Abstract
Tannic acid, a hydrolysable gallotannin present in plant tissues, consists of a central glucose molecule esterified with gallic acid molecules. Some microorganisms, including several Aspergillus species, can metabolize tannic acid by releasing gallic acid residues from tannic acid by secreting tannic acid specific esterases into the medium. The expression of these so-called tannases is induced by tannic acid or gallic acid. In this study, we identified a conserved transcriptional activator-repressor module involved in the regulation of predicted tannases and other genes involved in gallic acid metabolism. The transcriptional activator-repressor module regulating tannic acid utilization resembles the transcriptional activator-repressor modules regulating galacturonic acid and quinic acid utilization. Like these modules, the Zn(II)2Cys6 transcriptional activator (TanR) and the putative repressor (TanX) are located adjacent to each other. Deletion of the transcriptional activator (ΔtanR) results in inability to grow on gallic acid and severely reduces growth on tannic acid. Deletion of the putative repressor gene (ΔtanX) results in the constitutive expression of tannases as well as other genes with mostly unknown function. Known microbial catabolic pathways for gallic acid utilization involve so-called ring cleavage enzymes, and two of these ring cleavage enzymes show increased expression in the ΔtanX mutant. However, deletion of these two genes, and even deletion of all 17 genes encoding potential ring cleavage enzymes, did not result in a gallic acid non-utilizing phenotype. Therefore, in A. niger gallic acid utilization involves a hitherto unknown pathway. Transcriptome analysis of the ΔtanX mutant identified several genes and gene clusters that were significantly induced compared to the parental strain. The involvement of a selection of these genes and gene clusters in gallic acid utilization was examined by constructing gene deletion mutants and testing their ability to grow on gallic acid. Only the deletion of a gene encoding an FAD-dependent monooxygenase (NRRL3_04659) resulted in a strain that was unable to grow on gallic acid. Metabolomic studies showed accumulation of gallic acid in the ΔNRRL3_04659 mutant suggesting that this predicted monooxygenase is involved in the first step of gallic acid metabolism and is likely responsible for oxidation of the aromatic ring.