Identification and expression of a hydrophobic carboxylic acid reductase from Trichoderma virens

Carboxylic acid reductases (CARs) have been garnering attention in applications for the sustainable synthesis of aldehydes. Despite numerous discoveries, not all characteristics of CAR enzymes have been extensively studied or understood. Herein, we report the discovery and expression of a new CAR enzyme (TvirCAR2) from the ascomycetous fungus, Trichoderma virens. Tvircar2 is one of the five putative CARs identified from analyses of the T. virens genome. In silico, analyses showed that TvirCAR2 has a high hydrophobicity index and that its corresponding gene is part of a biosynthetic gene cluster predicted to synthesize hybrid polyketide synthases-nonribosomal peptide synthetase secondary metabolites. TvirCAR2 was highly expressed as soluble and insoluble forms in an Escherichia coli expression host. The solubility of the purified TvirCAR2 necessitated the addition of glycerol in the purification and assay buffers. Substrate screening via molecular docking showed that benzoic acid was a suitable substrate candidate. The TvirCAR2 enzyme catalyzed the reduction of benzoic acid with a specific activity of around 1.4 mu mol/h/mg. Homologs, which are predicted to exhibit similar hydrophobicity, are the CARs from Stachybotrys bisbyi (StbB), which is involved in the production of the meroterpenoid, ilicicolin B, and Trichoderma reesei (TrCAR), which is part of a similar but still uncharacterized biosynthetic gene cluster. We have identified, produced and characterized a new novel carboxylic acid reductase (CAR) from the filamentous fungus Trichoderma virens. We have identified features not previously seen or reported in other CARs, which supports the role of this CAR as a natural tailoring enzyme in a hybrid polyketide synthase/ nonribosomal peptide synthetase-linked pathway.