Biosyntheis Of 3-phenylpropanol In Escherichia Coli

3-phenylpropanol with the fragrance of flowers,is an important intermediate in the preparation of spices and medicines.Currently,it mainly relies on plant extraction and chemical synthesis,but there have some disadvantages such as low titer,long period and environmental pollution.In order to solve these problems,it is of great significance to construct a microbial cell factory for de novo synthesis of 3-phenylpropanol from renewable resources.In this study,a new artificial biosynthesis pathway of 3-phenylpropanol was designed and constructed based on the structural similarity between substrates or intermediates and products,as well as the group transfer relationship between compounds,and a biosynthesis platform of 3-phenylpropanol was established for the first time.In this study,based on the similarity of catalytic substrates,we designed two heterologous 3-phenylpropanol synthesis pathways with cinnamic acid as key intermediate which dependent on coenzyme A reductase and carboxylic acid reductase,respectively.By feeding experiments to investigate the feasibility of the pathway,the results show that the synthesis pathway based on carboxylic acid reductase has successfully achieved de novo biosynthesis of 3-phenylpropanol with a titer of 91 mg/L.To improve the titer of 3-phenylpropanol,we constructed a cinnamic acid-producing strain to increase the supply of cinnamic acid.First,the initial production of de novo synthesis of cinnamic acid by overexpression of phenylalanine ammonia lyase in E.coli was only 113 mg/L.In order to improve the titer of cinnamic acid,remove the bottlenecks in the process of cinnamic acid production,we improved the titer of cinnamic acid to 393 mg/L by enhancing the carbon flux of shikimate pathway.Then,by further increasing the expression levels of key genes in the shikimate pathway,the cinnamic acid production was increased to 499 mg/L.Finally,the titer of cinnamic acid was improved to 835 mg/L by knocking out the competitive pathways to maximize the carbon source into the target production pathway,and the titer is 7.39 times that of the original strain.Introducing the heterologous synthetic pathway of 3-phenylpropanol into the best cinnamic acid-producing strain,the shake flask titer of 3-phenylpropanol reached 476 mg/L,5.23 times that of the original strain.In this paper,we rationally designed and constructed a new artificial biosynthesis pathway of 3-phenylpropanol,realized the efficient biosynthesis of 3-phenylpropanol by eliminating the rate-limiting steps,increased the carbon flux of shikimate pathway and knocking out of competitive pathway,with the titer of 476 mg/L,which is the highest level reported so far.