Regulation Of Ergosterol Produced By Saccharomyces Cerevisiae And Activity Of Sterol Derivatives

Ergo sterol is the main sterol in fungi and is used as an important pharmaceutical chemical raw material for the conversion of vitamin D2 and the production of sterols.The biosynthesis of ergosterol in Saccharomyces cerevisiae is a complex process whose transcriptional regulation mechanism remains unclear.Therefore,it is important to elucidate the effect of transcriptional regulation on ergosterol synthesis.In this study,Saccharomyces cerevisiae was used as the research object.By constructing the transcription factor Ecm22 engineering bacteria,the effects of Ecm22 overexpression and truncated Ecm22 on the growth of Saccharomyces cerevisiae and ergosterol synthesis were investigated.The antibacterial activity of ergosterol derivatives was studied.1.The transcriptome analysis of Ecm22 overexpressing genetically engineered bacteria was used to determine the effect of transcription factor Ecm22 on the expression of ergosterol synthesis gene ERG.The results showed that overexpression of Ecm22 resulted in up-regulation of the majority of ERG gene expression levels in the ergosterol synthesis pathway,except for ERG4,ERG9,and ERG28.2.The effects of Ecm22 overexpression and truncated Ecm22 on the growth of Saccharomyces cerevisiae and ergosterol production were investigated.The results of the agar invasive test showed that the invasive growth of Ecm22 overexpressing engineered bacteria was more effective than the control group.The fluconazole tolerance test showed that the strai'n overexpressing Ecm22 could grow at 16 ?g/mL(fluconazole),while the control group could not grow.The results of shake flask fermentation showed that the output of ergosterol of Ecm22 overexpressing engineering bacteria reached 17.35 mg/g,which was 32%higher than that of the control group.In the truncated Ecm22,only the 1440 bp DNA fragment had the same effect on the growth and ergosterol content of Saccharomyces cerevisiae as the full-length gene.The other fragments had weaker effects on the growth of Saccharomyces cerevisiae and ergosterol than the full-length gene.3.Optimized the culture conditions of ergosterol produced by Saccharomyces cerevisiae.The optimal culture conditions were determined by orthogonal test:peptone 20 g/L,yeast powder 10 g/L,glucose 80 g/L,(NH4)2S04 6 g/L,MgS04 1.0 g/L,KH2P04 1.5 g/L,FeS04 0.1 g/L.In the 5 L fermentor,the ergosterol production of the genetically engineered bacteria reached a maximum of 32 mg/g(DCW),an increase of about 20%compared to the control.4.The research confirmed the antibacterial activity of ergosterol derivatives.The sterol derivative(ED)is formed by the reaction of ergosterol with an alcohol The TLC results show that the four derivatives ED1,ED2,ED3,and ED4 are complex mixtures.They have strong inhi-bitory effects on Pseudomonas aeruginosa,Staphylococcus aureus and Bacillus subtilis.In particular,ED1 has the most significant inhibitory effect on Pseudomonas aeruginosa,with a minimum inhibitory concentration of 8?g/mL.However,ED has no inhibitory effect on Escherichia coli,yeast,Rhizopus,and Aspergillus niger.