Construction And Optimization Of Glycyrrhetinic Acid Synthesis Pathway In Saccharomyces Cerevisiae

At present,with the expansion of the application range of licorice,the contradiction between supply and demand is very prominent.To explain the molecular mechanism of glycyrrhetinic acid formation at the genetic level,large-scale production of glycyrrhetinic acid by synthetic biology provides a new strategy to solve this problem.Integrate known gene components,knock out competitive pathway genes and optimize yeast heterologous cytochrome P450 expression system,and use modular design to maximize yeast metabolism to glycyrrhetinic acid synthesis pathway,construct high-yield glycyrrhetinic acid yeast strain,and realize microbial fermentation Produces glycyrrhizic acid.The research results are as follows:First,a glycyrrhetinic acid synthesis-related gene was heterologously integrated into the chromosome of Saccharomyces cerevisiae to construct a glycyrrhetinic acid strain Y1-154-8.Then,a preliminary optimization of GA heterologous production was achieved using a combination of new enzyme identification,MVA upstream pathway optimization,and fusion protein construction.The first generation of glycyrrhetinic acid Saccharomyces cerevisiae cells was constructed.In order to down-regulate the endogenous sterol and isoprenoid metabolic shunts that compete for glycyrrhetinic acid synthesis,the CRISPR/Cas9 technology was applied to the MVA metabolic pathway engineering,and the triterpenoid competitive branch pathway gene 3-ketoreductase was successfully knocked out.ERG27)and GGPP synthase gene(BTS1).The superior chassis strain Y0-3 was provided for the late addition of 2,3-oxidized squalene to the metabolic flux of triterpenoids.By heterologous expression of licorice-derived β-aromatic alcohol synthase(β-AS),the synthetic pathway of β-aromatic alcohol in S.cerevisiae cell Y0-3 was successfully constructed,and β-aromatic alcohol was produced by using Saccharomyces cerevisiae..By further overexpressing the key enzyme gene of yeast MVA pathway,the yeast metabolic flux was promoted to the direction of β-aromatic alcohol synthesis,and finally the Y2-C2-4 engineering strain was successfully obtained,reaching 10.3 mg·L-1.Through the high-density fermentation strategy,the yield of β-aromatic alcohol can reach 157.4 mg·L-1.The construction of this strain provides a superior chassis strain for further research on the synthesis of glycyrrhetinic acid and optimization of cytochrome P450 system.The first cytochrome oxidase gene CYP88D6,which heterologously expressed the glycyrrhetinic acid synthesis pathway,was found to have a transformation rate of 89% compared with the Arabidopsis NADPH-cytochrome P450 reductase.The cytochrome P450 CYP88D6 oxidation system was modified to successfully convert β-aromatic alcohol to 11-oxo-β-aromatic alcohol.In this study,we first optimized the MVA upstream module factor and cytochrome b5,and successfully constructed the first generation of glycyrrhetinic acid cell culture cell Y7.Then according to the results of the first step,the glycyrrhetinic acid synthesis pathway was optimized step by step: the inhibition of competition pathway,the enhancement of precursor supply and the optimization of cytochrome oxidase system,and the metabolic transformation of glycyrrhetinic acid engineering Saccharomyces cerevisiae.The target product of the research,glycyrrhetinic acid,has many antioxidation and anti-inflammatory effects,and the constructed glycyrrhetinic acid synthetic engineering bacteria can provide a template for the heterologous synthesis of more triterpenoid compounds.