Improving Thermotolerance Of Saccharomyces Cerevisiae Using Heat Shock Protein And Superioxide Dismutase Combination Gene Device

Saccharomyces cerevisiae could be seriously restrained with heat stress inevitably duringthe fermentation process. The reactive oxygen species (ROS) could increase with the hightemperature, wich excerabate the above phenomenon. In order to improve thermotoleranceof yeast globally, heat stress and heat induced oxidative stress should be defensed. Based onthe thermotolerant mechanisms of Thermoanaerobacter tengcongensis and Thermusthermophiles, the engineered S. cerevisiae containing heat shock protein and superioxidedismutase (SOD) combinating gene device was designed and constructed on the molecularlevel using the synthetic biology method, and successfully applied in the production ofethanol. The main topics and results of this paper were listed as follows:Heat-resistant parts and anti-oxidative parts were mined in T. tengcongensis,T.thermophiles and S. cerevisiae, which focused on heat shock proteins and SOD.16HSPgene devices and2SOD gene devices were assembled with the the strong constitutivepromoter (FBA1p) from S. cerevisiae as the regulatory part. And these gene devices weretransformed into yeast.The excellent HSP gene device, FBA1p-shsp-HB8was screened trough gradual andconstant high temperature experiment. Meanwhile, the excellent SOD gene device,FBA1p-sod-MB4was screened through defencing oxidative experiment. Then theengineered yeast containing HSP and SOD combinationgene device, SOD-MB4-sHSP-HB8was constructed by combining the above two gene devices. Meanwhile, the engineerdstrains (GroES-HB8-sHSP-HB8， SOD-MB4-SOD-HB8and SOD-MB4-GroS2-MB4)containing different combinationgene device were also constructed.All the engineered strains grew better than the control cultured at42°C. There is nodifference between the OD660of GroES-HB8-sHSP-HB8, SOD-MB4-SOD-HB8,SOD-MB4-GroS2-MB4and the engineered strains containing single gene device. However,SOD-MB4-sHSP-HB8showed the best thermotolerance owing to the HSP gene device andSOD gene device synergistic defensing both heat stress and heat induced stress.The function of HSP gene device, SOD gene device and HSP and SOD combinationgenedevice were verified through ethanol high temperature fermentation. All the engineeredstrains showed higher ethanol yield than the control cultured at30°C and40°C. Theincreasing ethanol yield level of SOD-MB4-sHSP-HB8was highest as time went by. And itshowed21%ethanol yield increase than control and the highest productivity at48h. The HSP gene devices, SOD gene device and HSP and SOD combinationgene devicewere transformed into indutrial S. cerevisiea. The ethanol yield of all the engineered strainswas higher than the control after35°C fermentation especially the engineered strain withcombinationgene device showed the excellent ethanol producing ability. These resultsprovide a new idea for the production of bio-based products by high temperaturefermentation.