Screening tests of fungal metabolites were performed for developing new types of antioxidants and synergists for tocopherol (Toc). Flavoglaucin has been found to be an excellent antioxidant and synergist. It is a phenolic compound isolated from mycelial mats ofEurotium chevalieri. Under autoxidation conditions, flavoglaucin remarkably synergized with Toc and stabilized many edible oils and fats. After the addition of flavoglaucin (0.05 %) the vegetable oils retained their original stabilities even after thermal treatment at 180 C for 25 hr. During the oxidation of lard containing Toc (0.04%) under the simulated deep‐fat frying conditions, the addition of flavoglaucin didn't retard the oxidative decomposition of Toc. However, the stability of lard always was higher in the presence of flavoglaucin than in its absence. Flavoglaucin is not mutagenic to Salmonella typhimurium TA 100 and TA 98.

No abstract

Dichlorvos treatment of aflatoxigenic Aspergillus parasiticus SYS-4 (NRRL 2999) or a versicolorin Aaccumulating mutant, NIAH-9, resulted in accumulation of versiconol acetate (VOAc) and versiconal hemiacetal acetate (VHA), whereas the production of aflatoxins, versicolorin A (VA), and versiconol (VOH) decreased. In feeding experiments using another non-aflatoxigenic mutant, NIAH-26, aflatoxins were newly produced from each of VHA, VOAc, VOH, versicolorin B (VB) and versicolorin C (VC). In these experiments, aflatoxin production from VHA or VOAc was inhibited by dichlorvos, whereas that from each of VOH, VB and VC was insensitive to dichlorvos. In cell-free experiments using the cytosol fraction of NIAH-26, VHA was converted to VC (or VB) and a substance tentatively identified as versiconal (VHOH). By further addition of NADH or NADPH to the same reaction mixture, VOAc and VOH were also formed together with VC (VB) and VHOH. VOH was produced from VOAc irrespective of nicotinamide adenine nucleotide. Also, the incubation of VOH in the presence of NAD or NADP led to the formation of VC (VB). The production of VC (VB) and VHOH from VHA, and that of VOH from VOAc was inhibited by dichlorvos, whereas the production of VOAc from VHA, and that of VC (VB) from VOH, was insensitive to dichlorvos. These results indicate that a metabolic grid catalysed by dehydrogenase and esterase among VHA, VOAc, VOH and VHOH, and a reaction from VHOH to VC (VB) are involved in aflatoxin biosynthesis. These enzyme activities were also detected when yeast extract peptone medium was used, or when A. uryzae SYS-2 was examined.

The substances belonging to the sterigmatocystin group bear a close structural relationship to aflatoxins. When demethylsterigmatocystin (DMST) was fed to Aspergillus parasiticus NIAH-26, which endogenously produces neither aflatoxins nor precursors in YES medium, aflatoxins B1 and G, were produced. When dihydrodemethylsterigmatocystin (DHDMST) was fed to this mutant, aflatoxins B2 and G2 were produced. Results of the cell-free experiment with S-adenosyl-[methyl-3H]methionine showed that first the C-6-OH groups of DMST and DHDMST are methylated to produce sterigmatocystin and dihydrosterigmatocystin (0methyltransferase I) and then the C-7-OH groups are methylated to produce 0-methylsterigmatocystin (OMST) and dihydro-0-methylsterigmatocystin (DHOMST) (O-methyltransferase II). However, no methyltransferase activity was observed when either OMST, DHOMST, 5,6-dimethoxysterigmatocystin, 5-methoxysterigmatocystin, or sterigmatin was incubated with the cell extract. Treatment of the cell extract with N-ethylmaleimide inhibited 0-methyltransferase I activity but not that of 0-methyltransferase II. Furthermore, these 0-methyltransferases were different in their protein molecules and were involved in both the reactions from DMST to OMST and DHDMST to DHOMST. The reactions described in this paper were not observed when the same mold had been cultured in YEP medium.

The pathway from averufin (AVR) to versiconal hemiacetal acetate (VHA) in aflatoxin biosynthesis was investigated by using cell-free enzyme systems prepared from Aspergillus parasiticus. When (1S,5S)-AVR was incubated with a cell extract of this fungus in the presence of NADPH, versicolorin A and versicolorin B (VB), as well as other aflatoxin pathway intermediates, were formed. When the same substrate was incubated with the microsome fraction and NADPH, hydroxyversicolorone (HVN) and VHA were formed. However, (1R,5R)-AVR did not serve as the substrate. In cell-free experiments performed with the cytosol fraction and NADPH, VHA, versicolorone (VONE), and versiconol acetate (VOAc) were transiently produced from HVN in the early phase, and then VB and versiconol (VOH) accumulated later. Addition of dichlorvos (dimethyl 2,2-dichlorovinylphosphate) to the same reaction mixture caused transient formation of VHA and VONE, followed by accumulation of VOAc, but neither VB nor VOH was formed. When VONE was incubated with the cytosol fraction in the presence of NADPH, VOAc and VOH were newly formed, whereas the conversion of VOAc to VOH was inhibited by dichlorvos. The purified VHA reductase, which was previously reported to catalyze the reaction from VHA to VOAc, also catalyzed conversion of HVN to VONE. Separate feeding experiments performed with A. parasiticus NIAH-26 along with HVN, VONE, and versicolorol (VOROL) demonstrated that each of these substances could serve as a precursor of aflatoxins. Remarkably, we found that VONE and VOROL had ring-opened structures. Their molecular masses were 386 and 388 Da, respectively, which were 18 Da greater than the molecular masses previously reported. These data demonstrated that two kinds of reactions are involved in the pathway from AVR to VHA in aflatoxin biosynthesis: (i) a reaction from (1S,5S)-AVR to HVN, catalyzed by the microsomal enzyme, and (ii) a new metabolic grid, catalyzed by a new cytosol monooxygenase enzyme and the previously reported VHA reductase enzyme, composed of HVN, VONE, VOAc, and VHA. A novel hydrogenation-dehydrogenation reaction between VONE and VOROL was also discovered.

From the culture filtrates of Aspergillus sydowi two new metabolites, designated as sydonic acid and hydroxysydonic acid, have been isolated. Sydonic acid, C15H22O4, forms colorless needles, mp 158•`159•Ž and hydroxysydonic acid, C15H22O5, forms colorless needles, mp 135-136°C. These metabolites are optically inactive. From the results of chemical and

Hydroxyaverantin (HAVN) was isolated from a mold, Emericella heterothallica IFO 30842. Aspergillus parasiticus NIAH-26, a UV-irradiated mutant of A. parasiticus SYS-4, produced neither aflatoxins nor precursors in yeast extract-sucrose (YES) medium. When the postmicrosome (cytosol) fraction of NIAH-26, which had been prepared from the culture in YES medium, was incubated with norsolorinic acid (NA) in the presence of NADH or NADPH, averantin (AVN) was produced. The reverse reaction from AVN to NA was promoted by the addition of NAD or NADP (dehydrogenase reaction). When the microsome fraction of NIAH-26 was incubated with AVN, HAVN was produced in the presence of NADPH (monooxygenase reaction). HAVN was, furthermore, oxidized to averufin (AVR) by the cytosol fraction of NIAH-26 in the presence of NAD or NADP (dehydrogenase reaction). In the feeding experiments with A. parasiticus NIAH-26, aflatoxins were produced from AVN, HAVN, NA, and AVR but not from averufanin or averythrin. These results indicate that the reaction sequence NA<-*AVN-*HAVN-*AVR is involved in the biosynthetic pathway of aflatoxins. The enzyme activities described here were dependent on the culture medium, and no enzyme activities were observed in the nonaflatoxigenic strain A. oryzae SYS-2 (IFO 4251).

Aspergillus parasiticus NIAH-26, a UV-irradiated mutant of A. parasiticus SYS-4 (NRRL 2999), produces neither aflatoxins nor precursors. When sterigmatocystin (ST) or O-methylsterigmatocystin was fed to this mutant in YES medium, aflatoxins B, (AFBI) and G, (AFG,) were produced. When dihydrosterigmatocystin (DHST) or dihydro-O-methylsterigmatocystin was fed to this mold, aflatoxins B2 (AFB2) and G2 (AFG2) were produced. The reactions from ST to AFB1 and DHST to AFB2 were also observed in the cell-free system and were catalyzed stepwise by the methyltransferase and oxidoreductase enzymes. In the feeding experiments of strain NIAH-26, the convertibility from ST to AFB,-AFG, was found to be remarkably suppressed by the coexistence of DHST in the medium, and the convertibility from DHST to AFB2-AFG2 was also suppressed by the presence of ST. When some other mutants which endogenously produce a small amount of aflatoxins (mainly AFB, and AFG,) were cultured with DHST, the amounts of AFB1 and AFG, produced were significantly decreased, whereas AFB2 and AFG2 were newly produced. In similar feeding experiments in which 27 kinds of mutants including these mutants were used, most of the mutants which were able to convert exogenous ST to AFBI-AFG1 were also found to convert exogenous DHST to AFB2-AFG2. These results suggest that the same enzymes may be involved in the both biosynthetic pathways from ST to AFB,-AFG, and DHST to AFB2-AFG2. The reactions described herein were not observed when the molds had been cultured in the YEP medium. Aflatoxins B1 (AFBj), B, (AFB,), G1 (AFG1), and G, (AFG,) are toxic secondary metabolities produced by certain strains of the common molds Aspei-gillius flai'iis and A. par-asiticits (4, 22). The biosynthetic pathway of AFB1 has been extensively studied (4, 5, 22, 24), and it has been suggested that AFB1 is produced from sterigmatocystin (ST) and O-methylsterigmatocystin (OMST); also, some enzyme activities relating to several steps in the biosynthetic pathway have been reported (2, 4, 26-28). However, there are many conflicting hypotheses concerning the biosynthetic pathways of other aflatoxins (5, 6, 13-16, 20, 23), and the precise relationship between AFB1 and other aflatoxins is still unclear. Maggon and Venkitasubramanian suggested that AFB., AFG1, and AFG, may be metabolically related to AFB1 by a direct interconversion process (23). In contrast, Dutton et al. suggested that AFB1 and AFB, may arise independently via a branched pathway (13). Dihydrosterigmatocystin (DHST) and dihydro-O-methylsterigmatocystin (DHOMST) are fungal secondary metabolites that have been isolated from A. Versicolor (Vuill .) Tiraboshi (10, 17) and A.flavus (10, 11), respectively. These compounds are dihydroderivatives of ST and OMST and seemed to be precursors of AFB,, because AFB, is a dihydro derivative of AFB1. In this study, the biosynthetic relationship among four kinds of aflatoxins (AFB1, AFG1, AFB2 and AFG,) has been examined by both feeding experiments and a cell-free system.