Work

SPIN-guided engineering of a novel (R)-amine transaminase from Fusarium albosuccineum for enantioselective synthesis of chiral piperidyl amines

Year: 2025

Type: article

Abstract: Aminotransferases are promising green biocatalysts for the synthesis of chiral amines, yet their limited catalytic efficiencies restrict broader industrial applications. In this study, a novel ( R )-amine transaminase, FalAT, was identified from Fusarium albosuccineum through genome mining. FalAT exhibited optimal activity at 30 °C and pH 7.0 and catalyzed the conversion of 1-Boc-3-piperidone to ( R )-1-Boc-3-aminopiperidine with >99 % enantiomeric excess. To efficiently enhance its catalytic performance, a Substrate–Protein Interaction Network (SPIN) strategy was implemented, integrating structure-guided analysis, molecular docking, virtual saturation mutagenesis, and dual energy–distance filtering. SPIN first screened and constructed a mutational library covering 70 amino acid positions, which was subsequently narrowed to 9 key residues, ultimately yielding 15 candidate mutants for experimental validation. Experimental results showed that five mutants exhibited higher catalytic activity than the wild-type enzyme, among which R126A was the most effective, displaying approximately a 4-fold increase in catalytic activity and a 13-fold enhancement in catalytic efficiency ( k ca t /K m = 2.05 s −1 mM −1 ). Molecular dynamics simulations revealed that the R126A mutation expanded the active-site cavity, alleviated steric hindrance, and strengthened hydrophobic interactions, thereby improving substrate binding and catalytic turnover. Furthermore, substrate profiling demonstrated that FalAT possesses moderate substrate promiscuity. Overall, the SPIN strategy significantly improved the catalytic performance of FalAT while markedly reducing experimental workload, providing an efficient and generalizable approach for the directed evolution of ( R )-amine transaminases for the green synthesis of chiral amines.

Source: Synthetic and Systems Biotechnology

DOI: 10.1016/j.synbio.2025.12.010