Synthetic amino acid elongation via transaminase engineering

Unnatural amino acids expand the chemical space of medicines, yet scalable biosynthetic routes remain limited. Here, we present a modular strategy to “grow” α-amino acids one carbon at a time by coupling directed evolution of Escherichia coli transaminases with α-ketoacid chain extension. Using a glutamate–auxotroph selection system, we revealed previously uncharacterized residues in TyrB and IlvE and identified beneficial substitutions at these positions that broaden substrate scope toward hydroxyl-, tert-butyl-, and thioether-bearing frameworks with extended side chains. When paired with an acetyl-CoA-dependent ketoacid extension module, these engineered enzymes enabled the first biosynthetic access to multiple chain-elongated unnatural α-amino acids, including neopentylglycine, C5 and C6 hydroxy amino acids, homomethionine, and homoglutamate directly from their −1 precursors. Moreover, the system demonstrates iterative capability, successfully extending homoserine through two consecutive cycles to generate both +1 and +2 derivatives. Homoglutamate, selected for its pharmaceutical relevance, reached titers up to 10 g/L in shake flasks, underscoring the scale-up potential of this approach. This work establishes a generalizable enzymatic framework for stepwise expansion of α-amino acid scaffolds and broadens access to noncanonical building blocks for peptide and bioproduction applications.