Scalable continuous evolution for the generation of diverse enzyme variants encompassing promiscuous activities

• Published in: Nature communications Vol. 11; no. 1; p. 5644
• Main Authors: Rix, Gordon, Watkins-Dulaney, Ella J., Almhjell, Patrick J., Boville, Christina E., Arnold, Frances H., Liu, Chang C.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 06.11.2020; Nature Publishing Group; Nature Portfolio
• Subjects: 45/23; 45/47; 45/70; 631/181/735; 631/61/338/552; 631/92/469; 82/58; 82/80; 82/83; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Biocatalysis; Biocatalysts; Catalysis; Directed evolution; Enzymes; Evolution; Evolution, Molecular; Humanities and Social Sciences; Indoles; L-Serine; multidisciplinary; Mutation; Protein Subunits - chemistry; Protein Subunits - genetics; Protein Subunits - metabolism; Science; Science (multidisciplinary); Substrate Specificity; Substrates; Thermotoga maritima - chemistry; Thermotoga maritima - enzymology; Thermotoga maritima - genetics; Tryptophan; Tryptophan - chemistry; Tryptophan - metabolism; Tryptophan synthase; Tryptophan Synthase - chemistry; Tryptophan Synthase - genetics; Tryptophan Synthase - metabolism
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-020-19539-6

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 ( Tm TrpB) through multi-mutation pathways in many independent replicates, selecting only on Tm TrpB’s primary activity of synthesizing l -tryptophan from indole and l -serine. We find that the resulting sequence-diverse Tm TrpB 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. Generating rich ortholog diversity for biocatalysts can be difficult due to the deep evolutionary processes involved. Here the authors use OrthoRep to rapidly evolve TrpB to produce sequence-diverse variants with altered substrate promiscuity.