A functionally-distinct carboxylic acid reductase (PcCAR4) unearthed from a repertoire of type IV CARs in the white-rot fungus Pycnoporus cinnabarinus

Carboxylic acid reductases (CARs) are attracting burgeoning attention as biocatalysts for organic synthesis of aldehydes and their follow-up products from economic carboxylic acid precursors. The CAR enzyme class as a whole, however, is still poorly understood. To date, relatively few CAR sequences have been reported, especially from fungal sources. Here, we sought to increase the diversity of the CAR enzyme class. Six new CAR sequences from the white-rot fungus Pycnoporus cinnabarinus were identified from genome-wide mining. Genome and gene clustering analysis suggests that these PcCARs play different natural roles in Basidiomycete systems, compared to their type II Ascomycete counterparts. The cDNA sequences of all six PcCAR genes were deduced and analysis of their corresponding amino acid sequence showed that they encode for proteins of similar properties and that they possess a conserved modular functional tri-domain arrangement. Phylogenetic analyses showed that all PcCARs cluster with together with the other type IV CARs. One candidate, PcCAR4, was cloned and over-expressed recombinantly in Escherichia coli. Subsequent biotransformation-based screening with a panel of structurally-diverse carboxylic acid substrates suggest that PcCAR4 possessed a more pronounced substrate specificity compared to previously reported CARs. These findings thus present a new functionally-distinct member of the CAR enzyme class.