In recent years there has been an increased focus on the sustainable production of natural products. (R)-Phenyllactyl-CoA is an important intermediate for various natural products like plant alkaloids. Commercial production by plants suffers from low yield and requires large amounts of arable land as well as expensive labor. In addition, phenyllactyl-CoA ligases from natural sources that have not been well characterized, making phenyllactyl-CoA a viable candidate for production in a metabolically engineered microbial host. Since the natural pathway is unknown, we have developed a pathway for the production of (R)-phenyllactyl-CoA in the genetically pliable host E. coli. Our biological scheme for the production of (R)-phenyllactyl-CoA consists of two parts: producing (R)-phenyllactate and engineering a CoA ligase to produce (R)-phenyllactyl-CoA. First, we altered the phenylalanine biosynthesis pathway by increasing phenylpyruvate production, restricting flux to phenylalanine, and expressing a non-native reductase to stereospecifically produce (R)-phenyllactate. Second, we established two methods of in vitro production of (R)-phenyllactyl-CoA: a coupled enzyme pathway using a CoA transferase as well as engineering a CoA ligase to accept phenyllactate. Future work includes further screening of CoA ligase mutants, investigating the costs and benefits of using a coupled enzyme pathway, and optimizing genomic integration to create a sustainable microbial production platform.

Biosynthesis of (R)-phenyllactyl-CoA in E. coli

2019 Spring National Meeting

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