Reshaping the Substrate-Binding Pocket of Leucine Dehydrogenase for Efficient Synthesis of <scp>l</scp>-Phenylglycine and Its Substituted Derivatives

l-Leucine dehydrogenase (LeuDH)-mediated direct asymmetric reduction amination of prochiral α-keto acids represents an ideal approach for the synthesis of l-phenylglycine and its derivatives. However, limited substrate acceptance hinders their applications. Herein, we systematically investigated the substrate acceptance of LeuDHs for benzoylformic acid and its monosubstituted derivatives, revealing the correlation between substrate structure and enzyme activity. Meanwhile, to efficiently augment the LeuDH overall catalytic activity toward monosubstituted benzoylformic acids, we reported a two-stage screening strategy using <i>o</i>-chlorobenzoylformic acid (<b>2e</b>) as the starting screening substrate. A superior mutant library with 10-127-fold enhanced catalytic efficiency toward <i>ortho-</i>(M2-1 (L40V/V294A) and M2-2 (E114V/V294G)) and <i>meta-</i> and <i>para-</i>(M2-4 (E114L/V294G)) substituted benzoylformic acids was generated, and following future backtracking analysis, mutant M2-3 (L40V/T134G) with further increased catalytic activity of <i>meta</i>-substituted substrates was obtained. Furthermore, gram-scale asymmetric synthesis of l-phenylglycine (<b>3a</b>), L-<i>p</i>-fluorophenylglycine (<b>3d</b>), and L-<i>o</i>-chlorophenylglycine (<b>3e</b>) was performed with high substrate loading (1 M) and space-time yields up to 1800, 2016, and 2208 g/L·day, respectively. This study provides efficient biocatalysts for the synthesis of l-phenylglycine and its derivatives and establishes a referable engineering workflow for the collective evolution of amino acid dehydrogenase against differently positioned substituted substrate panels.