Abstract:
L-Phenylalanine (L-Phe) is one of the most important amino acids in food and pharmaceutical industries. It is widely used as a nutritional supplement and a precursor for the synthesis of food additives. The market of L-Phe has been stimulated by increasing demand for the low-calorie sweetener, aspartame. In Escherichia coli, the key enzyme which catalyzes the first committed step of aromatic amino acid biosynthesis pathway is 3-deoxy-D-arabino-heptulosonate-7- phosphate synthase (DAHP-synthase). The enzyme has 3 isoforms, AroG, AroF and AroH, which are feedback inhibited by L-Phe, L-Tyr and L-Trp, respectively. AroG, encoded by aroG, is the major isoform contributed about 80% of the total DAHP activity. To investigate the feedback inhibition site of AroG, Leu175 was replaced by Asp (L175D) and Gln151 was replaced by Ala (Q151A), Leu (Q151L) and Asn (Q151N). In this study, each feedback resistant aroG was cloned with other pivotal genes in L-Phe biosynthesis pathway (aroB, aroL, phedh and tktA) into pRSFDuet-1 vector. The recombinant plasmid (pBLPTG*) were then co-expressed with pBAD33 vector containing a glycerol uptake gene (glpF) and an aromatic amino acid exporter gene (yddG) (pYF) into E. coli BL21(DE3). The highest production of L-Phe at 1.8 g/L was obtained when both pBLPTG*Q151L & pYF and pBLPTG*Q151N & pYF clones were cultured in glycerol medium for 6 days. These L-Phe yields were 7.7 fold higher than obtained from the control with aroG wild-type (pBLPTG & pYF), while L-Phe yield from pBLPTG*L175D & pYF and pBLPTG*Q151A & pYF were 2.7 and 2.5 fold, respectively. The results revealed that substitution of Leu and Asn at Gln151 could well reduce the feedback inhibition. After that the recombinant clone of pBLPTG*Q151L & pYF was selected for optimization of medium components using response surface methodology (RSM). The maximum L-Phe production at 2.03 g/L was obtained when the pBLPTG*Q151L & pYF was cultured in minimum medium containing 60 g/L glycerol and 42.4 g/L (NH4)2SO4. after induction with 0.02% arabinose at 37 °C for 6 days.
