Promiscuity Guided Evolution of Decarboxylative Aldolases for Synthesis of Tertiary γ‐Hydroxy Amino Acids

Many applications of enzymes benefit from activity on structurally diverse substrates. Here, we sought to engineer the decarboxylative aldolase UstD to perform a challenging C‐C bond forming reaction with ketone electrophiles. The parent enzyme had only low levels of activity, portending multiple rounds of directed evolution and a possibility that mutations may inadvertently increase the specificity of the enzyme for a single model screening substrate. We show how to intentionally guide UstD towards generality through multi‐generational directed evolution using substrate‐multiplexed screening (SUMS). Mutations outside of the active site that impact catalytic function were immediately revealed by shifts in promiscuity, even when the overall activity was lower. By re‐targeting these distal residues that couple to the active site with saturation mutagenesis, broadly activating mutations were readily identified. When analyzing active site mutants, SUMS identified both specialist enzymes that would have more limited utility as well as generalist enzymes with complementary activity on diverse substrates. These new UstD enzymes catalyze convergent synthesis of non‐canonical amino acids bearing tertiary alcohol side chains. This methodology is easy to implement and enables the rapid and effective evolution of enzymes to catalyze desirable new functions.