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Methodology Development in Directed Evolution: Exploring Options when Applying Triple-Code Saturation Mutagenesis.

Ge Qu1, Richard Lonsdale2,3, Peiyuan Yao1

  • 1Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 West 7th Avenue, Tianjin Airport Economic Area, Tianjin, 300308, China.

Chembiochem : a European Journal of Chemical Biology
|January 10, 2018
PubMed
Summary
This summary is machine-generated.

Directed evolution of enzymes using triple-code saturation mutagenesis (TCSM) created highly selective biocatalysts for asymmetric synthesis. This approach significantly reduced screening efforts, yielding effective R- and S-selective alcohol dehydrogenase variants.

Keywords:
alcohol dehydrogenasebiocatalysisdirected evolutionsaturation mutagenesisstereoselectivity

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Area of Science:

  • Biocatalysis
  • Organic Synthesis
  • Protein Engineering

Background:

  • Enzyme evolution is crucial for asymmetric transformations in organic synthesis.
  • Screening large mutant libraries is a major bottleneck in enzyme evolution.
  • Developing "small but smart" mutant libraries is key to overcoming screening limitations.

Purpose of the Study:

  • To compare two distinct strategies for triple-code saturation mutagenesis (TCSM) at multiresidue sites.
  • To evolve Thermoanaerobacter brockii alcohol dehydrogenase (TbADH) using reduced amino-acid alphabets.
  • To identify highly stereo- or regioselective enzyme variants with minimal screening.

Main Methods:

  • Application of triple-code saturation mutagenesis (TCSM) on TbADH.
  • Utilizing distinct reduced amino-acid alphabets for mutagenesis.
  • Screening variants using the prochiral ketone tetrahydrofuran-3-one as a substrate.
  • Molecular dynamics analyses to determine the origin of stereoselectivity.

Main Results:

  • Obtained highly R- and S-selective TbADH variants (92-99% ee).
  • Demonstrated the effectiveness of TCSM with reduced amino-acid alphabets for efficient enzyme evolution.
  • Achieved high selectivity with minimal screening efforts.
  • Provided insights into the origin of stereoselectivity via molecular dynamics and Bürgi-Dunitz trajectory analysis.

Conclusions:

  • TCSM with reduced amino-acid alphabets is an effective strategy for evolving selective enzymes.
  • This method significantly reduces the screening burden in directed evolution.
  • The evolved TbADH variants show high potential for asymmetric synthesis applications.