ACS synthetic biology · 2020
Engineering Carboxylic Acid Reductase (CAR) through a Whole-Cell Growth-Coupled NADPH Recycling Strategy.
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Kramer Levi, Le Xuan, Rodriguez Marisa, Wilson Mark A, Guo Jiantao, Niu Wei
Аннотация
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.