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

Protein engineering altered the reaction direction of TsRTA for AP. • Adjusting the catalytic center and entrance of TsRTA promoted AP binding. • A 10-site variant of TsRTA enabled biosynthesis of ( R )-1-PEA. • An acetone elimination module increased Mu10 productivity by 57.4%. ( 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. Download: Download high-res image (205KB) Download: Download full-size image