The Study Of Adh2and Ald6Gene Knockout Of Saccharomyces Cerevislae Mf1015Strains

Ethanol is an important foundation raw material for industry which is widely used in food, chemical, pharmaceutical, military and other industries. The reconstruction and breeding of high-yield Saccharomyces cerevisiae strain through genetic engineering approaches, can not only greatly elevate the efficiency of ethanol fermentation, reduce the energy consumption and raw material consumption, but also can significantly reduce the labor costs, leading to a significant economic benefits. Furthermore, since the ethanol fermentation process of S. cerevisiae is a basic life phenomenon, the gene engineering was be used to enhance the strain competence of ethanol fermentation,this technical method can explore the basic regulation of life science,at the same time has an extreme significance for application and theoretical research. Therefore, this research field has attracted the attention by microbiology, molecular biology research as well as ethanol fermentation industry.The alcohol dehydrogenase and aldehyde dehydrogenase enzymes of S.cerevisiae were encoded by adh2and ald6genes, respectively. Both of which are involved in the metabolism of ethanol fermentation of S.cerevisiae. During the fermentation process, alcohol dehydrogenase catalyzes the conversion of ethanol to acetaldehyde and aldehyde dehydrogenase regulates the oxidation process of acetaldehyde to acetic acid. In the present study, the adh2and ald6genes were successfully knocked-out in the original S. cerevisiae strain MF1015through the strategy of metabolic engineering. First the type a and type a stable haploid cell lines of S. cerevisiae strain MF1015were establishment, then the adh.2and ald6gene knocked-out strains of each haploid were successfully obtained using the strategy of homologous recombination. Finally, the mutant strain MF1015-Δadh2-Δald6was acquired by the mating of adh2and ald6double gene knockout type a and type a haploid strains.Research progress is as follows:The type a and type a haploid strains of S. cerevisiae strain MF1015were successfully constructed. The adh2and ald6gene knockout components were constructed and transformed into the type a and type a haploid. After screening of positive clones, the pSH65expression plasmid was transformed into the each haploid and the selection marker gene was deleted by Cre recombinase in the induction of galactose and the pSH65expression plasmid was missing by serially passage. The mutant strain MF1015-Δadh2-Δald6was acquired by mating of adh2and ald6double gene knockout type a and type a haploid strains. Sugar fermentation experiments showed that the ethanol production of the mutant strain increased8.59%and acetic acid production decreased9.75%compared with the original MF1015strain. The molasses supplementary fermentation experiments showed that, compared to the original MF1015strain, the acetic acid production of the mutant strain decreased about7.47%and the ethanol production and the sugar utilization rate increased5.39%and3.56%, respectively. Serially passaging experiments showed that the mutant strain MF1015-Δadh2-Δald6also keep a good genetic stability. All of these results indicated that the mutant strain MF1015-Δadh2-Δald6was a promising ethanol fermentational strain for the future industrial application.