Metabolic Engineering Of Escherichia Coli For Production Of L-homophenylalanine And 3-phenylpropanol

L-homophenylalanine（L-Hph）and 3-phenylpropanol are greatly needed L-phenylalanine derivatives for their wide range of application.L-Hph can be applied for the synthesis of angiotensin-converting enzyme inhibitors,which have been widely used for the treatment of high blood pressure and heart diseases.And 3-phenylpropanol is widely used in foods,beverages and cosmetics as a fragrance ingredient.The efficiency of biosynthetic pathway is crucial for construction of microbial cell factories.In this study,we designed and refactored biosynthetic pathways of L-homophenylalanine and 3-phenylpropanol,achieving efficient production of them in Escherichia coli.For production of L-homophenylalanine,we developed an integrative biosynthetic gene cluster mining strategy.We identified 38 L-Hph biosynthetic gene clusters（BGCs）,and integrated phylogenetic analysis with target metabolite abundance mapping to prioritize 4 candidate BGCs.We tested various combinations of 12 genes from candidate BGCs and obtained an effective L-Hph pathway in 4 rounds of iterative stepwise assessment.We carried out chromosome engineering,with integration of aro G^fbr-phe A^fbrfor derepression of the feedback inhibition,and deletion of tyr A for elimination of the competing pathway,achieving the L-phenylalanine overproducing chassis and de novo biosynthesis of L-Hph.In the fed-batch shake-flask fermentation,engineered E.coli produced a final L-Hph titer of 1.41 g/L.For production of 3-phenylpropanol,we carried out in silico retrobiosynthesis analysis.For 5 possible biosynthetic pathways of 3-phenylpropanol,we compared their pathway length,thermodynamic feasibility,the consumption of reducing power NAD（P）H and energy ATP,and participation of metabolite.And one pathway was prioritized.We refactored the heterologous pathway comprising At PAL2,Ca ER,Sru CAR and Ec PPTase by screening different enzymes from various sources.We continued to engineer the chromosome of L-phenylalanine overproducing chassis by multi-gene disruption for enhancing PEP supply and eliminating negative regulation.We introduced 3-phenylpropanol refactoring pathway for de novo production and found chassis strain with triple deletion of the pts G,pyk F and pyk A genes was most compatible with the refactoring pathway.After balancing metabolic strength of pathway genes and optimizing fermentation conditions,engineered E.coli produced847.97 mg/L of 3-phenylpropanol.We designed and refactored biosynthetic pathways of L-homophenylalanine and3-phenylpropanol in E.coli,respectively,and obtained strains capable of efficient production of them by metabolic engineering.The study confirmed the potential of our engineered strains for industrial production of L-homophenylalanine and 3-phenylpropanol,respectively.Moreover,further application of our strategy will lead to engineer metabolic pathways of other high-value products.