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Iterative Saturation Mutagenesis: A Powerful Approach to Engineer Proteins by Systematically Simulating Darwinian Evolution

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Directed Evolution Library Creation

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1179))

Abstract

Iterative saturation mutagenesis (ISM) is a widely applicable and powerful strategy for the efficient directed evolution of enzymes. First, one or more amino acid positions from the chosen enzyme are assigned to multi-residue sites (i.e., groups of amino acids or “multisites”). Then, the residues in each multisite are mutated with a user-defined randomization scheme to all canonical amino acids or a reduced amino acid alphabet. Subsequently, the genes of chosen variants (usually the best but not necessarily) are used as templates for saturation mutagenesis at other multisites, and the process is repeated until the desired degree of biocatalyst improvement has been achieved. Addressing multisites iteratively results in a so-called ISM scheme or tree with various upward branches or pathways. The systematic character of ISM simulates in vitro the natural process of Darwinian evolution: variation (library creation), selection (library screening), and amplification (template chosen for the next round of randomization). However, the main feature of ISM that distinguishes it from other directed evolution methods is the systematic probing of a defined segment of the protein sequence space, as it has been shown that ISM is much more efficient in terms of biocatalyst optimization than random methods such as error-prone PCR. In addition, ISM trees have also shed light on the emergence of epistasis, thereby rationally improving the strategies for evolving better enzymes. ISM was developed to improve catalytic properties such as rate, substrate scope, stereo- and regioselectivity using the Combinatorial Active-site Saturation Test (CAST), as well as chemical and thermal stability employing the B-Factor Iterative Test (B-FIT). However, ISM can also be invoked to manipulate such protein properties as binding affinity among other possibilities, including protein-protein interactions. Herein, we provide general guidelines for ISM, using CAST as the case study in the quest to enhance the activity and regioselectivity of the monooxygenase P450BM3 toward testosterone.

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Acknowledgments

We thank Rubén Agudo-Torres, Gheorghe-Doru Roiban, Zhi-Gang Zhang, and Pamela Torres-Salas for constructive discussions and the critical review of this manuscript. We are also grateful to the editors for their excellent editorial work. This work was supported by the Max Planck Society and the Arthur C. Cope Foundation.

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Authors and Affiliations

  1. Philipps-Universität Marburg, Fachbereich Chemie, Hans-Meerwein-Straße, 35032 Marburg, Germany

    Carlos G. Acevedo-Rocha & Manfred T. Reetz

  2. Philipps-Universität Marburg, Fachbereich Chemie, Junior Research Group Leader Chemical Biology, Hans-Meerwein-Straße, 35032 Marburg, Germany

    Sabrina Hoebenreich

  3. Organische Synthese, Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470, Mülheim, Germany

    Carlos G. Acevedo-Rocha & Manfred T. Reetz

Authors
  1. Carlos G. Acevedo-Rocha
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  2. Sabrina Hoebenreich
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  3. Manfred T. Reetz
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Corresponding author

Correspondence to Manfred T. Reetz .

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Editors and Affiliations

  1. Chemistry and Molecular Biosciences, The University of Queensland, St. Lucia, Brisbane, Queensland, Australia

    Elizabeth M.J. Gillam

  2. Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia, Canada

    Janine N. Copp

  3. School of Biological Sciences, Victoria University, Wellington, New Zealand

    David Ackerley

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Acevedo-Rocha, C.G., Hoebenreich, S., Reetz, M.T. (2014). Iterative Saturation Mutagenesis: A Powerful Approach to Engineer Proteins by Systematically Simulating Darwinian Evolution. In: Gillam, E., Copp, J., Ackerley, D. (eds) Directed Evolution Library Creation. Methods in Molecular Biology, vol 1179. Springer, New York, NY. https://doi.org/10.1007/978-1-4939-1053-3_7

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  • DOI: https://doi.org/10.1007/978-1-4939-1053-3_7

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  • Publisher Name: Springer, New York, NY

  • Print ISBN: 978-1-4939-1052-6

  • Online ISBN: 978-1-4939-1053-3

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