The pathway oxoaverantin (OAVN) 3 averufin (AVR) 3 hydroxyversicolorone (HVN) 3 versiconal hemiacetal acetate (VHA) is involved in aflatoxin biosynthesis, and the cypX and moxY genes, which are present in the aflatoxin gene cluster, have been previously suggested to be involved in this pathway. To clarify the function of these two genes in more detail, we disrupted the genes in aflatoxigenic Aspergillus parasiticus NRRL 2999. The cypX-deleted mutant lost aflatoxin productivity and accumulated AVR in the mycelia. Although this mutant converted HVN, versicolorone (VONE), VHA, and versiconol acetate (VOAc) to aflatoxins in feeding experiments, it could not produce aflatoxins from either OAVN or AVR. The moxY-deleted mutant also lost aflatoxin productivity, whereas it newly accumulated HVN and VONE. In feeding experiments, this mutant converted either VHA or VOAc to aflatoxins but did not convert OAVN, AVR, HVN, or VONE to aflatoxins. These results demonstrated that cypX encodes AVR monooxygenase, catalyzing the reaction from AVR to HVN, and moxY encodes HVN monooxygenase, catalyzing a Baeyer-Villiger reaction from HVN to VHA as well as from VONE to VOAc. In this work, we devised a simple and rapid method to extract DNA from many fungi for PCR analyses in which cell disruption with a shaker and phenol extraction were combined.Aflatoxins (AF) are a group of polyketide-derived secondary metabolites produced mainly by certain strains of the common molds Aspergillus flavus and Aspergillus parasiticus (16). Some other strains of Aspergillus nomius (13), Aspergillus pseudotamarii (8), Aspergillus bombycis (17), and Aspergillus ochraceoroseus (12) have also been reported to produce aflatoxins. These toxins are highly toxic and carcinogenic in animals and humans, leading to hepatotoxicity, teratogenicity, immunotoxicity, and even death (3). Among the naturally occurring aflatoxins, the four major ones are aflatoxin B 1 (AFB 1 ), AFB 2 , AFG 1 , and AFG 2 , of which AFB 1 is the most toxic and carcinogenic compound. Contamination of food and feed crops, such as wheat, corn, cotton, peanuts, and tree nuts, with AFB 1

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In the aflatoxin biosynthetic pathway, 5-oxoaverantin (OAVN) cyclase, the cytosolic enzyme, catalyzes the reaction from OAVN to (2S,5S)-averufin (AVR) (E. Sakuno, K. Yabe, and H. Nakajima, Appl. Environ. Microbiol. 69:6418-6426, 2003). Interestingly, the N-terminal 25-amino-acid sequence of OAVN cyclase completely matched an internal sequence of the versiconal (VHOH) cyclase that was deduced from its gene (vbs). The purified OAVN cyclase also catalyzed the reaction from VHOH to versicolorin B (VB). In a competition experiment using the cytosol fraction of Aspergillus parasiticus, a high concentration of VHOH inhibited the enzyme reaction from OAVN to AVR, and instead VB was newly formed. The recombinant Vbs protein, which was expressed in Pichia pastoris, showed OAVN cyclase activity, as well as VHOH cyclase activity. A mutant of A. parasiticus SYS-4 ‫؍(‬ NRRL 2999) with vbs deleted accumulated large amounts of OAVN, 5-hydroxyaverantin, averantin, AVR, and averufanin in the mycelium. These results indicated that the cyclase encoded by the vbs gene is also involved in the reaction from OAVN to AVR in aflatoxin biosynthesis. Small amounts of VHOH, VB, and aflatoxins also accumulated in the same mutant, and this accumulation may have been due to an unknown enzyme(s) not involved in aflatoxin biosynthesis. This is the first report of one enzyme catalyzing two different reactions in a pathway of secondary metabolism.Aflatoxins are toxic, carcinogenic, and mutagenic secondary metabolites mainly produced by certain strains of Aspergillus flavus and Aspergillus parasiticus. Contamination by aflatoxins in food and feed is a serious problem in many areas of the world. The biosynthetic pathway of aflatoxin has been studied extensively, and most of the steps have been clarified (reviewed in references 12, 16, 18, 19, and 29). Many enzymes and the genes encoding the enzymes have been isolated, and most of the enzyme genes were found to comprise a huge cluster over 70 kb long in the fungal genome (16,25,27,29).We recently reported that two enzymes are involved in the pathway from 5Ј-hydroxyaverantin (HAVN) to averufin (AVR); HAVN dehydrogenase catalyzes the conversion of HAVN to 5Ј-oxoaverantin (OAVN), and OAVN cyclase catalyzes the next reaction from OAVN to AVR (13). These enzymes have been purified and characterized. The identity of HAVN dehydrogenase with the gene product of adhA (3) was confirmed by tryptic digestion and matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis (13). The molecular masses of natural OAVN cyclase and denatured OAVN cyclase are 158 kDa and 79 kDa, respectively.In this study, we determined the amino acid sequence of the enzyme to find the gene encoding the OAVN cyclase. Surprisingly, the N-terminal sequence of OAVN cyclase was the same as a stretch of the versiconal (VHOH) cyclase sequence that was deduced from the reported vbs gene (15). VHOH cyclase has been purified independently by Lin and Anderson (10) and by McGuire et al. (11). VHOH cyclase catalyzes the co...

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In aflatoxin biosynthesis, aflatoxins G(1) (AFG(1)) and B(1) (AFB(1)) are independently produced from a common precursor, O-methylsterigmatocystin (OMST). Recently, 11-hydroxy-O-methylsterigmatocystin (HOMST) was suggested to be a later precursor involved in the conversion of OMST to AFB(1), and conversion of HOMST to AFB(1) was catalyzed by OrdA enzyme. However, the involvement of HOMST in AFG(1) formation has not been determined. In this work, HOMST was prepared by incubating OrdA-expressing yeast with OMST. Feeding Aspergillus parasiticus with HOMST allowed production of AFG(1) as well as AFB(1). In cell-free systems, HOMST was converted to AFG(1) when the microsomal fraction, the cytosolic fraction from A. parasiticus, and yeast expressing A. parasiticus OrdA were added. These results demonstrated (1) HOMST is produced from OMST by OrdA, (2) HOMST is a precursor of AFG(1) as well as AFB(1), and (3) three enzymes, OrdA, CypA, and NadA, and possibly other unknown enzymes are involved in conversion of HOMST to AFG(1).

Five carboxin-resistant mutants from Aspergillus oryzae were characterized by the sensitivities of their mycelial growth and succinate dehydrogenase (SDH) activity to carboxin and three related fungicides. Despite a significant resistance to carboxin, exhibited by all the mutants, their patterns of sensitivity to the other fungicides was distinct. This provides clues to the molecular interaction between SDH and these fungicides.

The dichlorvos–ammonia (DV–AM) method is a sensitive method for distinguishing aflatoxigenic fungi by detecting red (positive) colonies. In this study, the DV–AM method was applied for the isolation of aflatoxigenic and atoxigenic fungi from soil samples from a maize field in Mexico. In the first screening, we obtained two isolates from two soil subsamples of 20 independent samples and, in the second screening, we obtained two isolates from one subsample of these. Morphological and phylogenic analyses of the two isolates (MEX-A19-13, MEX-A19-2nd-5) indicated that they were Aspergillus flavus located in the A. flavus clade. Chemical analyses demonstrated that one isolate could produce B-type aflatoxins, while the other produced no aflatoxins. These results demonstrate that the DV–AM method is useful for the isolation of both aflatoxigenic and atoxigenic Aspergilli.