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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 dihydroquinazolines. In this study, we adapted the ABAO‐assay for the identification and comparison of Pichia pastoris clones with the ability to produce aldehydes from carboxylic acids. Specifically, CAR and PPTase from Mycobacterium marinum (MmCAR and MmPPTase) 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 Pichia pastoris clone 2.A7, regulating MmCAR and MmPPTase expression by two strong constitutive promoters, fully converted 5 mM of piperonylic acid within 2 h. [ABSTRACT FROM AUTHOR]

The selective and direct reduction of carboxylic acids into the corresponding aldehydes by chemical methods is still a challenging task in synthesis. Several reductive and oxidative chemical methods are known to produce aldehydes, but most of them require expensive reagents, special reaction conditions, are two‐step procedures and often lack chemoselectivity. Nature provides an elegant tool, so called carboxylic acid reductases (CARs) for the direct reduction of carboxylic acids to aldehydes. Discovery as well as engineering of novel CAR enzymes necessitates a robust, product selective high‐throughput assay (HTA). We report a simple and fast HTA that allows the substrate‐independent and chemoselective quantification of aldehydes (irrespective of their chemical structure) and is sensitive to the nM range. The HTA was validated by NMR and GC analyses and in microbial cells by reexamination of the substrate scope of CAR from Nocardia iowensis (CARNi). The results were fully consistent with reported data. [ABSTRACT FROM AUTHOR]

The enzymatic reduction of carboxylic acids is in its infancy with only a handful of biocatalysts available to this end. We have increased the spectrum of carboxylate-reducing enzymes (CARs) with the sequence of a fungal CAR from Neurospora crassa OR74A ( NcCAR). NcCAR was efficiently expressed in E. coli using an autoinduction protocol at low temperature. It was purified and characterized in vitro, revealing a broad substrate acceptance, a pH optimum at pH 5.5-6.0, a Tm of 45 °C and inhibition by the co-product pyrophosphate which can be alleviated by the addition of pyrophosphatase. The synthetic utility of NcCAR was demonstrated in a whole-cell biotransformation using the Escherichia coli K-12 MG1655 RARE strain in order to suppress overreduction to undesired alcohol. The fragrance compound piperonal was prepared from piperonylic acid (30 mM) on gram scale in 92 % isolated yield in >98% purity. This corresponds to a productivity of 1.5 g/L/h. [ABSTRACT FROM AUTHOR]