Association of aflatoxin biosynthesis and sclerotialdevelopment in Aspergillus parasiticus

Secondary metabolism in fungi is frequently associated with asexual and sexual development. Aspergillus parasiticus produces aflatoxins known to contaminate a variety of agricultural commodities. This strictly mitotic fungus, besides producing conidia asexually, produces sclerotia, structures resistant to harsh conditions and for propagation. Sclerotia are considered to be derived from the sexual structure, cleistothecia, and may represent a vestige of ascospore production. Introduction of the aflatoxin pathway-specific regulatory gene, aflR, and aflJ, which encoded a putative co-activator, into an O-methylsterigmatocystin (OMST)-accumulating strain,A. parasiticus SRRC 2043, resulted in elevated levels of accumulation of major aflatoxin precursors, including norsolorinic acid (NOR), averantin (AVN), versicolorin A (VERA) and OMST. The total amount of these aflatoxin precursors, NOR, VERA, AVN and OMST, produced by the aflR plus aflJ transformants was two to three-fold that produced by the aflR transformants. This increase indicated a synergisticeffect of aflR and aflJ on the synthesis of aflatoxin precursors. Increased production of the aflatoxin precursors was associated with progressive decrease in sclerotial size, alteration in sclerotial shape and weakening in the sclerotial structure of the transformants. The results showed that sclerotial development and aflatoxin biosynthesis are closely related. We proposed that competition for a common substrate, such as acetate, by the aflatoxin biosynthetic pathway could adversely affect sclerotial development in A. parasiticus. This revised version was published online in June 2006 with corrections to the Cover Date.     

Primary metabolism of Aspergillus parasiticus in relation to aflatoxin biosynthesis

Summary The uptake of various ¹⁴C labelled compounds like (1-¹⁴C) glucose, (1-¹⁴C) acetate, (2-¹⁴C) uracil, (1-¹⁴C) leucine and (¹⁴C–CH₃) methionine was studied in Aspergillus parasiticus. A comparative study of asparagine deficient, zinc deficient and SLS cultures revealed different growth patterns. High lipid levels under zinc and asparagine deficiency were observed. During the stationary phase the synthesis of proteins and DNA declined. The uptake of ¹⁴C labelled glucose, methionine and acetate was maximum in asparagine deficient cultures during the transitional and stationary phase of growth. Maximum uptake of labelled methionine and glucose occured during the exponential growth phase (45 h). The uptake of labelled leucine was highest under asparagine deficiency during the exponential and transitional phases but reached a minimum during stationary phase. The uptake of labelled uracil remained high throughout in the asparagine deficient cultures. The mechanism of inhibition of aflatoxin biosynthesis in the absence of zinc and asparagine seems to be different.     

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.     

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.     

Identification of genes differentially expressed during aflatoxin biosynthesis in Aspergillus flavus and Aspergillus parasiticus

A complex regulatory network governs the biosynthesis of aflatoxin. While several genes involved in aflatoxin production are known, their action alone cannot account for its regulation. Arrays of clones from an Aspergillus flavus cDNA library and glass slide microarrays of ESTs were screened to identify additional genes. An initial screen of the cDNA clone arrays lead to the identification of 753 unique ESTs. Many showed sequence similarity to known metabolic and regulatory genes; however, no function could be ascribed to over 50% of the ESTs. Gene expression analysis of Aspergillus parasiticus grown under conditions conducive and non-conductive for aflatoxin production was evaluated using glass slide microarrays containing the 753 ESTs. Twenty-four genes were more highly expressed during aflatoxin biosynthesis and 18 genes were more highly expressed prior to aflatoxin biosynthesis. No predicted function could be ascribed to 18 of the 24 genes whose elevated expression was associated with 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.     

Effect of miconazole on growth and aflatoxin production by Aspergillus parasiticus

At 5 M, miconazole prevented the growth of Aspergillus parasiticus Speare in a number of media. Sensitivity to miconazole was increased approximately 10-fold in a medium containing glycerol. At sub-inhibitory concentrations, miconazole stimulated aflatoxin synthesis on media which normally support toxin formation. Miconazole inhibited respiration and altered mitochondrial ultrastructure, suggesting that miconazole inhibits growth and stimulates aflatoxin production by depressing mitochondrial activity.     

Enzymatic function of the nor-1 protein in aflatoxin biosynthesis in Aspergillus parasiticus

The nor-1 gene is involved in aflatoxin biosynthesis in Aspergillus parasiticus and was predicted to encode a norsolorinic acid ketoreductase. Recombinant Nor-1 expressed in Escherichia coli converted the 1' keto group of norsolorinic acid to the 1' hydroxyl group of averantin in crude E. coli cell extracts in the presence of NADPH. The results confirm that Nor-1 functions as a ketoreductase in vitro.     

Effect of selected inhibitors on growth,pigmentation, and aflatoxin production by Aspergillus parasiticus

We have treated a wild type strain of Aspergillus parasiticus with several known aflatoxin inhibitors in hopes of finding specific metabolic blocks in the aflatoxin biosynthetic pathway. In defined medium, benzole acid (2 and 3 mg/ml), cinnamon (1 mg/ml), and sodium acetate (5 mg/ml) were fungitoxic. Benzoic acid (0.5 and 1 mg/ml), chlorox (5 l/ml), and dimethyl sulfoxide (5 l/ml) did not affect dry weight or mycelial pigmentation. Sodium benzoate (1, 2, 4 and 8 mg/ml) added after 2 days growth inhibited aflatoxin production in two defined media. We were unable to confirm previously published reports that an uncharacterized yellow pigment accumulates with benzoate-inhibition of aflatoxin biosynthesis.     

Effect of pyridazinone herbicides on growth and aflatoxin release by Aspergillus flavus and Aspergillus parasiticus

The influence of pyridazinone herbicides on aflatoxin production by Aspergillus flavus and A. parasiticus was studied in liquid media. Mycelia production was not affected by 20, 40, or 60 micrograms of herbicide per ml; however, aflatoxin production by A. parasiticus was higher in media with herbicide, whereas A. flavus produced lower aflatoxin levels.     

Chemoprevention by thyme oils of Aspergillus parasiticus growth and aflatoxin production

The essential oils from Thymus eriocalyx and Thymus X-porlock obtained by hydrodistillation were analyzed by GC/MS. The major components of T. eriocalyx and T. X-porlock oils were thymol (63.8, 31.7%), beta-phellandrene (13.30, 38.7%), cis-sabinene hydroxide (8.1, 9.6%), 1,8-cineole (2, 1.7%), and beta-pinene (1.31, 2%), respectively. Antifungal activities of the oils were studied with special reference to the inhibition of Aspergillus parasiticus growth and aflatoxin production. Minimal inhibitory (MIC) and minimal fungicidal (MFC) concentrations of the oils were determined. Static effects of the above oils against A. parasiticus were at 250 ppm and lethal effects of T. eriocalyx and T. X-porlock were 500 and 1000 ppm of the oils, respectively. Aflatoxin production was inhibited at 250 ppm of both oils with that of T. eriocalyx being stronger inhibitor. Transmission electron microscopy (TEM) of A. parasiticus exposed to MIC level (250 ppm) of the oils showed irreversible damage to cell wall, cell membrane, and cellular organelles. It is concluded that the essential oils could be safely used as preservative materials on some kinds of foods at low concentrations to protect them from fungal infections.     

Pyridine nucleotides and redox state regulation of aflatoxin biosynthesis in Aspergillus parasiticus NRRL 3240

In vivo regulation of lipid and aflatoxin biosynthesis by pyridine nucleotides and their derived functions was studied in Aspergillus parasiticus NRRL 3240. Aflatoxins, total lipids and pyridine nucleotide content were estimated under different growth conditions. Aflatoxin formation was highest in cultures grown in sucrose-low salts medium followed by asparagine- and zinc-deficient media. The lipid content of the cultures followed an inverse pattern. The levels of oxidized nucleotides decreased with age under all culture conditions employed. Concentrations of NADPH peaked before the onset of aflatoxin biosynthesis. For each medium used, the estimated catabolite reduction charge was constant at all stages of growth whereas the anabolic reduction charge varied. A direct relationship between the level of extracellular ammonium ions and anabolic reduction charge was established. A high anabolic reduction charge was associated with increased lipid biosynthesis rather than aflatoxin biosynthesis.     

Pyridine nucleotides and redox state regulation of aflatoxin biosynthesis in Aspergillus parasiticus NRRL 3240

In vivo regulation of lipid and aflatoxin biosynthesis by pyridine nucleotides and their derived functions was studied in Aspergillus parasiticus NRRL 3240. Aflatoxins, total lipids and pyridine nucleotide content were estimated under different growth conditions. Aflatoxin formation was highest in cultures grown in sucroselow salts medium followed by asparagine- and zinc-deficient media. The lipid content of the cultures followed an inverse pattern. The levels of oxidized nucleotides decreased with age under all culture conditions employed. Concentrations of NADPH peaked before the onset of aflatoxin biosynthesis. For each medium used, the estimated catabolite reduction charge was constant at all stages of growth whereas the anabolic reduction charge varied. A direct relationship between the level of extracellular ammonium ions and anabolic reduction charge was established. A high anabolic reduction charge was associated with increased lipid biosynthesis rather than aflatoxin biosynthesis.     

Decrease of growth and aflatoxin production in Aspergillus parasiticus caused by spices

Non-commercial spices and herbs Tetrapleura tetrapetra, Triumfetta cordifolia, Garcina kola, Monodora myristica and Xylopia aethiopica at 0.08 to 0.32% (w/v) decreased the mycelial weight of Aspergillus parasiticus NRRL 2999 in yeast extract/sucrose broth by up to 68%. Aflatoxin production, monitored with ELISA, was most effectively decreased, from 97 to 23 g/ml, when the extract of G. kola was added at 0.32% (w/v).     

Effect of pyridazinone herbicides on growth and aflatoxin release by Aspergillus flavus and Aspergillus parasiticus.

The influence of pyridazinone herbicides on aflatoxin production by Aspergillus flavus and A. parasiticus was studied in liquid media. Mycelia production was not affected by 20, 40, or 60 micrograms of herbicide per ml; however, aflatoxin production by A. parasiticus was higher in media with herbicide, whereas A. flavus produced lower aflatoxin levels.