Heterologous pathway for the production of l-phenylglycine from glucose by E. coli

Abstract

The aproteinogenic amino acid l-phenylglycine (l-Phg) is an important side chain building block for the preparation of several antibiotics and taxol. To biosynthesis l-Phg from glucose, an engineered Escherichia coli containing l-Phg synthetic genes was firstly developed by an l-phenylalanine producing chassis supplying phenylpyruvate. The enzymes HmaS (l-4-hydroxymandelate synthase), Hmo (l-4-hydroxymandelate oxidase) and HpgT (l-4-hydroxyphenylglycine transaminase) from Amycolatopsis orientalis as well as Streptomyces coelicolor were heterologously expressed in E. coli and purified to evaluate their abilities on l-Phg formation. HpgT conversing phenylglyoxylate to l-Phg uses an unusual amino donor l-phenylalanine, which releases another phenylpyruvate as the substrate of HmaS. Thus, a recycle reaction was developed to maximize the utilization of precursor phenylpyruvate. To amplify the accumulation of l-Phg, the effects of attenuating l-phenylalanine transamination was investigated. After deletion of tyrB and aspC, l-Phg yield increased by 12.6-fold. The limiting step in the l-Phg biosynthesis was also studied; the l-Phg yield was further improved by 14.9-fold after enhancing hmaS expression. Finally, by optimizing expression of hmaS, hmo and hpgT and attenuation of l-phenylalanine transamination, the l-Phg yield was increased by 224-fold comparing with the original strain.