Cookies on this website

We use cookies to ensure that we give you the best experience on our website. If you click 'Accept all cookies' we'll assume that you are happy to receive all cookies and you won't see this message again. If you click 'Reject all non-essential cookies' only necessary cookies providing core functionality such as security, network management, and accessibility will be enabled. Click 'Find out more' for information on how to change your cookie settings.

Directed evolution has emerged as one of the most effective protein engineering methods in basic research as well as in applications in synthetic organic chemistry and biotechnology. The successful engineering of protein activity, allostery, binding affinity, expression, folding, fluorescence, solubility, substrate scope, selectivity (enantio-, stereo-, and regioselectivity), and/or stability (temperature, organic solvents, pH) is just limited by the throughput of the genetic selection, display, or screening system that is available for a given protein. Sometimes it is possible to analyze millions of protein variants from combinatorial libraries per day. In other cases, however, only a few hundred variants can be screened in a single day, and thus the creation of smaller yet smarter libraries is needed. Different strategies have been developed to create these libraries. One approach is to perform mutational scanning or to construct "mutability landscapes" in order to understand sequence-function relationships that can guide the actual directed evolution process. Herein we provide a protocol for economically constructing scanning mutagenesis libraries using a cytochrome P450 enzyme in a high-throughput manner. The goal is to engineer activity, regioselectivity, and stereoselectivity in the oxidative hydroxylation of a steroid, a challenging reaction in synthetic organic chemistry. Libraries based on mutability landscapes can be used to engineer any fitness trait of interest. The protocol is also useful for constructing gene libraries for deep mutational scanning experiments.

Original publication

DOI

10.1007/978-1-4939-7366-8_6

Type

Chapter

Publication Date

01/2018

Volume

1685

Pages

87 - 128

Addresses

Department of Biocatalysis, Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470, Mülheim an der Ruhr, Germany. acevedor@gmx.de.

Keywords

Cytochrome P-450 Enzyme System, Directed Molecular Evolution, Protein Engineering, Mutagenesis, Site-Directed, Gene Library, Stereoisomerism, Synthetic Biology