Aflatoxin biosynthetic pathway: Elucidation by using blocked mutants of Aspergillus parasiticus

Two mutant strains of Aspergillus parasiticus, both deficient in aflatoxin production, were used to elucidate the biosynthetic pathway of this mycotoxin. One of the mutants, A. parasiticus ATCC 24551, was capable of accumulating large amounts of averufin, and the other, A. parasiticus 1-11-105 wh-1, accumulated versicolorin A. The averufin producing mutant efficiently converted ¹⁴C-labeled versiconal acetate, versicolorin A, and sterigmatocystin into aflatoxin B₁ and G₁, indicating that averufin preceded these compounds in the aflatoxin biosynthetic pathway. In the presence of dichlorvos (dimethyl 2,2-dichlorovinyl phosphate), a known inhibitor of aflatoxin biosynthesis, the conversion of versicolorin A and sterigmatocystin was unaffected, but the conversion of versiconal acetate was markedly inhibited. The mutant accumulating versicolorin A incorporated ¹⁴C-labeled acetate, averufin, and versiconal acetate into versicolorin A. In the presence of dichlorvos, however, the major conversion product was versiconal acetate. This strongly suggested that dichlorvos inhibited the conversion step of versiconal acetate into versicolorin A. This mutant resumed production of aflatoxin B₁ if sterigmatocystin was added to the resting cell cultures, indicating that the mutant was blocked at the enzymatic step catalyzing the conversion of versicolorin A into sterigmatocystin, and as a result was incapable of aflatoxin production. The experimental evidence is thus provided for the involvement and interrelationship of three anthraquinones (averufin, versiconal acetate, and versicolorin A) and a xanthone (sterigmatocystin) in aflatoxin biosynthesis. A pathway for the biosynthesis of aflatoxin B₁ is proposed to be: acetate →→→ averufin → versiconal acetate → versicolorin A → sterigmatocystin → aflatoxin B₁.     

The Aspergillus parasiticus estA-Encoded Esterase Converts Versiconal Hemiacetal Acetate to Versiconal and Versiconol Acetate to Versiconol in Aflatoxin Biosynthesis

In aflatoxin biosynthesis, the pathway for the conversion of 1-hydroxyversicolorone to versiconal hemiacetal acetate (VHA) to versiconal (VHOH) is part of a metabolic grid. In the grid, the steps from VHA to VHOH and from versiconol acetate (VOAc) to versiconol (VOH) may be catalyzed by the same esterase. Several esterase activities are associated with the conversion of VHA to VHOH, but only one esterase gene (estA) is present in the complete aflatoxin gene cluster of Aspergillus parasiticus. We deleted the estA gene from A. parasiticus SRRC 2043, an O-methylsterigmatocystin (OMST)-accumulating strain. The estA-deleted mutants were pigmented and accumulated mainly VHA and versicolorin A (VA). A small amount of VOAc and other downstream aflatoxin intermediates, including VHOH, versicolorin B, and OMST, also were accumulated. In contrast, a VA-accumulating mutant, NIAH-9, accumulated VA exclusively and neither VHA nor VOAc were produced. Addition of the esterase inhibitor dichlorvos (dimethyl 2,2-dichlorovinylphosphate) to the transformation recipient strain RHN1, an estA-deleted mutant, or NIAH-9 resulted in the accumulation of only VHA and VOAc. In in vitro enzyme assays, the levels of the esterase activities catalyzing the conversion of VHA to VHOH in the cell extracts of two estA-deleted mutants were decreased to approximately 10% of that seen with RHN1. Similar decreases in the esterase activities catalyzing the conversion of VOAc to VOH were also obtained. Thus, the estA-encoded esterase catalyzes the conversion of both VHA to VHOH and VOAc to VOH during aflatoxin biosynthesis.     

Enzymatic Conversion of Averufin to Hydroxyversicolorone and Elucidation of a Novel Metabolic Grid Involved in Aflatoxin Biosynthesis

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 (1′S,5′S)-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, (1′R,5′R)-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 (1′S,5′S)-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.     

The Aspergillus parasiticus estA-encoded esterase converts versiconal hemiacetal acetate to versiconal and versiconol acetate to versiconol in aflatoxin biosynthesis

In aflatoxin biosynthesis, the pathway for the conversion of 1-hydroxyversicolorone to versiconal hemiacetal acetate (VHA) to versiconal (VHOH) is part of a metabolic grid. In the grid, the steps from VHA to VHOH and from versiconol acetate (VOAc) to versiconol (VOH) may be catalyzed by the same esterase. Several esterase activities are associated with the conversion of VHA to VHOH, but only one esterase gene (estA) is present in the complete aflatoxin gene cluster of Aspergillus parasiticus. We deleted the estA gene from A. parasiticus SRRC 2043, an O-methylsterigmatocystin (OMST)-accumulating strain. The estA-deleted mutants were pigmented and accumulated mainly VHA and versicolorin A (VA). A small amount of VOAc and other downstream aflatoxin intermediates, including VHOH, versicolorin B, and OMST, also were accumulated. In contrast, a VA-accumulating mutant, NIAH-9, accumulated VA exclusively and neither VHA nor VOAc were produced. Addition of the esterase inhibitor dichlorvos (dimethyl 2,2-dichlorovinylphosphate) to the transformation recipient strain RHN1, an estA-deleted mutant, or NIAH-9 resulted in the accumulation of only VHA and VOAc. In in vitro enzyme assays, the levels of the esterase activities catalyzing the conversion of VHA to VHOH in the cell extracts of two estA-deleted mutants were decreased to approximately 10% of that seen with RHN1. Similar decreases in the esterase activities catalyzing the conversion of VOAc to VOH were also obtained. Thus, the estA-encoded esterase catalyzes the conversion of both VHA to VHOH and VOAc to VOH during aflatoxin biosynthesis.     

Function of the cypX and moxY Genes in Aflatoxin Biosynthesis in Aspergillus parasiticus

The pathway oxoaverantin (OAVN) → averufin (AVR) → hydroxyversicolorone (HVN) → 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.     

TheAspergillus parasiticus polyketide synthase genepksA,a homolog ofAspergillus nidulans wA,is required for aflatoxin B1 biosynthesis

Aflatoxins comprise a group of polyketide-derived carcinogenic mycotoxins produced byAspergillus parasiticus andAspergillus flavus. By transformation with a disruption construct, pXX, we disrupted the aflatoxin pathway inA. parasiticus SRRC 2043, resulting in the inability of this strain to produce aflatoxin intermediates as well as a major yellow pigment in the transformants. The disruption was attributed to a single-crossover, homologous integration event between pXX and the recipientA. parasiticus genome at a specific locus, designatedpksA. Sequence analysis suggest thatpksA is a homolog of theAspergillus nidulans wA gene, a polyketide synthase gene involved in conidial wall pigment biosynthesis. The conservedβ-ketoacyl synthase, acyltransferase and acyl carrier-protein domains were present in the deduced amino acid sequence of thepksA product. Noβ-ketoacyl reductase and enoyl reductase domains were found, suggesting thatpksA does not encode catalytic activities for processingβ-carbon similar to those required for long chain fatty acid synthesis. ThepksA gene is located in the aflatoxin pathway gene cluster and is linked to thenor-1 gene, an aflatoxin pathway gene required for converting norsolorinic acid to averantin. These two genes are divergently transcribed from a 1.5 kb intergenic region. We propose thatpksA is a polyketide synthase gene required for the early steps of aflatoxin biosynthesis.     

Limited survey on aflatoxin contamination in rice

Aflatoxins (AFS) are toxic and carcinogenic fungal metabolites. Aflatoxin B1 is the most toxic and has been classified as a Group I carcinogen by the International Agency for Research on Cancer (IARC). Samples of imported rice were analyzed for their AFS content. Finley ground rice subsamples were extracted with water/methanol (100:150 v/v) followed by purification with Immunoaffinity columns (IAC). AFS purified from extracts were determined with RP-HPLC-FLD using post column electrochemical derivatization with a Kobra Cell. Concentrations of aflatoxin B1 and total AFS in test rice samples were ≤0.123 and ≤2.58 µg/kg, respectively. Tween 80 improved recoveries (86 and 106%) of aflatoxin B1 and aflatoxin G1 from brown rice. Recoveries of Aflatoxin B2 and aflatoxin G2 were substantially reduced (non-detected to 27%) by Tween 80 used in IAC cleanup of brown rice extracts. Visible dense growth of Aspergillus parasiticus (food isolate) occurred at 25 °C but higher aflatoxin B1amounts (23.9–39.3 µg/kg) accumulated when the mold grew at 37 °C in rice seeds stored for three weeks. It could be concluded that levels of aflatoxin B1 and total AFS in rice samples were within the permissible amounts of the EU and other international legislations.     

Purification and Characterization of Two Versiconal Hemiacetal Acetate Reductases Involved in Aflatoxin Biosynthesis

Two versiconal hemiacetal acetate (VHA) reductase activities (designated I and II), which catalyzed the reaction from VHA to versiconol acetate (VOAc) during aflatoxin biosynthesis, were purified to apparent homogeneity from the cytosol fraction of the mycelia of Aspergillus parasiticus mutant NIAH-26 through the following chromatography steps: first, fractionation with ammonium sulfate and then fractionation in succession with phenyl-Sepharose, DEAE-Sepharose, Sephacryl S-300, hydroxylapatite, and Matrex gel Green A chromatography. VHA reductase I and VHA reductase II were completely separated at the end of the DEAE-Sepharose step. The apparent molecular masses of reductase I and reductase II were estimated (by gel filtration) to be approximately 390 kDa; their denaturing molecular masses were 39- and 40-kDa, respectively (by sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Their pI values were 6.6 and 6.0, respectively (as determined by isoelectric focusing), and the optimal pH values were 8.0 and 9.0, respectively, although both enzymes exhibited a broad optimal pH range of between 7.5 and 9.0. The K_m values of reductase I and reductase II for VHA were 35.4 and 25.4 μM, respectively. On the other hand, in the cell-free experiments involving either VHA reductase fraction and high-performance liquid chromatography, both (2′S)- and (2′R)-VOAc enantiomers were formed from racemic VHA and more of the 2′R isomer than the 2′S isomer was produced, indicating that the VHA reductase fractions have very similar stereospecificities to the substrate.     

Enzymatic conversion of averufin to hydroxyversicolorone and elucidation of a novel metabolic grid involved in aflatoxin biosynthesis

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 (1'S,5'S)-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, (1'R,5'R)-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 (1'S,5'S)-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.     

Aspergillus parasiticus Cyclase Catalyzes Two Dehydration Steps in Aflatoxin Biosynthesis

In the aflatoxin biosynthetic pathway, 5′-oxoaverantin (OAVN) cyclase, the cytosolic enzyme, catalyzes the reaction from OAVN to (2′S,5′S)-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.     

Detoxification of aflatoxin B1 by an aqueous extract from leaves of Adhatoda vasica Nees

The effectiveness of aqueous extracts of various medicinal plants in detoxification of aflatoxin B1 (AFB1) was tested in vitro by thin-layer chromatography and enzyme-linked immunosorbent assay (ELISA). Among the different plant extracts, the leaf extract of Vasaka (Adhatoda vasica Nees) showed the maximum degradation of AFB1 (≥98%) after incubation for 24 h at 37 °C. The aflatoxin detoxifying activity of the A. vasica leaf extract was significantly reduced by heating to 100 °C for 10 min or autoclaving at 121 °C for 20 min. Dialysis had no effect on aflatoxin detoxifying ability of A. vasica extract and the dialyzed extract showed similar level of detoxification of AFB1 as that of the untreated extract. A time course study of aflatoxin detoxification by A. vasica extract showed that 69% of the toxin was degraded within 6 h and ≥95% degradation was observed after 24 h of incubation. Detoxification of AFB1 by A. vasica extract was further confirmed by liquid chromatography–mass spectrometry (LC–MS) analysis. Phytochemical analysis revealed the presence of alkaloids in methanolic extract of A. vasica leaves. A partially purified alkaloid from A. vasica leaves by preparative TLC exhibited strong AFB1 detoxification activity.     

Enzyme reactions and genes in aflatoxin biosynthesis

Aflatoxins are highly toxic and carcinogenic substances mainly produced by Aspergillus flavus and Aspergillus parasiticus. Sterigmatocystin is a penultimate precursor of aflatoxins and also a toxic and carcinogenic substance produced by many species, including Aspergillus nidulans. Recently, the majority of the enzyme reactions involved in aflatoxin/sterigmatocystin biosynthesis have been clarified, and the genes encoding the enzymes have been isolated. Most of the genes constitute a large gene cluster in the fungal genome, and their expression is mostly regulated by a product of the regulatory gene aflR. This review will summarize the enzymatic steps and the genes in aflatoxin/sterigmatocystin biosynthesis.     

A metabolic grid among versiconal hemiacetal acetate, versiconol acetate, versiconol and versiconal during aflatoxin biosynthesis.

Dichlorvos treatment of aflatoxigenic Aspergillus parasiticus SYS-4 (NRRL 2999) or a verscolorin A-accumulating 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. oryzae SYS-2 was examined.     

5-Azacytidine inhibits aflatoxin biosynthesis in Aspergillus flavus

Aflatoxins, mainly produced by Aspergillus flavus and A. parasiticus, are a group of potent mycotoxins with carcinogenic, hepatotoxic, and immunosuppressive properties. Many studies have been devoted to investigating their biosynthesis mechanism since they were discovered half a century ago. 5-Azacytidine (5-AC), a derivative of the nucleoside cytidine and an inactivator of DNA methyltransferase, is widely used for studies in epigenetics and cancer biology, and has also been used for studying secondary metabolism in fungi. In this study, 5-AC was applied to investigate its effect on the development and aflatoxin biosynthesis of A. flavus. The results indicate that 5-AC inhibits the ability to produce aflatoxin and also causes a fluffy aconidial phenotype. Further studies revealed that 5-AC affects gene expression of A. flavus to a limited degree, and the unique homolog of DNA methyltransferase gene (DmtA) expressed constitutively during different developmental stages of A. flavus irrespective of 5-AC. This work may provide some basic data to elucidate the role of 5-AC in aflatoxin biosynthesis and the development of A. flavus.     

Induction of aflatoxin biosynthesis inAspergillus parasiticus by ascorbic acid-mediated lipid peroxidation

The effect ofl-ascorbic acid on the biosynthesis of aflatoxin inAspergillus parasiticus was studied. Ascorbic acid at lower concentrations did not inhibit the growth of fungus but markedly induced aflatoxin biosynthesis. At a concentration of 1000 ppm of ascorbic acid, 4.8-fold higher levels of aflatoxin were detected. Copper did not enhance the induction of toxin synthesis by ascorbic acid when added to the growth medium. Ascorbic acid at 1000 ppm was also found to induce aflatoxin synthesis in resting mycelia. Chloroform (1% vol/vol) was found to induce aflatoxin synthesis under similar conditions. Ascorbic acid in the presence of ferrous ion can cause lipid peroxidation, which in turn is responsible for the induction of aflatoxin synthesis. During the induction of aflatoxin synthesis by ascorbic acid, the uptake of carbon source (acetate) was not affected. This observation suggests that on ascorbic acid treatment a precursor or an intermediate of aflatoxin biosynthesis is synthesized in vivo and is responsible for the higher levels of toxin without increasing the uptake of acetate.