Engineering a Thermostable Keto Acid Decarboxylase Using Directed Evolution and Computationally Directed Protein Design

Lemuel M.J. Soh, Wai Shun Mak, Paul P. Lin, Luo Mi, Frederic Y.H. Chen, Robert Damoiseaux, Justin Siegel, James C. Liao

Research output: Contribution to journalArticle

8 Scopus citations

Abstract

Keto acid decarboxylase (Kdc) is a key enzyme in producing keto acid derived higher alcohols, like isobutanol. The most active Kdc's are found in mesophiles; the only reported Kdc activity in thermophiles is 2 orders of magnitude less active. Therefore, the thermostability of mesophilic Kdc limits isobutanol production temperature. Here, we report development of a thermostable 2-ketoisovalerate decarboxylase (Kivd) with 10.5-fold increased residual activity after 1h preincubation at 60 °C. Starting with mesophilic Lactococcus lactis Kivd, a library was generated using random mutagenesis and approximately 8,000 independent variants were screened. The top single-mutation variants were recombined. To further improve thermostability, 16 designs built using Rosetta Comparative Modeling were screened and the most active was recombined to form our best variant, LLM4. Compared to wild-type Kivd, a 13 °C increase in melting temperature and over 4-fold increase in half-life at 60 °C were observed. LLM4 will be useful for keto acid derived alcohol production in lignocellulosic thermophiles.

Original languageEnglish (US)
Pages (from-to)610-618
Number of pages9
JournalACS Synthetic Biology
Volume6
Issue number4
DOIs
StatePublished - Apr 21 2017

Keywords

  • directed evolution
  • high-throughput screening
  • isobutyl alcohol
  • keto acid decarboxylase
  • protein design
  • thermostability

ASJC Scopus subject areas

  • Biomedical Engineering
  • Biochemistry, Genetics and Molecular Biology (miscellaneous)

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