Improved Ethanol Production Through Genetic Manipulation Resulting In Deceasing Glycerol Formation Of Saccharomyces Cerevisiae

In this investigation we employed the concept of metabolic engineering and molecular biology techniques to construct recombinant strains of the yeast Saccharomyces cerevisiae, in which the FPS1 gene, encoding a glycerol channel protein, and the GPD1 and the GPD2 genes, encoding the glycerol 3-phosphate dehydrogenases, were deleted to minimize formation and increase intracellular accumulation of glycerol and improve ethanol production. In addition, we also over-expressed the GLT1 gene encoding the glutamate synthase to overcome the redox imbalancing problem in the modified strains. FPS1, GPD1 and GPD2 were deleted using one-step gene replacement method and GLT1 was over-expressed by two-step gene replacement method. Haploid cells of opposite mating type can mate to produce an a/αdiploid and, under certain conditions, a diploid cell will form a tetrad of four haploid spores. By mating genetically modified haploid mutant trains mentioned above and subsequent sporulating and dissecting, we obtained 10 mutant strains with different genotype. Anaerobic batch fermentation experiments were carried out with these strains.The results showed that the rate of growth and glucose consumption of the KAM-9 (fps1Δ::REPEAT gpd2Δ::REPEAT) strain were slightly slower than the original strain, and the KAM-13 (gpd2Δ::REPEAT PGK1-GLT1) strain was indistinguishable compared to the original strain when the same criteria were analyzed. On the other hand, when compared to the original strain, there were a 31.40% and 36.22% reduction in glycerol formation for KAM-9 and KAM-13, respectively, and ethanol production increased by 12.00% for KAM-9 and 15.56% for KAM-13, respectively. Meanwhile, a dramatic reduction in the formation of both acetate and pyruvic acid was also observed in all the recombinant yeast strains compared to the wild type. Strains KAM-10 (gpd1Δ::REPEAT gpd2Δ::REPEAT) did not produce glycerol but this strain showed a severe growth defect under experimental conditions and the ethanol yield decreased significantly compared to the wild type strain. A biochemical reaction network of engineered S. cerevisiae was assembled with metabolites, and a stoichiometric model was used to analyze metabolic fluxes in batch cultures of two different engineered strains. The result of metabolic fluxes analysis indicated that flux in KAM-13 is optimum level.Although the product of GPD2 and FPS1 are responsible for the synthesis and efflux of glycerol, which is important for osmoregulation and maintenance of cellular redox balance in yeast cells, the recombinant yeast strains, KAM-9 (fps1Δ::REPEAT gpd2Δ::REPEAT) and KAM-13 (gpd2Δ::REPEAT PGK1-GLT1), were able to maintain proper cellular osmoregulation and redox balance under anaerobic fermentation conditions, and verified the proposed concept of increasing ethanol production in S. cerevisiae by genetic engineering of glycerol synthesis and transport systems as well as reconstituted NADH metabolism.