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Directed evolution by using iterative saturation mutagenesis based on multiresidue sites.

Loreto P Parra1, Rubén Agudo, Manfred T Reetz

  • 1Department of Synthetic Organic Chemistry, Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470 Mülheim an der Ruhr (Germany); Fachbereich Chemie, Philipps-Universität Marburg, Hans-Meerwein-Strasse, 35032 Marburg (Germany); Department of Chemical and Bioprocesses Engineering, Pontificia Universidad Católica de Chile, Avenida Vicuña Mackenna 4860, Santiago (Chile).

Chembiochem : a European Journal of Chemical Biology
|October 19, 2013
PubMed
Summary
This summary is machine-generated.

Iterative saturation mutagenesis (ISM) with larger randomization sites and a reduced amino acid alphabet enhances protein engineering. This method successfully tuned enzyme activity and diastereoselectivity, proving its viability.

Keywords:
Baeyer-Villiger reactiondirected evolutionenzyme modelsiterative saturation mutagenesisstereoselectivity

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Area of Science:

  • Protein Engineering
  • Directed Evolution
  • Enzymology

Background:

  • Iterative saturation mutagenesis (ISM) combined with reduced amino acid alphabets is efficient for directed evolution.
  • Previous ISM studies limited randomization sites to 2-3 residues to manage library size.
  • Large library sizes are typically avoided to prevent oversampling when aiming for high coverage.

Purpose of the Study:

  • To apply ISM using randomization sites of five amino acid positions for the first time.
  • To investigate two distinct ISM pathways (A→B and B→A) using two such sites.
  • To employ a severely reduced five-member amino acid alphabet for building blocks.

Main Methods:

  • Utilized iterative saturation mutagenesis (ISM) with five-residue randomization sites.
  • Employed a minimal set of five structurally representative amino acids.
  • Applied the method to Baeyer-Villiger monooxygenase PAMO for proof-of-principle.

Main Results:

  • Achieved notable activity and 99% diastereoselectivity in PAMO-catalyzed oxidation of a non-native substrate.
  • Ensured 8-9% library coverage, lower than traditional statistics but sufficient for results.
  • Demonstrated successful tuning of enzyme activity and diastereoselectivity.

Conclusions:

  • The novel ISM strategy with larger randomization sites is viable for protein engineering.
  • This approach can efficiently engineer enzyme properties like activity and selectivity.
  • Further applications of this method can advance directed evolution efforts.