Construction Of Biosynthetic Pathway And Yield Optimization Of Cinnamylamine In E.coli

With the development of metabolic engineering and synthetic biology,the biosynthesis of aromatic compounds has attracted much attention.Cinnamylamine is an aromatic compound derived from L-phenylalanine.It is mainly used in the synthesis of bioactive molecules,active drugs,and energetic materials,and has great research value.Cinnamylamine can be synthesized chemically,but this method has the disadvantages of high pollution and high cost.At present,the biosynthesis method targeting the production of cinnamylamine has not been reported,and there is an urgent need for us to establish an efficient method for the biosynthesis of cinnamylamine.This article is devoted to the construction of the biosynthetic pathway from cinnamic acid to cinnamylamine in E.coli.Based on the biosynthesis of cinnamaldehyde and referring to the in vitro enzymatic synthesis of amine compounds,we used ?-transaminase to catalyze the transamination reaction to convert cinnamaldehyde into cinnamamine.In the end,we opened up the biosynthetic pathway from cinnamic acid to cinnamylamine in E.coli for the first time,and continued to increase the production of cinnamylamine by relying on a series of metabolic engineering strategies.The most important step in the synthesis of cinnamylamine is to find an efficient transaminase.Therefore,we first screened transaminase from different sources,and verified the catalytic activity of transaminase derived from Chromobacterium violaceum(Cv-?TA)and transaminase derived from Halomonas elongata(He-?TA)The results show that Cv-?TA catalyzes the transamination reaction from cinnamaldehyde to cinnamylamine.Based on the enzymes required for the synthesis of cinnamaldehyde in E.coli,we chose 4-coumaric acid-CoA ligase(Sc4CL)from Streptomyce scoelicolor and cinnamoyl-CoA reductase(AtCCR)from Arabidopsis thaliana to catalyze the reduction of cinnamic acid to cinnamaldehyde,and inserted the coding genes of Sc4CL,AtCCR and Cv-?TA into the pColA-Duet-1 plasmid to construct a production plasmid-pocE,to verify the feasibility of the synthetic route from cinnamic acid to cinnamylamine.However,we found that cinnamaldehyde is easily converted into cinnamyl alcohol during the actual fermentation process,and the emergence of this by-product seriously hinders the production of cinnamylamine.In order to solve this problem,we used the MG1655(RARE)strain which targeted to knock out several aldehyde ketone reductase genes and alcohol dehydrogenase genes,and replaced the carbon source to reduce the reducing power produced by the strain during fermentation.We found that the fermentation using LB medium without additional carbon source produced the least cinnamyl alcohol,which is beneficial to the synthesis of cinnamylamine.At the same time,we successfully realized the synthesis of cinnamic acid to cinnamylamine by replacing the vector with different copy numbers,and using the relatively high copy number pET-Duet-1 vector to obtain 8.63 mg/L cinnamylamine.There is still much room for improvement in the production of cinnamylamine.In order to optimize the production of cinnamylamine,we screened carboxylic acid reductases from different sources.cinnamylamine is most produced when using the heterologous expression of carboxylic acid reductase(NcCAR)derived from Neurospora crassa,phosphoubiquitinyl transferase(PPTase)derived from E.coli and Cv-?TA.When 100 mg/L cinnamic acid was added,42.60 mg/L cinnamylamine was synthesized.In order to improve the catalytic performance of the transaminase,based on the molecular docking results of cinnamaldehyde and CV-?TA,we mutated the Phe22,Tyr168 and Ala231 sites of the transaminase and constructed a library containing 57 mutants.Through the combined mutation method to find the best transaminase mutant.By comparing the yield and conversion rate,the Y168G mutant was selected to synthesize cinnamylamine.After that,we overexpressed the zwf gene to provide the required NADPH for the synthesis of cinnamic acid to cinnamaldehyde,and by optimizing the concentration of the cofactor PLP and the amino donor alanine,under the condition of sodium cinnamate as the precursor,the production of cinnamylamine was increased to 523.15 mg/L.