Research On Metabolic Engineering Of Ergosterol Synthesis In Saccharomyces Cerevisiae

Ergosterol is an important component of fungal cell membrane and an important precursor of steroid drugs such as vitamin D,and its market demand is large and in short supply.At present,ergosterol is mainly extracted from yeast and penicillium,which have problems,such as low content,high production cost,insufficient production capacity and so on.Some researchers have modified Saccharomyces cerevisiae by overexpression of 1～2genes of ergosterol synthesis pathway or physical and chemical mutagenesis,and there is a lack of systematic research on the whole synthesis pathway.The metabolic engineering of ergosterol synthesis in Saccharomyces cerevisiae is expected to greatly improve the yield of ergosterol.Focusing on the synthesis and transport pathway of ergosterol in Saccharomyces cerevisiae,the candidate key enzymes to promote ergosterol synthesis are analyzed and screened systematically,and the modular metabolic engineering is implemented through promoter replacement and lipid droplet modification,and combined with the optimization of culture conditions to promote the synthesis of ergosterol in Saccharomyces cerevisiae,which provides a metabolic engineering strategy for the production of ergosterol in Saccharomyces cerevisiae.The main results are as follows:（1）The candidate key enzymes of ergosterol synthesis and transport in Saccharomyces cerevisiae are screened and cloned.Based on the systematic analysis of the candidate key enzymes of ergosterol synthesis and transport in Saccharomyces cerevisiae,the key step enzymes of ergosterol synthesis,including HMG-Co A reductase,squalene synthase,squalene epoxidase and sterol reductase,are virtually screened by molecular docking.The results show HMG-Co A reductase（HMG1）,squalene synthase（ERG9）,squalene epoxidase（ERG1）and sterol reductase（ERG4）from Saccharomyces cerevisiae,which have strong affinity with corresponding substrates among 16～20 enzymes randomly selecting from different biological sources in Uni Prot database.The physical and chemical properties,transmembrane domains and binding force with substrates of candidate key enzymes are analyzed.ERG9,ERG4 and ERG1 from Saccharomyces cerevisiae can be directly used for metabolic engineering.HMG1 needs to truncate the ERAD-targeted domain to eliminate product feedback regulation and improve enzyme activity.（2）Metabolic engineering of ergosterol transport storage module and upstream and downstream modules of ergosterol synthesis pathway with ERG9 as key position in Saccharomyces cerevisiae promotes ergosterol synthesis.Firstly,the ergosterol transport storage module is optimized by replacing the promoter of ARE2 with P_HXT7 and knocking out FLD1 gene,so that over synthesized ergosterol can be transported and stored in lipid droplets.Secondly,the downstream module of ergosterol synthesis pathway is optimized by replacing the promoters of ERG1 and ERG4 with P_TEF1,so that enhances downstream metabolic flux and avoids accumulation of intermediates,which improves ergosterol synthesis,and increases the content by 69.9%and the yield by 66.6%,and there is no significant difference in the biomass of yeast.Finally,the upstream module of ergosterol synthesis pathway is optimized by integrating P_TEF1-t HMG1-T_ADH1,so that breaks through the speed limiting step,enrichs the supply of precursors,which promots the growth of yeast and increases biomass by 69.6%,ergosterol content by 66.9%and yield by 183.9%.（3）The fermentation performance of yeast engineering strain WYP9 is evaluated based on the optimization of culture conditions.The results of single factor experiment shows that sucrose and maltose are the appropriate carbon source,yeast extract is the appropriate nitrogen source,and the initial p H of medium affects the fermentation performance of yeast engineering strain WYP9.The optimized culture conditions are obtained by orthogonal test:42.75 g/L sucrose,45.00 g/L maltose,10.00 g/L yeast extract,p H=5.80.Compared with Saccharomyces cerevisiae before modification,the biomass of yeast engineering strain WYP9 is increased by 212.1%,the content of ergosterol is increased by 83.8%,and yield is increased by 473.5%.In this study,a metabolic engineering strategy for ergosterol production in Saccharomyces cerevisiae is proposed:replacing the promoter of ARE2 with P_HXT7,knocking out FLD1 gene,replacing the promoters of ERG1 and ERG4 with P_TEF1,integrating P_TEF1-t HMG1-T_ADH1 into YPRCdelta15 site to modify the pathway of ergosterol synthesis and transport in Saccharomyces cerevisiae,which greatly improves ergosterol production.