Stress Response Of Candida Glycerinogenes Under High Temperature And High-glucose Fermentation

High temperature and high-glucose fermentation can reduce the using of cooling water,save costs,minimize microbial contamination,and simplify production process.Candida glycerinogenes is an excellent stress-tolerant strain,compared with the model Saccharomyces cerevisiae,C.glycerinogenes shows obvious growth advantages and better stress-tolerance under combined heat and high-glucose stress.This strain was used as a research object to reveal its tolerance mechanism to the combined stress,which aims to provide new genes and basis for the improvement of the combined stress tolerance of the industrial microorganism.The research of this paper is as follows:The changes in genes expression levels of C.glycerinogenes and S.cerevisiae under different stress conditions（heat,high-glucose and the combined heat and high-glucose）were investigated by qRT-PCR.There were significant differences in response gene profiles between C.glycerinogenes and S.cerevisiae under two separate single-stresses.Under the combined stresse,most of the single-stress（heat or high-glucose）responsive genes expressions had no obvious changes in S.cerevisiae,while,C.glycerinogenes still up-regulated the single-stress responsive genes,suggesting that C.glycerinogenes have more extensive stress-responsive genes.Furthermore,using antisense RNA interference technology to down-regulate the potential key responsive genes,revealed that compatible solutes accumulation,glucose metabolism,membrane lipid metabolism related genes Dip5,Gpd1,Pfk1,Hxt4,Hxt6,and Ino4 are important for cell tolerance to the combined stresses.Most of the ribosomal function related genes,such as Rpa190,Nop53,Rex4,Mrt4,Nug1,and Nsr1,play a critical role in cell tolerance.These results indicated that the more systematic response mechanisms and the activated expression of ribosomal function related genes might be key and prerequisite factors for the excellent tolerance to the combined heat and high-glucose stress of C.glycerinogenes.To investigate the effects of the different combined stress responsive genes on yeast tolerance,the genes were expressed in S.cerevisiae.The results showed that the final biomass of strains S.cerevisiae/CgIno4,S.cerevisiae/CgPot1 and S.cerevisiae/CgYad9 was about 9%,11%,and 14%higher than the control strain at 40^oC with high-glucose stress condition,respectively.When the temperature rose to 42^oC,the biomass of that three strains was about18%,12%,and 11%higher than the control strain,respectively.Compared with the control strain,the biomass of the recombinant strains S.cerevisiae/CgIno4 and S.cerevisiae/CgYad9increased more than 10%under ethanol and acetic acid stress except for S.cerevisiae/CgPot1.Meanwhile,the lactic acid tolerance of these three strains also increased to varying degrees.These results indicated that the overexpression of the genes CgIno4,CgPot1 or CgYad9 can inprove the tolerance of S.cerevisiae to multiple stresses of the combined heat and high-glucose,ethanol,acetic acid or lactic acid simultaneously.The discovery of these multiple stress-tolerance related genes has enriched stress-tolerance related genetic resources.The ethanol production of recombinant S.cerevisiae under heat and high glucose concentration was further investigated.The final ethanol titer of strains S.cerevisiae/CgIno4,S.cerevisiae/CgPot1 and S.cerevisiae/CgYad9 was 40.0 g?L^-1,40.0 g?L^-1 and 41.0 g?L^-1,respectively,increased by 14%,14%and 17%than the control strain,respectively.Furthermore,the overexpression of the genes CgIno4 and CgYad9 increased the ethanol titer of the recombinant S.cerevisiae fermented in the presence of acetic acid,up to 37.8 g?L^-1 and38.9 g?L^-1,respectively,increased by 12%and 15%than the control strain,respectively.These results showed that the genes CgIno4,CgPot1,and CgYad9 can improve the ethanol fermentation performance of the recombinant strain under different stress conditions.