Selective Enzymatic Transformation to Aldehydes <i>in vivo</i> by Fungal Carboxylate Reductase from <i>Neurospora crassa</i>

Abstract

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 <i>Neurospora crassa</i> OR74A (<i>Nc</i>CAR). <i>Nc</i>CAR was efficiently expressed in <i>E. coli</i> using an autoinduction protocol at low temperature. It was purified and characterized <i>in vitro</i>, revealing a broad substrate acceptance, a pH optimum at pH 5.5-6.0, a <i>T</i>_m of 45 °C and inhibition by the co-product pyrophosphate which can be alleviated by the addition of pyrophosphatase. The synthetic utility of <i>Nc</i>CAR was demonstrated in a whole-cell biotransformation using the <i>Escherichia coli</i> 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.