One pot cascade synthesis of L-2-aminobutyric acid employing ω-transaminase from Paracoccus pantotrophus

• Discovery of a novel ω-transaminase with a large S pocket. • Regulation of RBS strength from the substrate side to ease the inhibition of key enzymes by intermediate products. • Comparison of catalytic preparation of l -ABA by different amino donor decomposition systems. ω-transaminase can mediate the asymmetric synthesis of chiral amines from aldehydes and ketones, which has important value in the synthesis of pharmaceutical intermediates. A novel ω-transaminase derived from Paracoccus pantotrophus ( Pp TA) was obtained and cloned in E. coli BL21(DE3) for expression. The enzyme has high activity for 2-ketobutyric acid and benzylamine, as well as for aromatic compounds with side chains longer than ethyl aliphatic hydrocarbons. Molecular simulation showed that the S-pocket in the active center is larger than those of other ω-transaminases. Thereafter, a whole-cell catalytic system was designed to prepare l -2-aminobutyric acid by cascading Pp TA and other enzymes. By using several strategies (regulation of RBS intensity, by-product decomposition and cofactor self-sufficiency), whole-cell cascade biocatalysis showed a high ee value (> 99%) and high yield (71%) in one pot reaction. This study therefore proposes an efficient biocatalyst for the synthesis of unnatural amino acids with the participation of ω-transaminase.