Functional Research Of Transcription Factors HAA1 And ASG1 From Candida Glycerinogenes

Candida glycerinogenes,an osmotolerant yeast isolated from the nature,can tolerate excellent environmental stress,especially low pH.The use of lignocellulosic hydrolyzate to produce ethanol is important for alleviating the energy crisis of oil depletion,however,the high acid concentration of that has toxic effect on the growth of Saccharomyces cerevisiae.Introduction of acid-responsive transcription factors in S.cerevisiae is one of the methods for the production of ethanol from lignocellulose.In this study,we applied the acid-responsive transcription factors and their promoter from C.glycerinogenes to S.cerevisiae,aiming at improving the tolerance of S.cerevisiae.The transcription factor genes HAA1 and ASG1 were cloned from C.glycerinogenes.Sequences alignment and structural analysis were carried out.To confirm the acid-responsive function,the growth of mutants C.glycerinogenes haa1?,C.glycerinogenes asg1? and complementary strains C.glycerinogenes haa1?/CgHAA1,C.glycerinogenes asg1?/CgASG1 were observed.The results showed that HAA1 and ASG1 were able to responde to acidic environment.At the same time,CgHAA1 and CgASG1 were overexpressed in C.glycerinogenes,the biomass of C.glycerinogenes/CgHAA1 increased by 13.5% in 110 mmol·L^-1 acetic acid?pH 3.2?.qRT-PCR indicated that HAA1 had stronger ability to regulate its downstream genes,and the transcription levels of acid-related CgTPO2 and CgHRK1 were upregulated by 4.9 and 3.9 folds,respectively.For the purpose of examining the effect of different acid-responsive transcription factors on the acid tolerance of the model yeast,overexpressed CgHAA1,CgASG1,ScHAA1 and ScASG1 in S.cerevisiae.The results of tolerance test showed that overexpressing strains of CgASG1 and CgHAA1 had better tolerance to acetic acid.Especially in 90 mmol·L^-1 acetic acid,the biomass of S.cerevisiae/CgHAA1 and S.cerevisiae/CgASG1 was higher than that of S.cerevisiae/ScHAA1 and S.cerevisiae/ScASG1,44.3% and 18.9%,respectively.qRT-PCR showed that the transcription levels of recombinant S.cerevisiae/CgHAA1 and S.cerevisiae/CgASG1 were higher.Compared to S.cerevisiae/ScHAA1,TPO2,TPO3 and YRO2 of S.cerevisiae/CgHAA1 improved by 1.3,1.3 and 1.1 folds respectively;Compared with S.cerevisiae/ScASG1,TPO3 and YRO2 of S.cerevisiae/CgASG1 improved by 1.3 and 1.6 folds respectively.The above results indicated that the transcription factors HAA1 and ASG1 from C.glycerinogenes show stronger ability to regulate the downstream acid tolerance genes.The ethanol accumulation of recombinant C.glycerinogenes and S.cerevisiae was further investigated in acetic acid environment.The ethanol accumulation of C.glycerinogenes/CgHAA1 was 40.3 g·L^-1 which improved by 9.1% comparing with the wild type;the ethanol yield of recombinant S.cerevisiae/CgASG1 was 34.3 g·L^-1,increased by 11.1%.Comparing with wild type in acetic acid,antioxidase activity assay found,the CAT activity of C.glycerinogenes/CgHAA1 and C.glycerinogenes/CgASG1 was increased by 76.3% and 50.8%,respectively;the SOD activity of recombinant strain increased by 50.7% and 17.2% respectively.The results showed that C.glycerinogenes/CgHAA1 had higher antioxidant enzyme activity under acid stress.The research indicated that overexpression of acid-responsive transcription factors HAA1 and ASG1 could effectively improve the fermentation performance of the recombinant strain under acidic condition.