Effect Of Medidator Subunit Med15 On Yeast Stress Tolerance

Microbial fermentation could use renewable feedstock to produce the desirable chemicals.During the industrial bioprocess,industrial strain may encounter complex environmental stress,such as p H and temperature extremes,oxidative or osmotic changes,solvents,and toxic metabolites.Thus,stress tolerance is a critical physiological parameter for industrial strains.However,the achievement of single or multi-gene modifications is limited,due to the complexity and multi-genic control of stress response.To address this problem,Mediator engineering has been developed to global alter the transcriptional regulatory network in Candida glabrata and Saccharomyces cerevisiae to regulate the stress response systems,and we also investigated the mechanisms underlying the yeast response to multidimensional stress.The main results are as follows:1.To elucidate the physiological function of the Mediator tail subunit Med15B in the response to osmotic stress,we constructed a deletion strain,med15B?,and overexpression strain,HTU?/Cg MED15B.First,under 1.2 M Na Cl condition,med15B?and HTU?/Cg MED15B significantly decreased the osmotic stress tolerance and their cell viability were deceased by 24.6%and 38.4%,respectively,compared with those of the parent strain?HTU??.Second,we found that the Na⁺/K⁺-ATPase activity was decreased by 45.3%and 26.2%in med15B?and HTU?/Cg MED15B,respectively,compared with those of HTU?,which resulted in the intracellular Na⁺/K⁺ratio increased by 61.7%and 72.4%.Finally,by testing the membrane fatty acid composition,we found that the content unsaturated and long-chain fatty acids was decreased in med15B?and HTU?/Cg MED15B,led to membrane integrity decreased by 19.5%and-8.0%,and membrane fluidity decreased by 12.7%and 18.2%,respectively.2.To elucidate the physiological function of Med15B in the response to osmotic stress,we first determined the tolerance of HTU??med15B?and HTU?/Cg MED15B at p H 2.0.Compared to HTU?,med15B?significantly decreased low p H tolerance and the cell viability was decreased by 26.5%,whereas HTU?/Cg MED15B increased low p H tolerance and the cell viability was increased by 2.3-fold.Second,transcriptome sequencing indicated that expression of lipid metabolism-related genes was significantly downregulated in the med15B?strain,whereas key genes of ergosterol biosynthesis showed abnormal upregulation.Third,changes of lipid related genes caused the ratio of unsaturated to saturated fatty acids,and total phospholipids content to decrease by 27.4%and 37.6%,respectively.Cells failed to synthesize fecosterol and ergosterol,leading to a 60.3-fold increase in the content of zymosterol.In HTU?/Cg MED15B,the UFA/SFA ratio,total phospholipids content and ergosterol content were increased by 18.7%,143.5%and 94.5%,respectively.Fourth,membrane integrity,fluidity,and H⁺-ATPase activity also increased by 69.2%,11.6%and 21.8%,respectively,whereas those parameters were increased by increased by 30.2%,6.9%and 51.8%,respectively,in HTU?/Cg MED15B.Furthermore,in the absence of p H buffering,pyruvate production was decreased by 23.4%in med15B?,increased by 61.2%in HTU?/Cg MED15B.3.To increase multiple stress tolerance of S.cerevisiae,transcriptome sequencing and phenotype analysis indicated that membrane lipid and transporter related genes ARV1,GIT1and NFT1 are crtical for acetic acid,H₂O₂ and Na Cl stress response,respectively.Furthmore,the KIX domain of Mediator tail subunit Med15 was identified here response to all three stress consitions by transcriptome sequencing and then engineered by protein engineering.The final Med15 mutation,S.cerevisiae Med15^V76R/R84K,led to 28%,21.4%and 27.8%increase in the half-maximal inhibitory concentration(IC₅₀)value for acetic acid,H₂O_2,and Na Cl,compared with that of S.cerevisiae BY4742-13 with its native Med15,respectively.4.To elucidate the mechanisms Med15^V76R/R84K mediated response to acetic acid,H₂O_2,and osmotic stress,first,we found that S.cerevisiae Med15^V76R/R84K enhanced interaction between Med15 and transcription factor Hap5 to improve ARV1 expression level by 4.3-fold,led to the proportion of fecosterol and ergosterol increased by 24.1%and 64.4%,respectively,and membrane fluidity decreased by 59.1%compared to that of S.cerevisiae BY4742-13.To improve oxidative tolerance,S.cerevisiae Med15^V76R/R84Kenhanced interaction between Med15and transcription factor Mga2 to improve GIT1 expression by 3.6 fold,led to the proportion of glycophosphatidylinositol?PI?increased by 44.3%and membrane integrity increased by 59.2%,compared to that of S.cerevisiae BY4742-13.To increase osmotic tolerance,S.cerevisiae Med15^V76R/R84K enhanced interaction between Med15 and transcription factor Aft1 to improve NFT1 expression level by 5.1 fold,led to the membrane transpoter Aft1 activity increased by41.3%and Na⁺/K⁺ratio decreased by 17.8%,which reduced membrane permeability 28.7%.