| lignocellulose provides the raw material to produce second-generation fuel ethanol,however,during pretreatment,some of inhibitors are generated,which hamper growth and fermentation of microorganism.This has been one of the bottleneck problems in the industrialization.Among these inhibitors,Phenolics are the most toxic and complex types.Vanillin is an esstial phenolic compound.The study on the resistance of Saccharomyces cerevisiae to vanillin have important theoretical significance and value for construct more stress-tolerant stain.In the previous study,we found that the deletion of multi-drug resistance transcription factor gene YRR1 can significantly improve the vanillin tolerance of S.cerevisiae by decreasing vanillin's inhibition on cell translational process.Based on this,this paper performed research on the molecular mechanism of the transcription factor Yrr1 p to regulate the tolerance of vanillin in S.cerevisiae.The main research results are:(1)Yrr1p homologous transcription factor and its activated transcription factor show no obvious effect on vanillin resistance.Yrr1p has two highly homologous transcription factor Yrm1 p and Pdr8 p in S.cerevisiae,which regulates similar target genes.,Yrm1 p and Pdr8 p were deleted in BY4741 and BY4741(yrr1?),respectively.However,these two factors did not change vanillin resistance of S.cerevisiae.It was indicating that these two homologous transcription factors cannot compensate the function of Yrr1 p in improving the resistance to vanillin.Additionally,the deletion of transcription factors activating YRR1 can theoretically reduce the expression of Yrr1 p,but in the absence of Pdr1 p and Pdr3 p,the results showing no effect on vanillin tolerance as above,it was suggesting that there may be other activation factors to regulated YRR1.(2)Vanillin mainly inhibits the synthesis of proteins related to ribosome and amino acid metabolism,and stimulates protein synthesis in energy metabolism pathways.Proteome analysis of S.cerevisiae BY4741 with or without vanillin stress showed that vanillin disturbtation caused 218 differentially expressed proteins.Down-regulated proteins were mainly enriched in ribosomes and phenylalanine,alanine,and aspartic acid metabolism pathways.The levels of key enzyme proteins involved in oxidative phosphorylation,glycolysis and pentose phosphate metabolic pathways were significantly increased.(3)The deletion of YRR1 causes the increase of proteins related to cell translation,basal transcription and stress response.The proteome analysis results of strains BY4741(yrr1?)and BY4741 showed that the deletion of Yrr1 p caused a change in the expression of 121 proteins.Up-regulated proteins were mainly enriched in r RNA processing,ribosome synthesis,translation regulation,basic transcription and stress response.Based on the biological functions of related proteins,12 candidate genes were screened for overexpression in BY4741.The results showed that only the transcription cofactor Mbf1 p,proteasome assembly factor Tma17 p,and stress-stress transcription factor Haa1 p showed increased cell tolerance to vanillin,and the maximum specific growth rate was increased by 15%,33% and 18% compared with parental stain,respectively.However,the overexpression of genes involved in the translational process that we are concerned about had no effect on improving vanillin tolerance.In proteome of vanillin disturbtation and YRR1-deletion disturbtation,the heat shock protein Hsp12 p and Rtc3 p involved in RNA metabolism changed anti-directional change,and the results showed that these two factors had no effect on improving vanillin tolerance.(4)Increasing expression of SPE1 involved in polyamine synthesis and ribosomal small subunit RPS21 A levels can enhance vanillin tolerance.Proteomic analysis results of strains BY4741(yrr1?)and BY4741 under vanillin stress showed that the Yrr1 p deletion caused 83 differentially expressed proteins,and the up-regulated 25 proteins were mainly enriched in ATP-binding cassette transporter,amino acid metabolism,purine metabolism,cell translation and protein modification-related biological process,based on the function and fold change of the relevant protein,we selected ornithine decarboxylase Spe1 p,ribosomal small subunit Rps21 ap and group Protein modification enzyme Swd2 p as candidate genes.Overexpression of these three factors in the BY4741 strain,respectively,compared with the parent strain,under vanillin stress,overexpression of SPE1 and RPS21 A showed improved cell tolerance to vanillin,the maximum specific growth rate was increased by 50% and 11.4%.The results of this study provide a further understanding of the molecular mechanism of high vanillin resistance by Yrr1 p deletion,and the discovery of a series of elements to vanillin resistance are useful to construct vanillin-resistant strains. |
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