Casting epPCR (cepPCR): A simple random mutagenesis method to generate high quality mutant libraries

4 years ago

Casting epPCR (cepPCR): A simple random mutagenesis method to generate high quality mutant libraries

Anna J. Ruff, Jianhua Yang, Ulrich Schwaneberg, Marcus Arlt

During the last decade, directed evolution has become a standard protein engineering strategy to reengineer proteins for industrial applications under high stress conditions (e.g., high temperature, extreme pH, ionic liquids, or organic solvents). The most commonly employed method for diversity generation to improve biocatalysts for these properties is random mutagenesis by error-prone polymerase chain reaction (epPCR). However, recent reports show that epPCR often fails to produce >70% of beneficial positions/amino acid exchanges which improve enzyme properties such as organic solvent or ionic liquid resistance. In this report, bsla (543 bp, small lipase gene from Bacillus subtilis) was divided into three fragments (147, 192, 204 bp). Each fragment was subjected to an epPCR with a high mutation load (22, 31, and 33 mutations per kb) in order to increase the number of identified beneficial positions while maintaining a fraction of active population which can efficiently be screened in agar plate or microtiter plate format. The use of this “casting epPCR” process termed as (cepPCR), doubles the number of identified beneficial positions (from 14% to 29%), when compared to standard epPCR for the BSLA enzyme model. A further increase to 39% of beneficial positions is obtainable through combination of cepPCR with the transversion biased sequence saturation mutagenesis (SeSaM) method. Furthermore, sequencing of up to 600 mutations per fragment provided valuable insights into the correlation of total throughput and number of identified beneficial positions as well as how an efficient balance of screening efforts to obtainable results can be achieved in directed evolution campaigns. Biotechnol. Bioeng. 2017;114: 1921–1927. © 2017 Wiley Periodicals, Inc. The combination of high mutational loads with small fragment sizes yields in cepPCR twice the number of beneficial BSLA positions and to significant more powerful method, when compared to standard epPCR protocols. Analysis of cepPCR libraries taught lessons on how throughput relates to the number of identified beneficial positions and how an efficient balance of screening efforts to obtainable results can be achieved in directed evolution campaigns.

Publisher URL: http://onlinelibrary.wiley.com/resolve/doi

DOI: 10.1002/bit.26327