Metabolic Engineering Of The Chorismate Pathway In Saccharomyces Cerevisiae To Enhance Tryptophol (IET) Production

Tryptophol（IET）is an important chemical and pharmaceutical intermediate,which is currently produced industrially by chemical synthesis methods,which are neither green nor environmentally friendly and produce many by-products.Biofermentation using the IET synthesis pathway in Saccharomyces cerevisiae can overcome these problems,but the yield of wild-type IET is very low due to the insufficient content of its precursor substance tryptophan.The aromatic amino acid pathway in which tryptophan is located contains three amino acid branches,and the current research focuses on the phenylalanine branch and tyrosine branch,while the tryptophan branch is less studied due to the complex metabolic process resulting in insufficient carbon metabolic flux.In this study,the chorismate pathway was metabolically engineered in S.cerevisiae with the aim of improving tryptophan production,and the problem of insufficient carbon flux in the tryptophan branch was solved,and high IET production with glucose as the only carbon source was achieved for the first time in S.cerevisiae.The main findings are as follows:（1）Establishment of a carbon flux detection system in the aromatic amino acid pathway.By adding tyrosine and phenylalanine precursors phenylpyruvate（PP）and 4-hydroxy-phenylpyruvate（4-HPP）and detecting the corresponding metabolism in the fermentation broth,we found that the amount of products 2-phenylethanol（PET）and 4-hydroxyphenylpyruvate（p PET）has a good linear relationship with the amount of precursor added,so these two products and IET were used to detect aromatic amino acids pathway carbon fluxe.（2）Metabolic engineering modularly modifies the branching acid pathway and the Ehrlich pathway.The Yeast Toolkit（YTK）was used to construct plasmids for the characterization of the relevant genes.The metabolic engineering transformation was divided into the following five modules:overexpression of substrate feedback insensitive mutant genes ARO4^K229L and TRP2^S65R,S76L of the key rate-limiting enzymes;overexpression of TRP1,TRP3,TRP4,TRP5 in the tryptophan branch;knockdown of ARO7,a key gene in the phenylalanine and tyrosine branches of the competitive pathway;stepwise overexpression of related genes in the Ehrlich pathway;overexpression of genes related to the supply of precursors of the aromatic amino acid pathway.The carbon metabolic flux of the tryptophan branch was significantly increased by the above modifications,and the IET yield reached 0.77 m M.（3）Optimization of fermenter production conditions.The following three media and supplementation methods were optimized in a 5 L fermenter:synthetic media,YPD media with simultaneous flow addition of glucose,and YPD media with simultaneous flow addition of glucose and regular addition of yeast extract.The final IET yield reached 6.80 m M(1.1 g·L^-1).This study is the first report of high production of tryptophol in S.cerevisiae using glucose as the sole carbon source,which solves the problem of insufficient carbon metabolic flux of tryptophan branch and provides theoretical support for the biosynthesis of other tryptophan branch-related compounds using tryptophan.