Random Mutagenesis‐Driven Improvement of Carboxylate Reductase Activity using an Amino Benzamidoxime‐Mediated High‐Throughput Assay

Carboxylic acid reductases (CARs) catalyze the direct adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide phosphate (NADPH) dependent reduction of carboxylic acids to their corresponding aldehydes. The identification and improvement of CARs by protein engineering is, however, severely limited by the lack of fast and generic methods to quantify aldehydes. Within this study, we applied a convenient high‐throughput assay (HTA) based on amino benzamidoxime (ABAO) that allows the substrate‐independent and chemoselective quantification of aldehydes. Random mutagenesis of the well‐known CAR from Nocardia iowensis (CARNi) to improve its activity for sterically demanding 2‐substituted benzoic acid derivatives was conducted in a KM‐dependent fashion, and the HTA applied in the presence of microbial cells. The study identified a hot spot in the active site of CARNi that increased the affinity to 2‐methoxybenzoic acid 9‐fold upon mutation from glutamine to proline (Q283P). The catalytic performance of CARNiQ283P appeared to be significantly improved also for other substrates such as 2‐substituted (2‐Cl, 2‐Br) as well as 3‐ and 4‐substituted benzoic acids (3‐OMe, 4‐OMe), and even aliphatic octanoic acid.