Aspergillus niger is an important filamentous fungus in industrial biotechnology for the production of citric acid and enzymes. In the late 1980s, the A. niger N400/NRRL3 strain was selected for both fundamental and applied studies in relation to several processes including gluconic acid and protein production. To facilitate handling of A. niger, the N400 wild-type strain was UV mutagenized in two consecutive rounds to generate N401 and N402. N402 was used as a reference laboratory strain and exhibits the phenotypes with reduced conidiophore stalk length and reduced radial growth. The conidiophore stalk length and radial growth of A. niger strain N400 were determined and compared to N401 and N402. The length of N400 conidiophore stalks (2.52 ± 0.40 mm) was reduced in N401 and N402 to 0.66 ± 0.14 mm and 0.34 ± 0.06 mm, respectively. Whereas N400 reached a colony diameter of 6.7 ± 0.2 cm after 7 days, N401 and N402 displayed reduced radial growth phenotype (4.3 ± 0.1 and 4.1 ± 0.1, respectively). To identify the mutations (dubbed cspA and cspB) responsible for the phenotypes of N401 and N402, the genomes were sequenced and compared to the N400 genome sequence. A parasexual cross was performed between N400 and N402 derivatives to isolate segregants which allowed cosegregation analysis of single nucleotide polymorphisms and insertions and deletions among the segregants. The shorter conidiophore stalk and reduced radial growth in N401 (cspA) was found to be caused by a 9-kb deletion on chromosome III and was further narrowed down to a truncation of NRRL3_03857 which encodes a kinesin-like protein homologous to the A. nidulans UncA protein. The mutation responsible for the further shortening of conidiophore stalks in N402 (cspB) was found to be caused by a missense mutation on chromosome V in a hitherto unstudied C2H2 transcription factor encoded by the gene NRRL3_06646. The importance of these two genes in relation to conidiophore stalk length and radial growth was confirmed by single and double gene deletion studies. The mutations in the laboratory strain N402 should be taken into consideration when studying phenotypes in the N402 background.

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.