Active site diversification of a non-canonical amino acid decarboxylase by merging substrate multiplexed screening with computationally guided recombination.

ABSTRACT: Recombination of active site mutations is a powerful strategy to alter enzyme activity. The vastness of sequence space, however, often limits screening-based engineering to single and double site libraries. Here, we explore focused recombination across five positions that enclose the active site of a tryptophan (Trp) decarboxylase. Our goal was to maximize the sequence diversity of enzymes that decarboxylate non-canonical amino acids (ncAAs) with minimal screening effort. We used substrate-multiplexed screening (SUMS) to distinguish recombinants that have impaired activity with all substrates from those that have altered specificity. In this way, we identified a larger fraction of active sequence space than could be found by single substrate screening alone. Wild-type primer doping during

SUBMITTER: McDonald AD 

PROVIDER: S-EPMC12541894 | biostudies-literature | 2025 Nov

REPOSITORIES: biostudies-literature