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The cAMP signal transduction pathway controls many processes in fungi. The pkaR gene, encoding the regulatory subunit (PKA-R) of cAMP-dependent protein kinase (PKA), was cloned from the industrially important filamentous fungus Aspergillus niger. To investigate the involvement of PKA in morphology of A. niger, a set of transformants which overexpressed pkaR or pkaC (encoding the catalytic subunit of PKA) either individually or simultaneously was prepared as well as mutants in which pkaR and/or pkaC were disrupted. Strains overexpressing pkaR or both pkaC and pkaR could not be distinguished from the wild-type, suggesting that regulation of PKA activity is normal in these strains. Absence of PKA activity resulted in a two- to threefold reduction in colony diameter on plates. The most severe phenotype was observed in the absence of PKA-R, i.e., very small colonies on plates, absence of sporulation and complete loss of growth polarity during submerged growth. Suppressor mutations easily developed in the DeltapkaR mutant and one of these mutants appeared to lack PKA-C activity. These data suggest that cAMP-dependent protein phosphorylation in A. niger regulates growth polarity and formation of conidiospores.

The external pH appeared to be the main factor governing oxalic acid production by Aspergillus niger. A glucose-oxidase-negative mutant produced substantial amounts of oxalic acid as long as the pH of the culture was 3 or higher. When pH was decreased below 2, no oxalic acid was formed. The activity of oxaloacetate acetylhydrolase (OAH), the enzyme believed to be responsible for oxalate formation in A. niger, correlated with oxalate production. OAH was purified from A. niger and characterized. OAH cleaves oxaloacetate to oxalate and acetate, but A. niger never accumulated any acetate in the culture broth. Since an A. niger acuA mutant, which lacks acetyl-CoA synthase, did produce some acetate, wild-type A. niger is apparently able to catabolize acetate sufficiently fast to prevent its production. An A. niger mutant, prtF28, previously isolated in a screen for strains deficient in extracellular protease expression, was shown here to be oxalate non-producing. The prtF28 mutant lacked OAH, implying that OAH is the only enzyme involved in oxalate production in A. niger. In a traditional citric acid fermentation low pH and absence of Mn2+ are prerequisites. Remarkably, a strain lacking both glucose oxidase (goxC) and OAH (prtF) produced citric acid from sugar substrates in a regular synthetic medium at pH 5 and under these conditions production was completely insensitive to Mn2+.

Summary: Aspergillus niger mutants relieved of carbon repression were isolated from an areA parental strain by selection of colonies that exhibited improved growth on a combination of 4-aminobutanoic acid (GABA) and D-glucose. In addition to derepression of the utilization of GABA as a nitrogen source in the presence of D-glucose, three of the four mutants also showed derepression of L-alanine and L-proline utilization. Transformation of the mutants with the A. niger creA gene, encoding the repressor protein CREA, re-established the areA phenotype on GABA/D-glucose, identifying the mutations as creA d. The creA gene mapped on chromosome IV by linkage analysis and contour-clamped homogeneous electric field hybridization. The creA mutants obtained were used to study the involvement of CREA in repression by D-glucose of arabinases and L-arabinose catabolism in A. niger. In wild-type A. niger, α-L-arabinofuranosidase A, α-L-arabinofuranosidase B, endo-arabinase, L-arabinose reductase and L-arabitol dehydrogenase were induced on L-arabinose, but addition of D-glucose prevented this induction. Repression was relieved to varying degrees in the creA mutants, showing that biosynthesis of arabinases and L-arabinose catabolic enzymes is under control of CREA.