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A New Screening Method for the Directed Evolution of Thermostable Bacteriolytic Enzymes
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Engineering a Thermostable Keto Acid Decarboxylase Using Directed Evolution and Computationally Directed Protein

Lemuel M J Soh, Wai Shun Mak1, Paul P Lin

  • 1Department of Chemistry, Biochemistry & Molecular Medicine, and the Genome Center, University of California Davis , One Shields Avenue, Davis, California 95616, United States.

ACS Synthetic Biology
|January 5, 2017
PubMed
Summary

Researchers engineered a more heat-stable keto acid decarboxylase (Kdc) enzyme, Kivd, for efficient production of higher alcohols like isobutanol. This enhanced enzyme, LLM4, functions effectively at higher temperatures, overcoming limitations of current methods.

Keywords:
directed evolutionhigh-throughput screeningisobutyl alcoholketo acid decarboxylaseprotein designthermostability

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Area of Science:

  • Biocatalysis
  • Protein Engineering
  • Metabolic Engineering

Background:

  • Keto acid decarboxylase (Kdc) is crucial for producing higher alcohols, such as isobutanol, from keto acids.
  • Mesophilic Kdc enzymes exhibit higher activity but lack the thermostability required for industrial applications.
  • Current Kdc activity in thermophilic organisms is significantly lower, hindering efficient high-temperature bioprocessing.

Purpose of the Study:

  • To engineer a thermostable 2-ketoisovalerate decarboxylase (Kivd) for improved isobutanol production.
  • To enhance the thermal stability of mesophilic Kdc enzymes for industrial viability.

Main Methods:

  • Directed evolution using random mutagenesis on Lactococcus lactis Kivd.
  • Screening of approximately 8,000 variants to identify improved single-mutation enzymes.
  • Recombination of top variants and further optimization using Rosetta Comparative Modeling.
  • Characterization of enzyme stability through melting temperature and half-life assays at elevated temperatures.

Main Results:

  • Development of a highly thermostable Kivd variant, LLM4.
  • LLM4 exhibits a 10.5-fold increase in residual activity after preincubation at 60 °C.
  • LLM4 shows a 13 °C increase in melting temperature and over a 4-fold longer half-life at 60 °C compared to wild-type Kivd.
  • Significant improvement in enzyme stability at elevated temperatures.

Conclusions:

  • The engineered LLM4 variant demonstrates enhanced thermostability, making it suitable for high-temperature applications.
  • LLM4 is a promising biocatalyst for efficient keto acid-derived alcohol production in lignocellulosic thermophiles.
  • This work overcomes the limitations of mesophilic Kdc thermostability in industrial bioprocesses.