Heterologous Synthesis Of Silybin By Microorganism

As a flavonolignan,silybin is the primary bioactive component of silymarin,which is a widely used drug for liver diseases and hepatoprotective reagent.Silybin is currently extracted from its original plant milk thistle.Because of thelimitation of season,climate,and breed,this method suffers from limited yield and unstable quality.These problems should be solved by using bioconversion or synthetic biology method to produce silybin.Currently,the complete silybin synthetic pathway is unclear,which hinders the microbial production of silybin.Consequently,we analyzed the key unknown step in silybin synthetic pathway.By improving catalase activity in the reaction system,the issues of H₂O₂ feedback inhibition and coniferyl alcohol over-oxidation were resolved.Based on the characteristics of taxifolin pathway and advantages of Yarrowia lipolytica as chasis,taxifolin pathway was modularized,reconstructed,validated,and optimized in Y.lipolytica.By auto-regulating fatty acid biosynthesis,taxifolin production was improved.Finally,an efficient silybin producing method was developed.Basic works of microbial production of silybin and its precursors were finished in this thesis,and the bioconversion methods for silybin and coniferyl alcohol production are also applicable.The main results of this work are summarized as follow:（1）The in planta silybin biosynthetic pathway was analyzed.The difference of the distribution of silybin and its precursors was discovered by analyzing the content of these chemicals among different tissues and organs.RNA-Seq showed that typical flavonoid and phenylpropanoid pathways exist in milk thistle.The expression levels of genes involved in theses pathways are different among different tissue s and organs.The biomimetic reactions showed that both peroxidase and laccase are involved in silybin synthesis.However,an ascorbate peroxidase APX1 in seed coat was confirmed to catalyze silybin synthesis after RNA-Seq,gene expression level analysis,peroxidase assay,and silybin synthesis analysis.Finally,silybin synthetic pathway and transportation manner were explained.（2）An efficient coniferyl alcohol synthesis method was developed.The coniferyl alcohol yield can be very low by reconstructing its original pathway in microorganisms.To solve this problem,an enzymatic cascade composed of PsVAO and catalase was developed,which efficiently converts eugenol to coniferyl alcohol.By in situ eliminating H₂O₂,feedback inhibition and coniferyl alcohol over-oxidation were relieved.By screening catalases of different organisms and classes,3 enzymes with high catalase activity but low peroxidase activity were introduced into the system.After condition optimization,22.9 g?L^-1 coniferyl alcohol was produced by feeding eugenol in 1.5 L scale,with molar yield of 78.7%and productivity of 0.5 g?L^-1?h^-1.Finally,the product was purified and analyzed.（3）A taxifolin producer was constructed usingY.Lipolytica as chasis.Based on the common issue of microbial production of flavonoid and characteristic of Y.lipolytica,Y.lipolytica was used to produce taxifolin.Taxifolin pathway was modularized,validated,and optimized,according to the rate-limiting factors of taxifolin pathway.After determining the rate-limiting steps,taxifolin pathway was optimized by increasing gene copy number.By enhancing chorismate and malonyl-CoA pathway,intracellular availability of these precursors and taxifolin synthesis were improved.Fermentation optimization showed that higher glucose content and adding cerulenin can improve taxifolin synthesis.Finally,taxifolin titer reached99.8 mg?L^-1 in fed-batch fermentation.（4）A CRISPRi based taxifolin precursor supply dynamic-regulating method was developed,and taxifolin production was improved.To solve the problem that only selective markers of URA3 and LEU2 are available in Y.lipolytica Po1f,methods for random and site-directed integration were developed and the efficiency was analyzed.The hybrid promoter（A1R1）_x2A3 was used in fatty acid synthesis auto-regulation,after analyzing the inducible strength and dynamic range of a panel of promoters.Different gRNAs were designed and analyzed for their efficiency of repressing the transcription of key genes for fatty acid synthesis.The effects of regulating fatty acid accumulation and taxifolin synthesis were also analyzed.The effects of regulating fatty acid and taxifolin synthesis by combinatory repressing multiple genes were also analyzed.Transcriptional repression of key genes resulted in decreased fatty acid accumulation,but increased taxifolin synthesis.Moreover,the transcriptional level was positively related with fatty acid accumulation while negatively related with taxifolin synthesis.These results validated that the CRISPRi based fatty acid synthesis auto-regulation method.The effect of improving taxifolin synthesis by dynamic-regulating fatty acid synthesis was confirmed by monitoring parameters of fermentation.（5）An efficient silybin synthesis method was developed.To develop efficient silybin synthesis method,peroxidases of different organisms and classes were analyzed.The results showed that silybin synthesis is not dependent on the origin and class of peroxidase.An enzymatic cascade composed of PsVAO and APX1 was developed,which converts eugenol and taxifolin into silybin and isosilybin,without producing any harmful by-product.The optimal pH,temperature,and substrate ratio,and relation between dissolved oxygen level and silybin synthesis were determined by condition optimization.Finally,2.58 g?L^-1 silybin and isosilybin were produced in 12 h in 1.5 L scale,with a taxifolin yield of 76.7%.