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

Abstract

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.

CasPER, a method for directed evolution in genomic contexts using mutagenesis and CRISPR/Cas9

“…In order to employ directed evolution, both the strategy for creating mutant libraries, as well as the assays for screening and selection of improved variants in high throughput, need to be considered (DeLoache et al, 2015;Körfer et al, 2016;Wong et al, 2004). Nowadays, methods for generation of sequence-variant libraries often include custom-designed DNA oligo library pools, multiplexed oligo assembly, or more or less randomized mutagenesis of template DNA fragments (Mutalik et al, 2013;Plesa et al, 2018;Yang et al, 2017). Screening and selection assays based on conditional growth, genetically-encoded biosensors, and reporter gene activities, has recently been applied to complement the efficiency of library generation Mundhada et al, 2016;Raman et al, 2014;Skjoedt et al, 2016).…”

Activity Improvement of Organophosphorus Hydrolase Enzyme by Error Prone PCR Method

“…In previous studies, error prone PCR method have been applied due to its simplicity, cost-effective, efficient and production of high similar genes for shuffling, by researcher in order to improve the features of proteins. [22][23][24][25][26] We succeeded to optimize the amount of Mg 2+ and Mn 2+ concentrations for epPCR amplification of oph gene. We selected 5.5 and 0.2 mM from Mg 2+ and Mn 2+ respectively, for high throughput PCR product.…”

A robust protocol for directed aryl sulfotransferase evolution toward the carbohydrate building block GlcNAc

“…Gene diversity by random mutagenesis is usually generated via epPCR methods (Shivange et al, ; Wong, Zhurina, & Schwaneberg, ). Mutant libraries with a higher quality (e.g., higher number of identified beneficial positions) can for instance be generated by sequence saturation mutagenesis (SeSaM) (Ruff, Kardashliev, Dennig, & Schwaneberg, ) or casting epPCR (cepPCR) (Yang, Ruff, Arlt, & Schwaneberg, ); both methods overcome the main limitations of epPCR including polymerase bias, high Ts/Tv ratio and lack of subsequent mutations (Yang et al, ; Zhao, Kardashliev, Joëlle Ruff, Bocola, & Schwaneberg, ). Microtiter plate (MTP)‐based screening systems with a colorimetric or fluorometric readout are still the standard format for high‐throughput screening (HTS) system in directed enzyme evolution (Martínez & Schwaneberg, ).…”