One‐Pot Biocatalytic Synthesis of D‐Amino Acids via a Deracemization Cascade Exploiting Amine Transaminase as a Thermodynamic Sink

Thermodynamically neutral equilibria often limit the synthetic utility of amino acid transaminases. An effective strategy to overcome this limitation is to design a catalytic cascade that circumvents the thermodynamic barrier by recycling the coproduct into the cosubstrate. Here, we present a whole‐cell deracemization cascade for the synthesis of D‐amino acids, in which the D‐amino acid transaminase (DAAT) reaction is thermodynamically driven by an R ‐selective amine transaminase (RATA). The cascade consists of three compartmentalized modules, each implemented in a distinct Escherichia coli whole‐cell biocatalyst: (i) an acceptor‐supply module (ASM) that converts the L‐enantiomer of racemic amino acid to α‐keto acid using L‐amino acid deaminase (LAAD); (ii) an amine‐transfer module (ATM) that produces D‐amino acid from the α‐keto acid using DAAT with D‐alanine; and (iii) a donor‐recycling module (DRM) that regenerates D‐alanine using RATA with α‐methylbenzylamine (α‐MBA). Integration of the three modules enabled one‐pot deracemization of amino acids within the overlapping substrate scope of LAAD and DAAT, affording the desired D‐amino acids in yields exceeding 90%. This study demonstrates a whole‐cell cascade system that leverages highly exergonic α‐MBA‐mediated conversion of pyruvate to D‐alanine to overcome the unfavorable DAAT equilibrium, providing an efficient route for synthesizing D‐amino acids from inexpensive racemic precursors.