| Bioconversion of lignocellulosic materials,which are abundant and renewable,for production of biofuel and other valuable products has been the subject of intense study.The pretreatment is an unavoidable process to break the crosslinking of cellulose,hemicelluloses and lignin in lignocellulosic biomass,and then facilitate enzymatic hydrolysis of cellulose and release monosaccharides.However,a range of chemicals blocking cell growth and fermentation were generated.These compounds included weak acids,furans and phenolics.Among them,phenolic compounds are more toxic than others and their toxicity varies depending on the functional groups and molecular weight.For instance,vanillin,a low-molecular-mass guaiacyl phenol,inhibits cell viability at very low concentration.Saccharomyces cerevisiae is a competitive microorganism for biofuel production due to better fermentative performance,high tolerance to ethanol and easy to be genetic manipulated.In our previous work,ethyl methanesulfonate(EMS)mutagenesis and adaptive evolution in hydrolysate were performed on an S.cerevisiae industry diploid strain NAN-27 and we obtained a series of strains that resistant to hydrolysate.Among them,a strain named EMV-8 exhibited significantly higher vanillin tolerance than other strains and their parent strain NAN-27.The genome sequence of EMV-8 and NAN-27 were performed.Over 450 non-synonymous SNPs in CDS(coding sequence)—involved in 275 genes and 44 InDels were detected.In present work,the genes of oxidoreductase,oxidative stress and DNA repair response with SNPs were select as studying objects.The genes relevant to vanillin tolerance were identified and the related molecular mechanisms were studied.(1)Screening the candidate genes related to vanillin resistanceThe PCR and Sanger sequence were performed on the 19 genes we selected,and SNPs in 15 genes were confirmed.Then these genes were respectively deleted in the lab strain BY4741 and the growth phenotypes of gene deletion strains were studied by spot dilution growth assay under vanillin stress or not.The results showed that four deletion mutants BY4741(gcy1?),BY4741(mbf1?),BY4741(msn1?)and BY4741(wtm2?)had similar growth characters with BY4741 on YPD,but grew slower on the plates containing vanillin,which indicated that they are more sensitive to vanillin than the wild type strain.Therefore,these genes may relate to vanillin tolerance.Then,the four genes and their mutant with SNPs were overexpressed in the corresponding deletion strains,and the lab strain BY4741 with the empty plasmid pJFE3 was used as the control.The resistance characteristics affected by the overexpression of the genes and mutations were evaluated in liquid SC-URA medium containing vanillin.The results showed that when 6 mmol L-1 vanillin was present,the maximum specific growth rates of strains overexpressing of GCY1WT and GCY1A181E respectively increased by 49%and 54%compared with the control BY4741(pJFE3).Moreover,the specific vanillin consumption rates were respectively 36%and 34%higher than the control.Moreover,the strain overexpressing of YPR1,which is the paralog of GCY1,also grew markedly faster than the control BY4741(pJFE3).The specific vanillin consumption rate was increased by 30%,because of the overexpession of YPR1.The maximum specific growth rates of strains overexpressing of MBF1WT and MBF1P148L were respectively 48%and 44%faster than the control strain BY4741(pJFE3).Meanwhile,there were negligible differences of the specific consumption rate for vanillin between strain overexpressing of MBF1 and not.The similar growth patterns of strains overexpressing of GCY1WT and GCY1AI81E,as well as strains overexpressing of MBF1WT and MBF1P148L suggested that these two SNPs do not affect the vanillin tolerance related the function of GCY1 and MBF1.Besides,the compensation of MSN1WT,MSN1S171P/P190Q,WTM2WT and WTM2Q8L did not moderate the sensitive phenotype of the strains BY4741(msn1?)and BY4741(wtm2?).(2)Study on the mechanism of GCY1 enhancing vanillin toleranceThe improved specific vanillin consumption rates of strains overexpression of GCY1WT and GCY1A18IE implied that Gcy1p may have vanillin reductase activity or supply the reduced coenzyme.First,the enzyme assay results showed that Gcylp has NADPH-dependent vanillin reductase activity.Second,overexpression of GCY1 did not affect[NAD(P)H]/[NAD(P)+].These result indicated that Gcy1p enhancing vanillin tolerance of S.cerevisiae does not by increasing the supply of reduced coenzyme but by directly reducing vanillin to less toxic vanillyl alcohol.Moreover,Ypr1p,the paralog of Gcy1p,also has NADH-and NADPH-dependent vanillin reductase activity.Then,we mutated the tyrosine(Y)-56 to phenylalanine(F),which is the active site of Gcy1p.The result showed that the vanillin catalytic activity of mutant Gcy1pY56F was almost abolished,but the growth rates of mutant strains were still faster than that of the control BY4741(pJFE3).This suggested that Gcylp has other function to enhance vanillin tolerance except to be a vanillin reductase.It is reported that Gcy1p has mRNA binding activity and it can bind to the mRNAs of 44 genes.According to the reported mechanisms of vanillin tolerance,three types of the 44 genes were selected out,and their effect on vanillin tolerance was evaluated.Among them,overexpression of genes related to ribosomal protein and chromatin organization did not affect the vanillin tolerance.Meanwhile,Pex5p,a protein related to the organization of peroxisome and Trs85p,a protein involved in pexophagy and vesicle transport,were proved to be related to the vanillin resistance.These results indicated that Gcylp may affect S.cerevisiae vanillin tolerance by binding to the mRNAs of PEX5 and TRS85 and then affecting their expression.Fluorescence quantitative PCR showed that overexpression of GCY1 did not change the mRNA levels of PEX5 and TRS85.Studying on how Gcy1p affect the mRNAs of PEX5 and TRS85 is ongoing.(3)Study on the mechanism of MBF1 enhancing vanillin toleranceMbf1p is a transcriptional coactivator and it enables the interactions of TATA element-binding protein(TBP)with a regulatory factor to transcriptionally regulate the expression of the downstream genes.The aspartate-112 is necessary for the binding between Mbf1p and TBP.And it is reported that substitution of alanine(A)for aspartate-112(D)resulted in a weak binding to TBP and reducing the expression of downstream genes.Our results showed that when subjected to 6 mmol L-1 vanillin,the growth rates of the strains overexpression of MBF1D112A and MBF1P148L/D112A were similar to that of the strains overexpression of MBF1WT and MBF1P148L,which suggested that the effect of Mbf1p on vanillin tolerance is not cause by the binding to TBP.In conclusion,in this study,we found that overexpression of GCY1 and MBF1 enhanced the vanillin tolerance of S.cerevisiae.Gcy1p had vanillin reductase activity.Therefore,it can directly reduce vanillin to less toxic vanillyl alcohol.Furthermore,Gcy1p bound to the mRNAs of PEX5 and TRS85,which regulate the processes of peroxisome organization,and pexophagy/vesicle transport,therefore affect the vanillin resistance.Mbf1p also has positive effect on vanillin tolerance,but this dose not caused by the binding to TBP. |
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