Identification of Thermus aquaticus DNA polymerase variants with increased mismatch discrimination and reverse transcriptase activity from a smart enzyme mutant library

Govindan Raghunathan,Andreas Marx

doi:10.1038/s41598-018-37233-y

Abstract

DNA polymerases the key enzymes for several biotechnological applications. Obviously, nature has not evolved these enzymes to be compatible with applications in biotechnology. Thus, engineering of a natural scaffold of DNA polymerases may lead to enzymes improved for several applications. Here, we investigated a two-step approach for the design and construction of a combinatorial library of mutants of KlenTaq DNA polymerase. First, we selected amino acid sites for saturation mutagenesis that interact with the primer/template strands or are evolutionarily conserved. From this library, we identified mutations that little interfere with DNA polymerase activity. Next, these functionally active mutants were combined randomly to construct a second library with enriched sequence diversity. We reasoned that the combination of mutants that have minuscule effect on enzyme activity and thermostability, will result in entities that have an increased mutation load but still retain activity. Besides activity and thermostability, we screened the library for entities with two distinct properties. Indeed, we identified two different KlenTaq DNA polymerase variants that either exhibit increased mismatch extension discrimination or increased reverse transcription PCR activity, respectively.

Highlights

The invention of the polymerase chain reaction (PCR) is one of the most important innovations in biotechnology
We aimed at constructing a focused library of KlenTaq DNA polymerase variants by rationally selecting target residues for mutation that were located in the proximity of the active site and make contact with primer/template DNA complex
Upon saturation mutagenesis at target residues and fluorescence-based screening, functional mutants should be identified. These mutants should be shuffled in single reaction to construct a combinatorial library that was again screened for variants with improved mismatch discrimination and reverse transcription activity, respectively

Summary

Introduction

The invention of the polymerase chain reaction (PCR) is one of the most important innovations in biotechnology. We identified KlenTaq DNA polymerase variants that have significant reverse transcriptase activity allowing direct reverse transcription combined with PCR from RNA targets[22]. These enzymes are applicable in reverse transcription PCR applications. Over the years, when searching for improved DNA polymerases, we failed to identify DNA polymerases with increased mismatch discrimination from unfocused libraries while we failed to obtain DNA polymerases with reverse transcription activity from focused libraries. We identified two different KlenTaq DNA polymerase variants that either exhibit increased mismatch extension discrimination or increased reverse transcription PCR activity, respectively

Methods

Results

Conclusion