| Autor: |
Rix G; Department of Molecular Biology and Biochemistry, University of California, Irvine, CA, USA., Watkins-Dulaney EJ; Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA., Almhjell PJ; Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA, USA., Boville CE; Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA.; Aralez Bio, Emeryville, CA, USA., Arnold FH; Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA.; Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA, USA., Liu CC; Department of Molecular Biology and Biochemistry, University of California, Irvine, CA, USA. ccl@uci.edu.; Department of Biomedical Engineering, University of California, Irvine, CA, USA. ccl@uci.edu.; Department of Chemistry, University of California, Irvine, CA, USA. ccl@uci.edu. |
| Abstrakt: |
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. |
