Enhancement of L-phenylalanine production by mutagenesis of pheA gene in escherichia coli

Abstract

L-Phenylalanine (L-Phe) is an essential amino acid required for dietary meal of human. It is wildly used in chemical synthesis of various pharmaceutical and food industrial products. Nowaday, demand for L-Phe has increased dramatically due to the demand of the low-calorie sweetener aspartame. L-Phe can be synthesized by chemical synthesis, enzymatic synthesis or microbial fermentation. In Escherichia coli, the production of L-Phe is under the aromatic amino acid biosynthesis pathway by which accumulation of L-Phe can partially inhibited some enzyme in its own biosynthesis pathway. Bifunctional enzyme chorismate mutase / prephenate dehydratase (CMPD) encoded by pheA gene is a key enzyme that is sensitive to feedback inhibition mediated by allosteric binding of L-Phe. CMPD contains two catalytic domains for CM and PD activities as well as one regulation (R) domain for binding with L-Phe. To improve the production of L-Phe by metabolic engineering, feedback-resistant pheA (pheAfbr) located at L300D, M302D, L318D, L344D, L359D and L359P were obtained by PCR-driven overlap extension mutagenesis technique. Then pheAfbr was co-expressed with phedh, tktA, glpF, aroB, aroL and yddG gene which encoded phenylalanine dehydrogenase, transketolase, glycerol facilitator, 3-dehydroquinate synthase, shikimate kinase II and aromatic amino acid exporter, respectively. Among these mutated clones, pPTFBLYAL359D clone produced the highest concentration L-Phe in 200 mL shake flask culture containing glycerol as a carbon source about 135 mg/L which were calculated as 3.78 times in comparison to the L-Phe content of wild-type (pPTFBLYA). Fed-batch in fermentation a 5 L stirred-tank fermenter (37 oC, an aeration rate of 1 vvm, an agitation speed of 400 rpm) was with L-Phe production was carried out using pPTFBLYAL359D clone with addition of new medium at 54 h, the yield of L-Phe production of 176 mg/L was obtained. The maximum L-Phe production of pPTFBLYAL359D clone in the fermenter was 1.3-fold higher than that in shake flask.