resistance gene was deleted by Cre/loxp system. The resulted recombinant strainGDMS1produced74.9g ethanol L-1and3.56g glycerol L-1, and showed5.5%increase inethanol yield and48%decrease in glycerol yield when acetic acid was added beforeinoculation compared to the wild type. The intracellular NADH concentration of recombinant strain S.cerevisiae GDMS1is0.0178mmol/g-DCW, lower than0.0273mmol/g-DCW forrecombinant strain GDS1, but higher than that of the parent yeast.