Fast and Flexible Synthesis of Combinatorial Libraries for Directed Evolution

Methods in Enzymology
Joanna C SadlerAndrew Currin

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 kcat. 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.

Citations

Aug 17, 2019·Scientific Reports·Nicole G H LeferinkNigel S Scrutton
May 18, 2021·Biotechnology Advances·Andrew CurrinRainer Breitling

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