Heterologous Biosynthesis And Optimization Of Valerenic Acid In Engineered Saccharomyces Cerevisiae

Valerenic acid is the most important one of valerian extract.It is a short-chain fatty acid both sesquiterpene derivative.Valerenic acid has a therapeutic effect on diseases such as insomnia and epilepsy.The focus of interest began to shift to the use of synthetic biology and genetic engineering technology to construct a strain of Saccharomyces cerevisiae that produces valerenic acid heterologously.First,import the valerena-4,7(11)-diene synthase gene(VDS)and another excavated valerena-4,7(11)-diene synthase gene Vo TPS which to compare with VDS.VDS was finally selected to obtain a valereradiene-producing strain.By regulating the endogenous MVA pathway of Saccharomyces cerevisiae,overexpressing ERG8,ERG12 and ERG19,and then introducing the VDS module to increase the production of valereradiene.Then study the fusion expression system of protein tags MBP and SPY with VDS and ERG20,4 copies of MBP-VDS-ERG20 can increase the yield of valeriandiene to 75.0 mg/L.Then,Vo CYP71AR3,Vo CYP71DAR1,Vo CYP71DAR2,Ls GAO2 and At CPR1 were imported into the strain of high-titer valeriandiene.The titer of valerenic acid was 2.8 mg/L.Three Linkers of different lengths were selected for the fusion expression of Ls GAO2 and At CPR1,and the results showed that the co-expression one was better.Considering the limit of P450,we tried homology modeling and molecular docking,and screened out a total of 12 sites for alanine scanning mutations,but no better results were found.Through fermentation optimization in shake flasks and supplementation of sugar and ethanol in a 5 L fermentor,the titer of valerenic acid was increased to 6.8 mg/L.This study realized the heterologous synthesis of valerenic acid in Saccharomyces cerevisiae.Through the regulation of endogenous MVA pathway and the protein tag(MBP),the production of valerena-4,7(11)-diene was increased.A strain producing valerenic acid was constructed by expressing P450 and CPR.Although P450 mutation did not achieve better results,it could lay the foundation for further protein modification.