Engineering Carboxylic Acid Reductase (CAR) through a Whole-Cell Growth-Coupled NADPH Recycling Strategy

Levi Kramer, Xuan Le, Marisa Rodriguez, Mark A. Wilson, Jiantao Guo, Wei Niu

Research output: Contribution to journalArticlepeer-review

3 Scopus citations

Abstract

Rapid evolution of enzyme activities is often hindered by the lack of efficient and affordable methods to identify beneficial mutants. We report the development of a new growth-coupled selection method for evolving NADPH-consuming enzymes based on the recycling of this redox cofactor. The method relies on a genetically modified Escherichia coli strain, which overaccumulates NADPH. This method was applied to the engineering of a carboxylic acid reductase (CAR) for improved catalytic activities on 2-methoxybenzoate and adipate. Mutant enzymes with up to 17-fold improvement in catalytic efficiency were identified from single-site saturated mutagenesis libraries. Obtained mutants were successfully applied to whole-cell conversions of adipate into 1,6-hexanediol, a C6 monomer commonly used in polymer industry.

Original languageEnglish (US)
Pages (from-to)1632-1637
Number of pages6
JournalACS Synthetic Biology
Volume9
Issue number7
DOIs
StatePublished - Jul 17 2020

Keywords

  • 1,6-hexanediol
  • 2-methoxybenzoate
  • adipate
  • carboxylic acid reductase
  • enzyme engineering
  • redox growth coupling

ASJC Scopus subject areas

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

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