Cloning And Functional Study Of Saccharomyces Cerevisiae Srp40³⁹ Gene

The toxic effects of isobutanol on cells limit the maximum yield of isobutanol in S.cerevisiae.Therefore,construction of engineered strains with higher tolerance toward isobutanol is of great significance for isobutanol production.In this work,mutant EMS39screened by our colleagues and wild-type W303-1A were used as the parental strains.The srp40³⁹,wsc2³⁹and spt6³⁹mutant genes were cloned from mutant EMS39,and the SRP40,WSC2,SPT6,AQY1,OLE1,CWP1 and CWP2 genes were cloned from wild-type W303-1A.We will explore effects of these genes on isobutanol tolerance of yeast cells.Firstly,we overexpressed these genes in wild-type W303-1A,and then tolerance of these strains was tested under the half lethal concentration of isobutanol of 1%（V/V）.Our results showed that single overexpression of AQY1,OLE1,CWP1 or wsc2³⁹genes had negative effects on isobutanol tolerance.Meanwhile,single overexpression of SRP40,SPT6or spt6³⁹genes had little effects on isobutanol tolerance.While single overexpression of srp40³⁹,WSC2 and CWP2 gene could improve isobutanol tolerance,and strain W303-1A-srp40³⁹overexpressing mutant gene srp40³⁹had the highest tolerance to isobutanol.In order to study mechanisms of mutant gene srp40³⁹affecting isobutanol tolerance,we carried out DNA sequencing of mutant gene srp40³⁹.DNA sequencing results showed that open reading frame of mutant gene srp40³⁹contained five mutation sites,including two synonymous mutations caused by base substitution mutations at 570 bp and711 bp,one base substitution mutation（T→C）at 442 bp resulted in amino acid substitution S148P,one base deletion at 1018 bp caused a frameshift mutation,and one base substitution mutation at 1050 bp was located in the frameshift mutation.Subsequently,the properties and structures of Srp40³⁹protein were analyzed and simulated,and we found that Srp40³⁹protein has a similar structure to pleural-1 of diatoms.Secondly,in order to further explore effects and mechanisms of mutant gene srp40³⁹on yeast cell’s tolerance,strain W303-1A-srp40³⁹was fermented in CM medium containing1%isobutanol,1.3%isobutanol,8%ethanol and 0.5%isoamyl alcohol,respectively.Cell densities(OD₆₀₀)and growth were measured,and specific growth rates（μ）from 2 h to 10 h were calculated.Furthermore,strain W303-1A-srp40³⁹was subjected to heat shock at 55℃for 4 minutes,and then dilution experiments were carried out to observe their growth on the plate.To further determine its heat resistance,strain W303-1A-srp40³⁹was fermented at38℃.In CM medium with 1%isobutanol,cell density of strain W303-1A-srp40³⁹was1.12-fold higher than that of strain W303-1A-SRP40 and 1.10-fold higher than that of control strain W303-1A-22 at 24 h.The specific growth rate of strain W303-1A-srp40³⁹was 1.1-fold higher than that of control strain and 1.07-fold higher than that of strain W303-1A-SRP40.In CM medium with 1.3%isobutanol,the cell density of strain W303-1A-srp40³⁹was 1.06-fold higher than that of strain W303-1A-SRP40 and 1.10-fold higher than that of control strain W303-1A-22.The specific growth rate of strain W303-1A-srp40³⁹was 1.1-fold higher than that of control strain and strain W303-1A-SRP40.In CM medium with 8%ethanol and 0.5%isoamyl alcohol,the cell density of strain W303-1A-srp40³⁹was 1.12-fold higher and 1.01-fold higher than that of strain W303-1A-SRP40,1.17-fold and 1.07-fold higher than that of strain W303-1A-22 at24 h,respectively.The specific growth rate of strain W303-1A-srp40³⁹was increased by1.37-fold and 1.07-fold than that of strain W303-1A-SRP40,and 1.31-fold and 1.09-fold higher than that of control strain W303-1A-22,respectively.Strain W303-1A-srp40³⁹still grew better than the other two strains after heat shock at 55℃.And when fermented at38°C,cell density of strain W303-1A-srp40³⁹was increased by 1.16-fold and 1.23-fold than that of strain W303-1A-SRP40 and W303-1A-22 at 48h,respectively.Specific growth rate of strain W303-1A-srp40³⁹was 1.07-fold higher than that of control strain W303-1A-22,and there was no significant difference with strain W303-1A-SRP40.Transcriptome analysis revealed that the reduction of tryptophan anabolism and respiratory activity might be one of the main factors responsible for the higher isobutanol tolerance of strain W303-1A-srp40³⁹.Finally,the mutant gene srp40³⁹was overexpressed in the strain with optimized isobutanol and isoamyl alcohol metabolic pathway to explore the effect of the mutant gene srp40³⁹on the yield of isobutanol and isoamyl alcohol.Our results showed that overexpression of the mutant gene srp40³⁹could increase isobutanol production,but had little effects on the ethanol and isoamyl alcohol production.The highest isobutanol yield of strain W303-1A-V2-V3-ARO10-srp40³⁹was 0.38 g/L,which increased 50%than that of control strain W303-1A-V2-V3-ARO10-22 and increased 42%than that of strain W303-1A-V2-V3-ARO10-SRP40.Through transcriptome analysis,we found that the increase of isobutanol production was mainly related to the reduction of tryptophan catabolism,phenylalanine catabolism and tyrosine catabolism in the isobutanol metabolic pathway.