Protein domain library generation by overlap extension (PDLGO): a tool for enzyme engineering

Area of Science:

• Biotechnology and Molecular Biology
• Enzyme Engineering
• Protein Engineering

Background:

• Traditional error-prone PCR (epPCR) introduces random mutations across entire genes, making targeted protein domain mutagenesis challenging.
• Existing methods often require flanking restriction sites, limiting flexibility in selecting mutation regions.
• Efficient tools for directed evolution of enzymes, such as esterases, are crucial for improving their catalytic properties.

Purpose of the Study:

• To introduce an upgraded epPCR method, protein domain library generation by overlap extension (PDLGO), for targeted mutagenesis within specific gene regions.
• To validate PDLGO by generating a focused mutant library of the esterase EstE from Xanthomonas vesicatoria.
• To identify enhanced esterase variants using a high-throughput screening platform.

Main Methods:

• Development of PDLGO, a three-step DNA polymerase-catalyzed reaction enabling targeted random mutations.
• Generation of a random mutant library of EstE using PDLGO, focusing mutations on catalytic domains.
• High-throughput screening of the EstE variant library using E. coli expressing variants in microplates with integrated pH sensors.

Main Results:

• PDLGO successfully confined random mutations exclusively to the intended catalytic domains of EstE, leaving other domains unaltered.
• A variant (P286H) with significantly increased catalytic activity was identified from the generated library.
• The integrated pH sensor microplate system proved effective as a substrate-independent high-throughput screening tool.

Conclusions:

• PDLGO offers a precise and flexible method for generating targeted mutant libraries within specific protein domains.
• The combination of PDLGO and pH sensor microplates provides a rapid and efficient toolbox for the directed evolution of esterases.
• This approach facilitates the discovery of enzyme variants with improved catalytic functions for biotechnological applications.