Protein-engineered shift of the reaction equilibrium in transaminase reaction direction enables the asymmetric synthesis of (R)-1-phenylethylamine.

(R)-1-Phenylethylamine [(R)-1-PEA] is an important chiral compound widely used in pharmaceuticals, chemical synthesis, and materials science. ω-Transaminases are valuable biocatalysts for the asymmetric amination of ketone substrates under mild conditions and represent an attractive tool for the green synthesis of chiral amines. However, in natural (R)-selective transaminases, acetophenone (AP) is more commonly produced as the deamination product of (R)-1-PEA, whereas its amination as an amino acceptor is generally inefficient. In this study, an (R)-selective ω-transaminase from Thermomyces stellatus (TsRTA), which originally catalyzed only the deamination of (R)-1-PEA to AP, was selected as the engineering target. Protein engineering was performed to shift its reaction equilibrium, thereby enabling the asymmetric synthesis of (R)-1-PEA from AP. To overcome the thermodynamic limitation caused by acetone accumulation, an "acetone elimination-cofactor regeneration" coupled module was introduced to improve the whole-cell catalytic efficiency for (R)-1-PEA synthesis using AP as the substrate. Under optimal reaction conditions (35 °C, pH 10.0, 0.5 mM PLP, an isopropylamine/AP molar ratio of 4:1, 200 mM formate, and 0.2 mM NAD+), the whole-cell catalytic system produced 67 mM (R)-1-PEA from 100 mM AP within 8 h. This study lays a foundation for the scalable preparation of (R)-1-PEA and provides a useful reference for regulating the reaction direction of transaminases.