Fast and flexible synthesis of combinatorial libraries for directed evolution

Joanna C. Sadler, Lucy Green, Neil Swainston, Douglas B. Kell, Andrew Currin*

*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

Directed evolution (DE) is a powerful tool for optimizing an enzyme's properties toward a particular objective, such as broader substrate scope, greater thermostability, or increased k cat . A successful DE project requires the generation of genetic diversity and subsequent screening or selection to identify variants with improved fitness. In contrast to random methods (error-prone PCR or DNA shuffling), site-directed mutagenesis enables the rational design of variant libraries and provides control over the nature and frequency of the encoded mutations. Knowledge of protein structure, dynamics, enzyme mechanisms, and natural evolution demonstrates that multiple (combinatorial) mutations are required to discover the most improved variants. To this end, we describe an experimentally straightforward and low-cost method for the preparation of combinatorial variant libraries. Our approach employs a two-step PCR protocol, first producing mutagenic megaprimers, which can then be combined in a “mix-and-match” fashion to generate diverse sets of combinatorial variant libraries both quickly and accurately.

Original languageEnglish
Title of host publicationMethods in Enzymology
EditorsNigel Scrutton
PublisherAcademic Press Inc.
Pages59-79
Number of pages21
Volume608
ISBN (Print)9780128151488
DOIs
Publication statusE-pub ahead of print - 24 May 2018

Publication series

NameMethods in Enzymology
Volume608
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

Keywords

  • combinatorial libraries
  • directed evolution
  • DNA mutagenesis
  • enzyme evolution
  • synthetic biology
  • variant libraries

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