A Four-Step Enzymatic Cascade for Efficient Production of L- Phenylglycine from Biobased L-Phenylalanine

Abstract Enantiopure amino acids are of particular interest in the agrochemical and pharmaceutical industries. Here, we reported a multi-enzyme cascade for efficient production of L-phenylglycine (L-Phg) from biobased L-phenylalanine (L-Phe). We first attempted to engineer Escherichia coli for expressing L-amino acid deaminase (LAAD) from Proteus mirabilis , hydroxymandelate synthase (HmaS) from Amycolatopsis orientalis , ( S )-mandelate dehydrogenase (SMDH) from Pseudomonas putida , the endogenous aminotransferase (AT) encoded by ilvE and L-glutamate dehydrogenase (GluDH) from E. coli . However, 10 mM L-Phe only afforded the synthesis of 7.21 ± 0.15 mM L-Phg. The accumulation of benzoylformic acid suggested that the transamination step might be rate-limiting. We next used leucine dehydrogenase (LeuDH) from Bacillus cereus to bypass the use of L-glutamate as amine donor, and 40 mM L-Phe gave 39.97 ± 3.84 mM (6.04 ± 0.58 g/L) L-Phg, reaching 99.9% conversion. In summary, this work demonstrated a concise four-step enzymatic cascade for the L-Phg synthesis from biobased L-Phe, with a potential for future industrial applications. Graphical abstract a concise four-step enzymatic cascade for the L-phenylglycine synthesis from biobased L-phenylalanine was devised. 40 mM L-phenylalanine afforded the synthesis of 39.97 ± 3.84 mM (6.04 ± 0.58 g/L) L-phenylglycine, reaching 99.9% conversion.