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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