Co-Expression of feedback resistant enzymes in phenylalanine biosynthesis pathway to increase phenylalanine production

Abstract

L-Phenylalanine (L-Phe) is an important commercial amino acid. It is widely used in food and pharmaceutical industries. Currently, the requirement of L-Phe is increased according to the great demand for the low-calorie sweetener, aspartame. In Escherichia coli, the synthesis of L-Phe is controlled by the multi-hierarchical regulations. AroG isoform of 3-deoxy- D-arabino-heptulosonate-7-phosphate synthase (DAHP synthase) and chorismate mutase/prephenate dehydratase (PheA), two important enzymes, are feedback inhibited by LPhe. Co-expression of feedback-resistant pheA (pheA[superscript L359D]) with other pivotal genes in L-Phe biosynthesis pathway: aroB, aroL, phedh, tktA, aroG, pheA, yddG, and glpF in pRSFDuet-1 (pPTFBLYA[superscript L359D]) elevated L-Phe production of E. coli BL21(DE3) 3.78 fold in comparison to that of wildtype (pPTFBLYAwt). In this research, wildtype aroG (aroGwt) and feedback resistant aroG genes (aroGL175D, aroG[superscript Q151L], aroGQ151A and aroG[superscript Q151N]) were cloned into pRSFDuet-1 and then transformed into E. coli BL21(DE3). AroGQ151N clone gave the highest specific activity of DAHP synthase in the presence of 20 mM L-Phe. Therefore, E. coli BL21(DE3) containing pBLPTA[superscriptL359D]Gwt & pYF and pBLPTA[superscript L359D]GQ151N & pYF were constructed and their production of LPhe in 6% glycerol medium were determined in comparison to pBLPT & pYF clone. The presence of PheA[superscript L359D] and AroG[superscript Q151N] elevated L-Phe production 8.7 fold while the clone containing AroG[superscript Q151N] produced L-Phe 1.2 fold higher than AroGwt clone.