Transcriptome Differential Analysis Of Saccharomyces Cerevisiae Under Isoamyl Alcohol Stress

Isoamyl alcohol is an unpleasant smelling colorless liquid higher alcohol,which is highly diluted to produce a fruity aroma and is one of the main byproducts of alcohol fermentation.Isoamyl alcohol is commonly used in the production of a variety of cleaning products and is a precursor to a variety of chemicals used in laboratories.In recent years,isoamyl alcohol as a new fuel with high energy density has attracted wide attention.Because isoamyl alcohol can be purified by microbial fermentation for industrial production,more and more researches have tried to improve the yield and purity of isoamyl alcohol by modifying microorganisms.S.cerevisiae can naturally produce isoamyl alcohol by degrading leucine through Ehrlich pathway,it is one of the main host strains used in the laboratory at present.In 2017,Jifeng Yuan et al.introduced an artificial protein scaffold to close proximity to dihydroxy acid dehydrase andα-IPM synthetase.Finally,the isoamyl alcohol yield was more than twice of the original,which was 522.76±38.88 mg/L,which was much higher than the yield when using Escherichia coli as host cells,but still far from meeting the demand of industrial production.We hypothesized that isoamyl alcohol,as a higher alcohol,would have an effect on microbial hosts such as E.coli or S.cerevisiae at certain concentrations.This may be the direct cause of affecting microbial isoamyl alcohol production.Firstly,through real-time detection of the growth of S.cerevisiae under different concentrations of isoamyl alcohol pressure,it was determined that isoamyl alcohol can produce toxicity to S.cerevisiae at low concentrations.Transcriptome analysis was performed on S.cerevisiae without isoamyl alcohol and with growth inhibition rates of about 3%（0.5 g/L isoamyl alcohol）and 25%（2.5 g/L isoamyl alcohol）.Each sample produced an average of 6.38 Gb of data.A total of 5,405 genes were detected,among which5,362 genes were identified.Under 0.5 g/L isoamyl alcohol stress,there were 3631differentially expressed genes between the sample and the control group,among which1813 up-regulated genes and 1818 down-regulated genes;Under 2.5 g/L isoamyl alcohol stress,there were 3543 differentially expressed genes between the sample and the control group,among which 1779 up-regulated genes and 1764 down-regulated genes.GO enrichment analysis was performed for up-regulated and down-regulated genes.Vacuole transport,sulfur metabolism and amino acid biosynthesis were the most up-regulated terms.The terms with more down-regulated gene enrichment had intrinsic components of membrane,nitrogen compound metabolism and transport,gene expression,metal ion transmembrane transport,mannose-biosynthesis process,fatty acid elongase activity,FAD transmembrane transport enzyme activity.Under isoamyl alcohol stress,S.cerevisiae may enter the nitrogen starvation state,induce the expression of related genes to competitively inhibit translation promoter,resulting in decreased translation efficiency,and then affect the expression of normal functional proteins of S.cerevisiae.At the same time,most of the genes related to cell wall were down-regulated under isoamyl alcohol stress,such as NCW2,CCW12,BGL2and SUN4,but some chitin related genes were up-regulated.It was speculated that isoamyl alcohol stimulated S.cerevisiae to produce a large number of pseudomycelia,resulting in changes in the proportion of cell wall components.Among the genes related to cell membrane,most genes related to cell membrane stability and mechanical strength were down-regulated,such as ELO1,ERG2 and OPI3.However,the expression of regulatory genes（RTG1,INO1）and some transporter-related genes（PMC1）on the membrane were up-regulated.Also up-regulated was the gene FAA1,which was utilized by S.cerevisiae by exogenous fatty acids activated by Faa1p.KEGG analysis revealed that the glycolysis pathway and ATP biosynthesis related genes were up-regulated during the citric acid cycle.It is speculated that more ATP is needed to provide energy for cell life activities under isoamyl alcohol stress.The pentose phosphate pathway is also upregulated in genes associated with NADPH biosynthesis.It is speculated that under isoamyl alcohol stress,more NADPH is needed to provide reducing agents for various synthesis reactions of cells and participate in various anabolic reactions,such as the synthesis of lipids,fatty acids and nucleotides to maintain cell life activities.The gene deletion strains of the selected key genes（BY family）were cultured under different isoamyl alcohol concentrations.In the medium without isoamyl alcohol,the growth of ccw12△,bgl2△and ncw2△was severely inhibited.elo1△,erg2△,faa1△and zwf1△grow more slowly than BY4741 without isoamyl alcohol,but they still grow normally.elo1△,erg2△,faa1△and zwf1△grow normally in the condition without isoamyl alcohol,although they grow slowly compared with BY4741.With the increase of isoamyl alcohol concentration,they showed stronger sensitivity than BY4741,and their growth was severely inhibited.The growth state of sun4△,opi3△,ftr1△and fet3△also decreased compared with BY4741.However,with the increase of isoamyl alcohol concentration,the sensitivity of the strain to isoamyl alcohol did not improve further.The down-regulation of cell wall related genes may be one of the reasons for the decreased life activity of S.cerevisiae under isoamyl alcohol pressure.At the same time,the increase of membrane permeability leads to the destruction of the balance of ions inside and outside the cell.Ca²⁺transporter genes related to ATP and NADPH synthesis were up-regulated under isoamyl alcohol stress,which helped S.cerevisiae resist isoamyl alcohol stress.This study was the first to investigate the toxicity of isoamyl alcohol on S.cerevisiae.Transcriptome analysis was used to identify the target locations and key potential genes for isoamyl alcohol to inhibit S.cerevisiae cells.Combined with the function of related genes,the toxicity mechanism of isoamyl alcohol was preliminarily discussed and speculated,which provided a theoretical basis for the construction of high isoamyl alcohol tolerance microorganisms in the future and promoted the progress of isoamyl alcohol biosynthesis technology.