Enhancing The Robustness Of Engineered Saccharomyces Cerevisiae Through The Regulation Of Cellular Protein Homeostasis

The robustness of yeast can still maintain its physiological function stable when yeast sustains environmental stress.In the process of industrial fermentation,improving the robustness,especially the heat performance of fermentation strains can significantly reduce energy consumption,improve product yield and ultimately reduce the cost of fermentation.This paper is aimed at regulating the inner balance of protein,improving the robustness and activity of saccharomyces cerevisiaes by enhancing function of the inner balance network of protein,inquiring the corresponding mechanism of the level of transcription and protein and opening up a new approach to improve the robustness of saccharomyces cerevisiae.The main results were as follows:First,building strains which the ubiquitin proteasome had remoulded,including the mutant engineering strains S.c-P?-G of missing N terminal in ?3 subunit,the mutant engineering strains S.c-FP?-G which the genomes overexpression of missing N terminal in ?3 subunit,the engineered strains S.c-Pre9-P and S.c-P?-P of saccharomyces cerevisiae within and without N terminal in ?3 subunit which overexpressed by plasmid;then building the functional components of ovsrexpression of ubiquitin enzymes E2,E3 and its corresponding engineering bacteria S.c-U+R;To examine the heat resistant of the five engineering strains of saccharomyces cerevisiae by using three kinds of high temperature fermentation mode(constant temperature 40 ?,constant temperature 42 ? and gradient temperature(37?-43?).Compared respectively with the strains WT/V((including the empty plasmid strains)),S.c-Pre9-P,S.c-U+R,S.c-P?-G,S.c-FP?-G and strains without antibiotics stress,the OD value of the engineering strains S.c-P?-P which cultured 84 h in 40?increased by 120.0%,220.8%,6.01%,220.8%,33.9% and 35.7%.When cultured in 42?,the strain amount of other strains without obvious growth,even gradual decline except the strain amount of S.c-P?-P increased obviously in 24 h.When cultured in gradient temperature,the engineering strains S.c-P?-P still showed that the optimal growth advantage,and the engineering strains S.c-Pre9-P still showed the worst heat resistance.TTC and MDH enzyme activity results showed that S.c-P?-P with the highest cell vitality.The intravenous drip plate experiment results showed that,compared with WT/V,the abilities of DNA damage stress resistance,osmotic stress resistance,antioxidant stress resistance,thermal shock stress resistance,cell wall damage stress resistance,ethanol stress resistance of S.c-U+R and S.c-P?-P were improved significantly;compared with WT,the abilities of antioxidant stress resistance,thermal shock stress resistance,cell wall damage stress resistance were enhanced obviously.By comparing the transcriptional ability of chitin synthase of each strains found that over-expression ubiquitin enzymes E2,E3 and reconstructive proteasome(amputated N terminal in ?3 subunit)could obviously improve the transcriptional ability of chitin synthase,and then enhanced the abilities of thermal shock stress resistance,cell wall damage stress resistance;meanwhile,the sulfur flavin T dyeing experiment results showed that the ubiquitin proteasome with modification had stronger ability of protein degradation in cells.The bifunctional components RSP5 + UBC4 were characterized by transforming to the industrial fermentation bacterium angel yeast host showed that functional components had good universality,function stability and industrial application potential,could improve the cells' activity,heat resistance and cells' alcohol production,meanwile the functional components could effectively improve the ubiquitin level of cells.