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

  • Carlos G. Acevedo-Rocha
  • Sabrina Hoebenreich
  • Manfred T. Reetz
Part of the Methods in Molecular Biology book series (MIMB, volume 1179)


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.

Key words

Protein engineering Directed evolution Saturation mutagenesis Library quality Library statistics Screening effort CAST B-FIT P450-BM3 Steroids 



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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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Carlos G. Acevedo-Rocha
    • 3
    • 1
  • Sabrina Hoebenreich
    • 2
  • Manfred T. Reetz
    • 1
    • 3
  1. 1.Philipps-Universität Marburg, Fachbereich Chemie35032 MarburgGermany
  2. 2.Philipps-Universität Marburg, Fachbereich Chemie, Junior Research Group Leader Chemical Biology35032 MarburgGermany
  3. 3. Organische Synthese, Max-Planck-Institut für Kohlenforschung45470, MülheimGermany

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