Development Of New Methods For Genetic Manipulation In Saccharomyces Cerevisiae And Construction Of Strains With Improved Ethanol Yield

Due to the imminent decline in the availability of global fossil energy(oil,coal and natural gas)and increasing concern over the issue of environment protection, partial substitution of fossil fuel with bioethanol has become an important renewable energy strategy. For technical reasons, bioethanol production is mainly achieved at present through fermentation of starch- or sugar-based feedstock by Saccharomyces cerevisiae. The cost of feedstock accounts for a large portion of the total production cost in starch- or sugar-based ethanol fermentation, which, to a large extent, hampers the development of the bioethanol industry. Hence, increasing the sugar-ethanol conversion rate and reducing the energy consumption of the process (through shortening the cycle of fermentation and implementation of high gravity fermentation) are key subjects of starch- and sugar-based ethanol industry development. However, the sugar to ethanol conversion rate, the speed of fermentation and the ethanol tolerance are complex traits under the control of multiple genes, which are difficult to alter with classical breeding, metabolic engineering and other genetic manipulation methods with specific genes or pathways as targets. Therefore, employing the whole genome engineering approach will be an effective way to manipulate yeast strains.In this study, yeast shuttle vector YCplac33 was used to clone the HO gene under the control of a galactose inducible promoter, generating plasmid YCplac33-GHK. This plasmid was transformed into the strain W303. After inducted with galactose, the mating type of the cells carrying this plasmid was switched, which was verified by diagnostic PCR. Cells with appropriate mating type were selected and mated, producing polyploid yeast strains HLH33 and HLH34. At the end of high gravity fermentation, ethanol production of HLH33 and HLH34 improved by 1.29% amd 3.57%, compared to the reference strain W303. The results indicated that strains with increased ploidy produced more ethanol and exhibited enhanced ethanol and osmotic stress tolerance.Treatment of the polyploid strain HLH34 with methyl benzimidazole-2-yl-carba-mate (MBC) induced chromosome loss, resulting in the isolation of an aneuploid strain HLH34-M. The remaining sugar at the end of high gravity fermentation, decreased 58.70%, while the ethanol yield increased 5.33%, compared to the reference strain W303. Moreover, ethanol and osmotic stress tolerance of HLH34-M was significantly improved. These results demonstrated that manipulating yeast cell ploidy could improve its fermentation performance significantly.Using standard recombinant DNA technology, the SPT15 and SPT3 genes, encoding two general transcription factors, were each cloned into the YEplac195 vector, generating plasmid YEplac195-SPT15 and YEplac195-SPT3, respectively. A mutant allele of SPT15, spt15(Phe177Ser,Tyr195His,Lys218Arg) , was created by PCR mediated site-directed mutagenesis and coloned into the same vector, creating plasmid YEplac195-spt15. Two additional plasmids, YEplac195-SPT15-SPT3 and YEplac195-spt15-SPT3 were also constructed. In all plasmids constructed in this work, the SPT15/spt15 gene was always under the control of the strong TEF1 promoter, while the SPT3 gene was under the control of the strong PGK1 promoter. The plasmids were transformed into strain W303, then the fermentation performance, ethanol and osmotic stress tolerance tolerance of the tranformants were investigated. Overexpression of SPT15 and spt15 resulted in 1.14% and 0.23% increase in ethanol yield, respectively compared to the control strain W303; Cooverexpression of spt15 and SPT3 enhanced the strain's ethanol yield 2.05%; On the other hand, cooverexpression of spt15 and SPT3 enhanced the strain's ethanol yield 1.70%; Overexpression of SPT3 enhanced the strain's ethanol yield 1.25%, and the strain's ethanol and osmotic stress tolerance was better than other strain's. These results indicated that, under our experimental condition, modulation of the expression level of the SPT15 and SPT3 gene could to some extent improve yeast strain's fermentation performance.Diploid W303 strain were treated with Ethyl methane sulphonate (EMS) of experimentally determined optimal dose,generating a population of yeast cells with high level genetic variation; Genome shuffling was carried out within the mutagenised cell population via sexual recombination, which resulted in the isolation of the strain HLHS3-7. The results showed that, for strain HLHS3-7, the ethanol yield increased by 8.88% and the cycle of fermentation shortened 10 h, while the remaining sugar decreased by 64.37% at end of high gravity fermentation and ethanol and osmotic stress tolerance was significantly improved, compared to that of the control strain W303. These results demonstrated that the strategy employed in this work is valuable for creating yeast strains with desired multiplex traits.