Construction of an enzymatic-chemical cascade involving engineered amine dehydrogenases for the synthesis of chiral non-α-amino acids by substrate engineering and enzyme evolution

Chiral non-α-amino acids are highly marketable compounds. However, present enzymatic synthesis predominantly targets the generation of β- and γ-amino acids, lacking biosynthetic routes for non-α-amino acids with amino substitutions at more remote positions due to specific polar interactions with substrate's carboxyl group. Substrate engineering via esterification is a crucial method for carboxyl group modification. Consequently, keto esters were selected as substrates, and asymmetric reductive amination of these esters with varying substitution positions was accomplished using amine dehydrogenase from Bacillus badius (F-BbAmDH). The strategy of coupling substrate engineering with enzyme evolution was applied to enhance the compatibility between substrate and enzyme, thereby increasing the activity on β-, γ-, δ-, and ε-keto esters by 13, 8, 9, and 40 times, respectively. Molecular dynamics simulations and kinetic parameters analysis revealed that substrate engineering and enzyme evolution boost nonpolar interactions and improve substrate binding affinity within the enzyme's active site. Ultimately, diverse non-α-amino acids was synthesized employing exceptional mutants, yielding substantial conversion and enantioselectivity exceeding 99 %. The synthesis encompassed β- andγ-amino acids, as well as δ- and ε-amino acids with more distal substitution positions. Consequently, an eco-friendly, enzymatic-chemical cascade for the synthesis of non-α-amino acids with distinct substitution patterns was successfully established.