NADH Driven Enzymatic Carboxylic Acid Reduction

Carboxylic acid reductases (CARs) are versatile biocatalysts for aldehyde production from carboxylic acids. The redox cofactor utilized by CARs is NADPH and no explicitly NADH-dependent CARs have been described so far. Especially in view of cell free applications which necessitate cofactor recycling, NADH-dependent enzymes are more appealing due to higher cofactor stability, lower cost, and more flexibility in the choice of recycling systems. We aimed to extend the current toolbox of CARs with NADH-dependent enzymes and explored a yet untapped family of putative CARs. A CAR derived from Clostridium bornimense was readily produced in Escherichia coli and its cofactor preference studied in detail and compared to that of known CARs. Most surprisingly, many of the work-horse enzymes like Mycobacterium marinum CAR are able to utilize NADH as a cofactor. In conclusion, NADPH remains the preferred cofactor for all CARs studied herein, but conversions can reach almost the same levels with both cofactors in some cases.