L-phenylalanine production by recombinant Escherichia coli under regulation of T7 and ara promoters

Abstract

L-Phenylalanine (L-Phe) is mainly used as a reactant for production of important substances in medical and food industry such as aspartame. An increase in demand for aspartame leads to the improvement of L-Phe production in microorganisms, especially Escherichia coli by metabolic engineering. In this research, L-Phe production was increased by cloning four important genes in L-Phe biosynthesis pathway (aroB, aroL, phedh and tktA) into pRSFDuet-1 vector. After that the recombinant plasmid was co-transformed with pBAD33 vector containing a glycerol uptake gene (glpF) and an aromatic amino acid exporter gene (yddG) into E. coli BL21(DE3). Response surface methodology (RSM) was applied to optimize concentration of glycerol and ammonium sulfate. The highest production of L-Phe at 746 mg/L was obtained when the recombinant clone was cultured in minimum medium containing (g/L): glycerol 31; (NH₄) ₂SO₄ 63; MgSO₄·7H₂O 0.3; CaCl₂·2H₂O 0.015; KH₂PO₄4 3.0; K₂HPO₄ 12; NaCl 1; FeSO₄·7H₂O/Na-citrate 0.075 /1.0; thiamine·HCl 0.0075 and trace element solution (containing (g/L): Al₂ (SO₄)3·18H₂O 2.0; CoSO4·7H₂O 0.75; CuSO₄·5 H₂O 2.5; H3BO3 0.5; MnSO₄·H₂O 24; Na₂MoO₄·2H₂O 3.0; NiSO₄·6H₂O 2.5 and ZnSO₄·7 H₂O 15) 1.5 ml/L pH 7.0 at 37 °C after induction by 0.02% arabinose for 240 hours.