Effect Of Overexpression Of TOS3?SAK1?ELM1 On Multiple Stress Tolerance Of Saccharomyces Cerevisiae

Saccharomyces cerevisiae is one of the important industrial microorganisms,which has the characteristics of fast fermentation speed and high ethanol production.However,Saccharomyces cerevisiae cells are inevitably subjected to multiple environmental stresses during the alcohol fermentation process,such as high sugar stress caused by high concentration of substrates in the early fermentation process,high temperature stress caused by slow heat dissipation of the substrate during the fermentation process,and ethanol accumulation stress in the late fermentation period.This study took Saccharomyces cerevisiae AEY15 as the research object,and explored the effects of overexpression and delete of Tos3,Sak1,Elm1 protein kinase encoding genes TOS3,SAK1,ELM1 on stress tolerance of Saccharomyces cerevisiae.(1)Overexpression and delete of TOS3,SAK1,ELM1 genes,respectively,and the growth performance of the mutant strains and the survival rate under ethanol,high temperature,and high sugar stress were determined.It was found that the survival rate of the mutant strains overexpressing SAK1 under high temperature stress,the survival rate of the mutant strain overexpressing ELM1 was increased by 12.6% compared with the parent strain under high sugar stress.Under high sugar and high temperature stress,delete and overexpression of TOS3 had no effect on the survival rate of the strain.Under ethanol stress,whether delete or overexpression of the three genes can improve the survival rate of the strain,indicating that the three protein kinases have different regulatory mechanisms in the stress response of Saccharomyces cerevisiae.Sak1 and Elm1 are respectively under high temperature and high sugar stress.The balance between the positive regulators,endogenous Tos3,Sak1 and Elm1 is necessary to regulate the response to ethanol stress.(2)Preliminary study on the mechanism of Sak1 regulating the response of Saccharomyces cerevisiae to high temperature stress.It was found that the overexpression of SAK1 mutant strains increased the intracellular trehalose content by 20.3% and the ergosterol content by 1.3 times,as well as trehalose and ergosterol.The expression levels of key synthetic genes were up-regulated to varying degrees.Trehalose and ergosterol are important biological protective agents for cells,and they participate in a variety of regulatory mechanisms to improve cell membrane homeostasis.Sak1 protein kinase may accumulate protective molecules to respond to high temperature stress by regulating the metabolic pathways of trehalose and ergosterol.(3)Preliminary research on the mechanism of Elm1 regulating the high sugar stress response of Saccharomyces cerevisiae.It was found that the overexpression ELM1 mutant strain increased the intracellular trehalose content by 10.1%,the ergosterol content by 80%,and the oleic acid(C18:1)compared with the parent strain.The content has increased by 4%,and the expression levels of key genes in the synthesis of trehalose,ergosterol,and unsaturated fatty acids have been increased to varying degrees.The increase in oleic acid content strengthens the cell membrane permeability barrier and improves cell tolerance.After the high gravity fermentation,the residual sugar content of the fermentation substrate was reduced by 10.7%,and the alcohol production was increased by 6.4%.Elm1 protein kinase may respond to high glucose osmotic stress by regulating the metabolic pathways of various protective factors in Saccharomyces cerevisiae.This study systematically analyzed the effects of overexpression and knockout of three upstream global regulatory proteins Tos3,Sak1 and Elm1 encoding genes on ethanol,high temperature,and high sugar tolerance of Saccharomyces cerevisiae for the first time.The research results can not only improve the regulation mechanism of Saccharomyces cerevisiae tolerance,but also the breeding of new resistant yeast strains lays a theoretical foundation,and can also improve the global regulatory mechanism of yeast Tos3,Sak1 and Elm1,and provide a reference for the study of the role of protein kinases in other life activities of Saccharomyces cerevisiae.