Strain-specific activation of aspergillic acid biosynthesis in Aspergillus oryzae NRRL 3483

Aspergillic acid, a hydroxamate-containing pyrazinone derived from leucine and isoleucine, is a hallmark metabolite of Aspergillus flavus, which also produces the potent carcinogen aflatoxin. Its biosynthesis is governed by the recently characterized asa gene cluster. While A. flavus consistently produces aspergillic acid, its domesticated relative Aspergillus oryzae has long been considered a non-producer. Here, we compared the asa cluster activity and metabolite output in A. flavus NRRL 3357 and two A. oryzae strains (RIB40 and NRRL 3483) under amino acid-rich conditions. In A. flavus, asa genes were strongly induced in casein peptone medium, resulting in robust production of aspergillic acid, deoxyaspergillic acid, and iron-chelating ferriaspergillin. A. oryzae RIB40 showed no transcriptional activation or metabolite production. By contrast, A. oryzae NRRL 3483 exhibited clear asa cluster induction and detectable accumulation of deoxyaspergillic acid and trace aspergillic acid sufficient to form ferriaspergillin. Notably, the disproportionate buildup of deoxyaspergillic acid revealed a bottleneck in downstream tailoring steps. These results demonstrate that A. oryzae retains strain-specific, conditionally inducible aspergillic acid biosynthesis, highlighting evolutionary attenuation of secondary metabolism in domesticated fungi. This work establishes a framework for dissecting regulatory and enzymatic constraints in the asa pathway, highlighting the potential of hidden biosynthetic clusters for natural product discovery and biotechnological applications.