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

doi:10.1021/acssynbio.0c00290

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 journal › Article › peer-review

30 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 language	English (US)
Pages (from-to)	1632-1637
Number of pages	6
Journal	ACS Synthetic Biology
Volume	9
Issue number	7
DOIs	https://doi.org/10.1021/acssynbio.0c00290
State	Published - 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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10.1021/acssynbio.0c00290

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

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.",

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author = "Levi Kramer and Xuan Le and Marisa Rodriguez and Wilson, {Mark A.} and Jiantao Guo and Wei Niu",

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AU - Rodriguez, Marisa

AU - Wilson, Mark A.

AU - Guo, Jiantao

AU - Niu, Wei

PY - 2020/7/17

Y1 - 2020/7/17

N2 - 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.

AB - 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.

KW - 1,6-hexanediol

KW - 2-methoxybenzoate

KW - adipate

KW - carboxylic acid reductase

KW - enzyme engineering

KW - redox growth coupling

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

Abstract

Keywords

ASJC Scopus subject areas

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