Mechanisms On Improved Stress Tolerance By Overexpression Of Zinc Responsive Genes In Saccharomyces Cerevisia

Saccharomyces cerevisiae has been widely studied for bioethanol production utilizing lignocellulosic biomass.However,growth inhibition and low ferementation efficiency due to the high concentration of inhibitors,derived from process of lignocellulosic biomass pretreatment,are great challenges.Acetic acid is one of the most common inhibitors in hydrolysate,which severely inhibits growth of S.cerevisiae,and subsequently leads to low cell viability as well as low ethanol yield and productivity.Besides,yeast cells also suffer from other environmental stresses during cellulosic ethanol fermentation,such as high temperature,low p H,osmotic pressure,oxidative stress,and other inhibitors in the hydrolysate.Therefore,it is of great significance to improve the tolerance of the engineered strains towards acetic acid as well as other stresses.Previous studies in our group showed that acetic acid stress tolerance of S.cerevisiae can be effectively improved by appropriate zinc sulfate supplement.Transcriptomic analysis showed that the transcription of arginine hydrolase gene CAR1 and adenine encoding gene ADE17 was significantly up-regulated,which are key genes involved in amino acid metabolism and purine biosynthetic pathway,respectively.Subsequently,overexpression of these two genes led to an improved tolerance of S.cerevisiae.However,the molecular mechanism underlying tolerance improvement by zinc regulation under acetic acid stress remains unclear.In this thesis,to further construct robust yeast strains and explore the regulatory mechanism of zinc-response related genes under acetic acid stress,effects of overexpression of CAR1 and ADE17 on stress tolerance,as well as involvement of the zinc responsive transcription factor Zap1 p in the regulation of these two genes were investigated.In previous studies,the overexpression strains4126-CAR1 and BADE17 using the host strains of S.cerevisiae Sc4126 and BY4741,respectively,were constructed.In this study,it was found that CAR1 overexpressing strain contained higher ATP content and cytoplasma membrane integrity under acetic acid stress,suggesting that overexpression of CAR1 might alleviate intracellular acidification stress,relieving membrane injury and oxidative damage.Although there is no Zap1 p binding site in the CAR1 promoter region,the potential binding site of Com2 p,which is putatively regulated by Zap1 p,was identified.Consequently,involvement of transcription factor Com2 p in acetic acid tolerance was studied.The results demonstrated that disruption of COM2 or the Com2 p binding site in the CAR1 promoter region decreased the tolerance to acetic acid stress when compared to the parental strain BY4741,and more severe effect was observed when the COM2 binding site was modified.We proposed that Zap1 p may regulate CAR1 transcription by modulating Com2 p under acetic acid stress.In addition,the effects of ADE17 overexpression on environmental stress tolerance were studied.Compared with many other key gene overexpression strains,BADE17 showed the strongest resistance to diverse stresses.Laboratory adaptive evolution in the presence of multiplex inhibitors was performed,and the lag phase of the selected adapted strains BADE17-2 and BADE17-4 strains was shortened by 23 h compared to that of the control strain.Subsequently,it was found that comparing with the non-stressed control,ZAP1 transcription was down-regulated,whereas ADE17 transcription was up-regulated,indicating a possible negative effect of Zap1 p on ADE17 transcription under acetic acid stress.Intriguingly,transcription of ADE17 was inhibited significantly when ZAP1 was knocked out.It was deduced that down-regulation of ZAP1 could activate ADE17 expression,but knockout of ZAP1 might trigger the negative regulation of ADE17 by other regulators.Moreover,it was showed that transcription of ADE17 in the mutant with Zap1 p binding site mutation within the ADE17 promoter was increased under either the non-stressed condition or acetic acid stress condition.Thus,the negative effect of Zap1 p on ADE17 was further validated.In conclusion,our results provide insights in regulation of zinc responsive genes related to stress tolerance by the zinc-responsive transcription regulator Zap1 p,and will benefit the construction of robust yeast strains with efficient ethanol fermentation using lignocellulosic biomass.