Promiscuity guided evolution of a decarboxylative aldolase for synthesis of chiral tertiary alcohols

Enzymes play an increasingly important role in synthetic biology and organic synthesis. Many potential applications benefit from promiscuous activity with a diverse array of substrates. Here, we show how to intentionally guide an enzyme towards generality through multi-generational directed evolution using substrate-multiplexed screening (SUMS). We demonstrate the advantages of promiscuity-guided evolution in a challenging context, engineering the decarboxylative aldolase UstD to perform a C-C bond forming reaction with ketone electrophiles. 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.