Metabolic Engineering Of Saccharomyces Cerevisiae For De Novo Biosynthesis Of δ-Tocotrienol

Vitamin E is an important fat-soluble vitamin and consists of eight natural isomers,namelyα,β,γ,δ-tocopherol andα,β,γ,δ-tocotrienol.Among them,δ-tocotrienol has been widely concerned for its more significant anti-inflammatory and anticancer effects.At present,the production of tocotrienols still relies on plant extraction.Plant extraction faces with the limitation of resources and a long growth cycle.Moreover the content ofδ-tocotrienol in plants is generally low,which limits the large scale production ofδ-tocotrienol.Therefore,biosynthesis ofδ-tocotrienol by microbial cell factory has the advantages of renewable raw materials,a short growth cycle,and environmental protection,which is an attractive alternative production method.This paper took recombinant Saccharomyces cerevisiae S6 as the starting strain,constructed aδ-tocotrienol de novo biosynthesis pathway through modular engineering strategy,and further improved the production performance of the engineered strain by a variety of metabolic engineering strategies.The main research contents are as follows:（1）De novo biosynthesis ofδ-tocotrienol in S.cerevisiae.Firstly,the expression and localization of hydroxyphenylpyruvate dioxygenase（HPPD）from A.thaliana,homogentisate geranylgeranyl transferase（HGGT）from Synechocystis sp.PCC 6803,and tocopherol cyclase（TC）from A.thaliana in S.cerevisiae were determined.Then,the chassis cell forδ-tocotrienol de novo synthesis was constructed,and the titer ofδ-tocotrienol was 18.1μg·L^-1 during fermentation in the medium containing tyrosine.Finally,by introducing geranylgeranyl diphosphate（GGPP）synthetase（Crt E）from Taxus x media and the mutant farnesyl pyrophosphate（FPP）synthase(FPS^F112A)from Gallus gallus to enhance the mevalonic acid module,the titer ofδ-tocotrienol reached 117.9μg·L^-1.（2）Identification and elimination of rate-limiting steps in theδ-tocotrienol biosynthesis pathway.By gradually overexpressing HPPD,HGGT,and TC,it was found that some rate-limiting steps may exit from the precursor homogentisic acid（HGA）toδ-tocotrienol.Overexpression of HGGT can increase the titer of its product 2-methyl-6-geranylgeranyl benzoquinol（MGGBQ）,but overexpression of TC cannot promote the conversion of MGGBQ toδ-tocotrienol.Hence,the signal peptide prediction and removal of TC were performed to relieve the rate-limiting step between MGGBQ andδ-tocotrienol.When the signal peptide removal length was 47 aa,theδ-tocotrienol titer reached 283.9μg·L^-1.（3）Engineering of substrate channeling to improveδ-tocotrienol biosynthesis.Three strategies including direct fusion,scaffold-free assembly,and synthetic scaffold were used to construct the substrate channeling in theδ-tocotrienol biosynthesis module,to improve the synthesis efficiency ofδ-tocotrienol.First,the fusion expression of HGGT and t TC through a short protein linker effectively increased theδ-tocotrienol titer.Then,by module assembly of HPPD and the HGGT-t TC complex using protein scaffold SH3,theδ-tocotrienol titer was increased to 760.4μg·L^-1,which was 2.56 times that of the control strain.（4）Combinatorial strategies to optimize the synthesis ofδ-tocotrienol.Firstly,shikimate biosynthesis module was strengthened by a"push-pull-block"strategy to remove the dependence ofδ-tocotrienol synthesis on tyrosine addition,and theδ-tocotrienol titer reached796.1μg·L^-1 without exogenous tyrosine addition.Then,the t TC protein was modified,and the mutant t TC^N331P with a significantly improved catalytic activity was constructed,and introducing this mutant make theδ-tocotrienol titer reach 1455.5μg·L^-1.Finally,the secretory production ofδ-tocotrienol was achieved by overexpression of the transporter PDR11,and theδ-tocotrienol titer reached 3262.2μg·L^-1 with the optimized two-phase extraction and fermentation conditions.