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

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Scientific reports. 2019, 9, 590. eISSN 2045-2322. Available under: doi: 10.1038/s41598-018-37233-y
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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.

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ISO 690RAGHUNATHAN, Govindan, Andreas MARX, 2019. Identification of Thermus aquaticus DNA polymerase variants with increased mismatch discrimination and reverse transcriptase activity from a smart enzyme mutant library. In: Scientific reports. 2019, 9, 590. eISSN 2045-2322. Available under: doi: 10.1038/s41598-018-37233-y
BibTex
@article{Raghunathan2019-01-24Ident-44896,
  year={2019},
  doi={10.1038/s41598-018-37233-y},
  title={Identification of Thermus aquaticus DNA polymerase variants with increased mismatch discrimination and reverse transcriptase activity from a smart enzyme mutant library},
  volume={9},
  journal={Scientific reports},
  author={Raghunathan, Govindan and Marx, Andreas},
  note={Article Number: 590}
}
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