A High-Throughput Screening Method to Reengineer DNA Polymerases for Random Mutagenesis

A screening system for directed evolution of DNA polymerases employing a fluorescent Scorpion probe as a reporter has been developed. The screening system has been validated in a directed evolution experiment of a distributive polymerase from the Y-polymerase family (Dpo4 from Sulfolobus solfataricus) which was improved in elongation efficiency of consecutive mismatches. The engineering campaign yielded improved Dpo4 polymerase variants one of which was successfully benchmarked in a sequence saturation mutagenesis experiment especially with regard to the desirable consecutive transversion mutations (>2.5-fold increase in frequency relative to a reference library prepared with Dpo4 WT). The Scorpion probe screening system enables to reengineer polymerases with low processivity and fidelity, and no secondary activities (i.e. exonuclease activity or strand displacement activity) to match demands in diversity generation for directed protein evolution.