Enzyme orthologs sharing identical primary functions can have different promiscuous activities. While it is possible to mine this natural diversity to obtain useful biocatalysts, generating comparably rich ortholog diversity is difficult, as it is the product of deep evolutionary processes occurring in a multitude of separate species and populations. Here, we take a first step in recapitulating the depth and scale of natural ortholog evolution on laboratory timescales. Using a continuous directed evolution platform called OrthoRep, we rapidly evolve the Thermotoga maritima tryptophan synthase β-subunit (TmTrpB) through multi-mutation pathways in many independent replicates, selecting only on TmTrpB’s primary activity of synthesizing l-tryptophan from indole and l-serine. We find that the resulting sequence-diverse TmTrpB variants span a range of substrate profiles useful in industrial biocatalysis and suggest that the depth and scale of evolution that OrthoRep affords will be generally valuable in enzyme engineering and the evolution of biomolecular functions.

Tryptophan synthase (TrpS) has emerged as a paragon of noncanonical amino acid (ncAA) synthesis and is an ideal biocatalyst for synthetic and biological applications. TrpS catalyzes an irreversible, CC bond forming reaction between indole and serine (Ser) to make L-tryptophan (Trp); native TrpS complexes possess fairly broad specificity for indole analogs, but are difficult to engineer to extend substrate scope or to confer other useful properties due to allosteric constraints and their heterodimeric structure. Directed evolution freed the catalytically relevant TrpS β-subunit (TrpB) from allosteric regulation by its TrpA partner and has enabled dramatic expansion of the enzyme's substrate scope. This review examines the long and storied career of TrpS from the perspective of its application in ncAA synthesis and biocatalytic cascades.

Supporting information for this article is given via a link at the end of the document.

Enzyme orthologs sharing identical primary functions can have different promiscuous activities. While it is possible to mine this natural diversity to obtain useful biocatalysts, generating comparably rich ortholog diversity is difficult, as it is the product of deep evolutionary processes occurring in a multitude of separate species and populations. Here, we take a first step in recapitulating the depth and scale of natural ortholog evolution on laboratory timescales. Using a continuous directed evolution platform called OrthoRep, we rapidly evolved the Thermotoga maritima tryptophan synthase β-subunit (TmTrpB) through multi-mutation pathways in many independent replicates, selecting only on TmTrpB's primary activity (synthesizing L-tryptophan from indole and L-serine). We find that the resulting sequence-diverse TmTrpB variants span a range of substrate profiles useful in industrial biocatalysis and suggest that the depth and scale of evolution that OrthoRep affords will be generally valuable in enzyme engineering and the evolution of new biomolecular functions.

Supporting information for this article is given via a link at the end of the document.