Engineering of Escherichia coli for the Economic Production L-phenylalanine in Large-scale Bioreactor

Abstract

As an essential aromatic amino acid, L-phenylalanine (L-Phe) has a lot of applications as precursors for pharmaceuticals, artificial sweetner, etc. Previously, through metabolic engineering, we developed an Escherichia coli strain (YHP05) capable of overproducing L-Phe. In this study, we further engineered E. coli strain to develop an economic bioprocess for L-Phe production in large scale bioreactor. First, we developed E. coli YHP06 harboring pYHP which cultivation does not require supplementation of amino acids for cell growth. Next, the strong and constitutive gene expression systems for L-Phe biosynthesis were sequentially introduced into chromosome of E. coli, which allowed the antibiotic-free and inducer-free cultivation. Among the engineered strains, the H-02 strain showed the highest production of L-Phe (3.6 g/L) in the flask cultivation. The production of L-Phe in the engineered H-02 strain was also evaluated in lab-scale (5 L), and L-Phe as high as 30.7 g/L could be produced with 0.81 g/L/h of productivity. Finally, the pilot-scale (30 L) fed-batch cultivation was performed, and we could achieve the production of L-Phe as high as 38.6 g/L with 1.07 g/L/h of productivity.