Genetic Safeguard against Mycotoxin Cyclopiazonic Acid Production in Aspergillus oryzae

Abstract

Aspergillus oryzae is a fungus widely used in traditional Japanese fermentation industries. Its inability to produce mycotoxins, due to mutation or transcriptional repression of the genes responsible for their biosynthesis, is consistent with the hypothesis that A. oryzae is a domesticated species derived from A. flavus, a wild species that is a well-known producer of aflatoxin. In contrast, the cyclopiazonic acid (CPA) biosynthetic gene (cpa) cluster in A. oryzae contains genes that have been lost in A. flavus. Through targeted gene inactivation, isolation of the corresponding metabolite, and evaluation of biological activity of the metabolite, we demonstrated that an A. oryzae-specific gene-cpaH-mediates the conversion of CPA into the less toxic 2-oxocyclopiazonic acid, a new analogue of CPA. The detoxifying properties of cpaH, which have been lost in the A. flavus pathway, reflect the relationship of the two species.

Functional Analysis of the Cyclopiazonic Acid Biosynthesis Gene Cluster inAspergillus oryzaeRIB 40

“…1A). 1) We also found that cpaA is truncated by a telomere at the chromosomal end in CPA-nonproducing strain A. oryzae RIB 40 (Fig. 1A), 2) and that cpaH, which is deleted from the cpa cluster in A. flavus, is responsible for the synthesis of a new derivative of CPA, 2-oxoCPA, in CPA-producing strain A. oryzae NBRC 4177 (Fig.…”

“…The HPLC conditions were as in our previous study. 1) Chromatograms of the substrate solution fed to the fungal cultures showed a single peak of cAATrp at 19.9 min (data not shown). We confirmed that no Northern blot analysis of cpaA, cpaD, cpaO, and cpaH in the cpa cluster in A. oryzae RIB 40, NBRC 4177, and ÁcpaR, a gene disruption strain of cpaR in A. oryzae NBRC 4177.…”

“…1B). 1) 2-oxoCPA is less toxic than CPA, 1) and these results led us to consider that multiple genetic safeguards ensure or strengthen the safety of A. oryzae, although its close relative, A. flavus, has potent toxigenicity. In the CPA-nonproducing strain, A. oryzae RIB 40, truncation of cpaA appears to be one of the determinants of CPA nonproducibility.…”

“…1A). 1) To determine whether any cpa cluster genes besides truncated cpaA in A. oryzae RIB 40 are under the same genetic control as CPA-producing strain A. oryzae NBRC 4177, first we analyzed the expression levels of cpa cluster genes by Northern blot. 3.0% sucrose) at 30 C for 48 h with rotary shaking at 150 rpm.…”

“…1B). 1) cAATrp was later found to be a reaction product of PKS-NRPS encoded by the cpaA homolog in A. flavus, which shows 92% amino acid sequence identity with cpaA in A. oryzae NBRC 4177. 7) Hence the culture supernatant of A. oryzae NBRC 4177 ÁcpaD, instead of authentic cAATrp, was used as substrate in our feeding experiment.…”