Many pathways in laboratory evolution can lead to improved enzymes: How to escape from local minima

Gumulya, Yosephine, Sanchis, Joaquin and Reetz, Manfred T. (2012) Many pathways in laboratory evolution can lead to improved enzymes: How to escape from local minima. ChemBioChem, 13 7: 1060-1066. doi:10.1002/cbic.201100784

Author Gumulya, Yosephine
Sanchis, Joaquin
Reetz, Manfred T.
Title Many pathways in laboratory evolution can lead to improved enzymes: How to escape from local minima
Journal name ChemBioChem   Check publisher's open access policy
ISSN 1439-4227
Publication date 2012-05-07
Sub-type Article (original research)
DOI 10.1002/cbic.201100784
Open Access Status Not Open Access
Volume 13
Issue 7
Start page 1060
End page 1066
Total pages 7
Place of publication Weinheim, Germany
Publisher Wiley - V C H Verlag GmbH & Co. KGaA
Language eng
Abstract Directed evolution is a method to tune the properties of enzymes for use in organic chemistry and biotechnology, to study enzyme mechanisms, and to shed light on Darwinian evolution in nature. In order to enhance its efficacy, iterative saturation mutagenesis (ISM) was implemented. This involves: 1) randomized mutation of appropriate sites of one or more residues; 2) screening of the initial mutant libraries for properties such as enzymatic rate, stereoselectivity, or thermal robustness; 3) use of the best hit in a given library as a template for saturation mutagenesis at the other sites; and 4) continuation of the process until the desired degree of enzyme improvement has been reached. Despite the success of a number of ISM-based studies, the question of the optimal choice of the many different possible pathways remains unanswered. Here we considered a complete 4-site ISM scheme. All 24 pathways were systematically explored, with the epoxide hydrolase from Aspergillus niger as the catalyst in the stereoselective hydrolytic kinetic resolution of a chiral epoxide. All 24 pathways were found to provide improved mutants with notably enhanced stereoselectivity. When a library failed to contain any hits, non-improved or even inferior mutants were used as templates in the continuation of the evolutionary pathway, thereby escaping from the local minimum. These observations have ramifications for directed evolution in general and for evolutionary biological studies in which protein engineering techniques are applied.
Keyword Directed evolution
Enzyme catalysis
Fitness landscapes
Iterative saturation mutagenesis
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status Non-UQ

Document type: Journal Article
Sub-type: Article (original research)
Collection: School of Chemistry and Molecular Biosciences
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Citation counts: TR Web of Science Citation Count  Cited 38 times in Thomson Reuters Web of Science Article | Citations
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