Random Saturation Mutagenesis to Generate Highly Diverse Libraries for Directed Evolution

Directed evolution improves the function of enzymes by diversifying the wild-type to generate variants and searching for the desired functions. When prior knowledge about the wild-type is limited, random mutagenesis is employed to introduce mutations at arbitrary positions. However, conventional methods exhibit significant bias in the distribution of amino acid substitutions, resulting in inaccessible chemical properties, limiting the chemical diversity, and hindering discovery of substantially improved variants. To overcome such limitations, we develop Random Saturation Mutagenesis (RSM) that uniformly introduces all amino acid substitutions across the gene at controlled rates by using overlapping site-mutagenesis oligo pools. Sequencing results illustrate the high quality and diversity of RSM libraries as compared to conventional libraries. We demonstrate the utility of RSM by improving thermotolerance of LipaseA. With only one round of mutagenesis, we identify two variants with over 40°C improvement in thermotolerance, demonstrating the potential of RSM to transform and accelerate directed evolution.