Improvement of l-phenylalanine production in Escherichia coli by methabolic engineering

Abstract

L-Phenylalanine (L-Phe), an essential amino acid, is mainly used as a reactant for production of important substances in medicine and food industry such as aspartame. An increase in demand for aspartame leads to the improvement of L-Phe production in well-known microorganisms, especially Escherichia coli.This work aims to improve L-Phe production from glycerol by metabolic engineering process. Phenylalanine dehydrogenase gene (phedh) from Bacillus lentus was cloned into pRSFDuet-1 (pPheDH) and expressed in E. coli BL21 (DE3) to increase the conversion of phenylpyruvate to L-Phe in the last step of L-Phe biosynthesis pathway. The recombinant pPheDH clone had limited ability to produce L-Phe when the cells were grown in the minimal medium containing glycerol, a by-product from biodiesel production, as carbon source, possibly due to poor glycerol uptake, low ability to excrete L-Phe, or tightly regulated steps in L-Phe biosynthesis. Accordingly, various genes encoding pivotal proteins involving in L-Phe biosynthesis pathway, glycerol uptake and L-Phe excretion were co-expressed with phedh gene. These genes were aroB, aroL, glpF, glpK, pheA, tktA and yddG which encode 3-dehydroquinate synthase, shikimate kinase II, glycerol facilitator, glycerol kinase, chorismite mutase/prephenate dehydratase, transketolase and aromatic amino acid exporter, respectively. The recombinant pPFBLY clone containing phedh, tktA, glpF, aroL and yddG genes had the highest L-Phe production rate of 3.36 mg/L/h which was 2.1-fold higher than that of pPheDH clone and the L-Phe concentration of 429 mg/L was attained at 240 h after the optical density at 600 mm reached 0.6. In a 5 L stirred-tank fermenter (37℃, an aeration rate of 1 vvm, an agitation speed of 400 rpm), the L-Phe production was carried out using pPYF cline. For batch fermentation, the maximum concentration of L-Phe (366 mg/L) was achieved at 56 h. In fed-batch fermentation with addition of new glycerol medium at 40 h and 60 h, the highest yield of L-Phe production of 445 mg/L was obtained. Moreover, one time feeding of glycerol at 40 h resulted in the maximum L-Phe titer of 545 mg/L. The maximum L-Phe production of pPYF clone in the fermenter was nearly 2.0-fold higher than in shake flask.