Amino benzamidoxime (ABAO) based assay to identify efficient aldehyde-producing Pichia pastoris clones

The chemoselective synthesis of aldehydes is a challenging task. Nature provides carboxylic acid reductases (CARs) as elegant tools for the direct reduction of carboxylic acids to their respective aldehydes. The discovery of new CARs and strains that efficiently produce these enzymes necessitates a robust high‐throughput assay with selectivity for aldehydes. We recently reported a simple assay that allows the substrate independent and chemoselective quantification of aldehydes (irrespective of their chemical structure). The assay utilized amino benzamidoxime (ABAO), which forms UV‐ active and fluorescent dihydro‐quinazolines. In this study, we adapted the ABAO‐assay for the identification and comparison of <i>Pichia pastoris</i> clones with the ability to produce aldehydes from carboxylic acids. Specifically, the CAR and PPTase from <i>Mycobacterium marinum</i> (<i>Mm</i>CAR) were co‐expressed using different bidirectional promoters (BDPs). A library of 598 clones was screened for piperonal production with the ABAO‐assay and the results were validated by HPLC quantification. 1 OD unit of the best <i>Pichia pastoris</i> clone 2.A7, regulating <i>Mm</i>CAR and <i>Mm</i>PPTase expression by two strong constitutive promoters, fully converted 5 mM of piperonylic acid within 2 h.