Synthesis Of Aromatic Amino Acid Derivatives By Metabolic Engineering Of Escherichia Coli

Aromatic amino acid derivatives are a class of compounds with special physiological activities derived from aromatic amino acids,which contain extremely high commercial value.Such compounds usually have complex chemical structures and chiral centers,and plant extraction is often expensive due to the low content and the need to occupy a large area of arable land.In chemical production,it is often difficult to separate chiral isomers,resulting in low purity and residual solvents,which will lead to low biological safety.The green production method of recombinant microorganism fermentation fills the above shortcomings.In recent years,significant progress has been made in the synthesis of aromatic amino acid derivatives through strain modification.However,some aromatic amino acid derivatives still have problems of low yield or even unable to be synthesized due to unbalanced metabolic flux and unresolved synthetic pathways.In this study,the biosynthesis of3-phenylpropanol and S-equol was taken as an example,and the efficient synthesis of 3-phenylpropanol was achieved by redirecting carbon metabolic flux,and the first de novo biosynthesis of S-equol in Escherichia coli was achieved by rationally designing an artificial biosynthetic pathway.3-Phenylpropanol is widely used in cosmetics,food and other fields due to its unique aromatic odor,and is clinically used in the treatment of cholelithiasis due to its bile secretion-promoting and mild spasmolytic effects.On the basis of establishing its biosynthetic pathway through pathway screening,the metabolic flux balance was achieved by adjusting the expression intensity of the downstream enzymes of the metabolic pathway.The optimal fermentation mode was established by comparing the chassis cells,medium components and culture methods.Overexpression of the upstream key genes of the metabolic pathway enhanced the shikimate pathway and knocked out the competitive pathway.Reducing carbon source diversion and enhancing cofactor supply increased the yield of 3-phenylpropanol to 1005 mg/L,which is the highest yield reported so far.Due to its estrogenic properties and strong antioxidant activity,S-equol is widely used to prevent cancer,reduce the risk of cardiovascular disease,and relieve menopausal symptoms among others.In this study,the codon optimization,N-terminal truncation,replacement of hydrophilic peptides,and replacement of connection methods were performed on isoflavone synthase Ge2-HIS and its redox chaperone Cr CPR2 to improve the conversion efficiency of liquiritigenin.Finally,56.7mg/L daidzein was produced from 200 mg/L liquiritigenin.By making P212A-directed mutation of another key enzyme of the pathway,dihydrodaidzein DHDR,and optimizing the expression intensity of dzr,ifc A,ddr,and tdr to balance metabolic flux,finally,55 mg/L S-equol was produced from 150 mg/L daidzein.and 2.8 mg/L S-equol was synthesized de novo by co-culture with the upstream host bacteria,which was the first de novo synthesis of S-equol in E.coli.This study provides valuable ideas for the construction and optimization of the biosynthesis of aromatic amino acid derivatives,and lays a foundation for the industrial production of 3-phenylpropanol and S-equol.