Fig. 3: ALDE optimization trajectory on the ParPgb active site.

The optimization campaign started with (A) constructing an initial library with mutations at all five sites under study using NNK degenerate codons, randomly selecting 384 for screening for product formation, and mapping to sequences using LevSeq. This was followed by two rounds of ALDE–(B) Round 1 and (C) Round 2. In Round 1 and Round2, exact genes were ordered as ENFINIA DNA produced by Elegen Corp. and screened for product formation. For each round, we present the distribution of amino acids sampled at each site and the distribution of yields for the cis and trans products, with a few of the top-performing variants labeled. Overall improvement in (D) cis – trans Yield, (E) Total Yield, and (F) cis/trans Selectivity over several rounds of ALDE for the best variant in each round and the mean across variants in each round. The best variant in each round, defined by the obejctive of cis – trans Yield is labeled. Error bars indicate the standard deviation across variants in the round. Yields were measured in biological triplicate. Source data are provided as a Source Data file.