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.     

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.     

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.     

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.     

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.     

Aflatoxins in sunflower seeds: Effect of zinc in aflatoxin production by two strains of Aspergillus parasiticus

Growth and aflatoxin production by Aspergillus parasiticus NRRL 2999 and Aspergillus parasiticus RC 12 were studied both in sunflower seed and a synthetic culture medium (with and without zinc enrichment).On a synthetic culture medium the strains behaved in different ways according to the zinc concentration.In sunflower seed medium the influence of zinc was not so evident. Thus the results show that the influence of zinc is not the same for different strains and substrates.     

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.     

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.     

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.     

Effect of phytate on aflatoxin formation by Aspergillus parasiticus and Aspergillus flavus in synthetic media

The effect of phytate on the production of aflatoxins by Aspergillus parasiticus and Aspergillus flavus grown on synthetic media was examined. In the absence of pH control (initial pH 4.5–6.5) for A. parasiticus, phytate (14.3 mM) caused a six-fold decrease in aflatoxins in the medium and a ten-fold decrease in those retained by the mycelia. When the initial pH of the medium was adjusted to 4.5 no effect on aflatoxin production was observed. With A. flavus or A. parasiticus grown on media with a higher initial pH value (6 to 7), the presence of phytate in the media caused an increase in aflatoxin production. These results are inconsistent with previous studies which indicated that phytate depresses aflatoxin production by rendering zinc, a necessary co-factor for aflatoxin biosynthesis, unavailable to the mold.     

Nitrogen metabolism in Aspergillus parasiticus NRRL 3240 and A. flavus NRRL 3537 in relation to aflatoxin production

The relationship between nitrogen assimilation, metabolism and aflatoxin formation has been investigated in a toxigenic and a non-toxigenic strain of Aspergillus parasiticus. Ammonia from the medium is mainly assimilated via NADP-requiring glutamate dehydrogenase. During growth NAD-requiring glutamate dehydrogenase followed an inverse pattern of activity with respect to NADP glutamate dehydrogenase. Alpha-ketoglutarate, the product of NAD glutamate dehydrogenase, stimulated acetate incorporation into aflatoxins. Glutamine synthetase, ornithine transcarbamylase, both utilizing glutamate as substrate were assayed under different growth conditions. An important regulatory role for glutamine synthetase is suggested. The metabolic route of asparagine utilization was also investigated. Both the known pathways, glutamate oxaloacetate transaminase and glutamate pyruvate transaminase are operative simultaneously.     

Nitrogen metabolism in Aspergillus parasiticus NRRL 3240 and A. flavus NRRL 3537 in relation to aflatoxin production

The relationship between nitrogen assimilation, metabolism and aflatoxin formation has been investigated in a toxigenic and a non-toxigenic strain of Aspergillus parasiticus. Ammonia from the medium is mainly assimilated via NADP-requiring glutamate dehydrogenase. During growth NAD-requiring glutamate dehydrogenase followed an inverse pattern of activity with respect to NADP glutamate dehydrogenase. Alpha-ketoglutarate, the product of NAD glutamate dehydrogenase, stimulated acetate incorporation into aflatoxins. Glutamine synthetase, ornithine transcarbamylase, both utilizing glutamate as substrate were assayed under different growth conditions. An important regulatory role for glutamine synthetase is suggested. The metabolic route of asparagine utilization was also investigated. Both the known pathways, glutamate oxaloacetate transaminase and glutamate pyruvate transaminase are operative simultaneously.     

Cloning of the Aspergillus parasiticus apa-2 gene associated with the regulation of aflatoxin biosynthesis.

An Aspergillus parasiticus gene, designated apa-2, was identified as a regulatory gene associated with aflatoxin biosynthesis. The apa-2 gene was cloned on the basis of overproduction of pathway intermediates following transformation of fungal strains with cosmid DNA containing the aflatoxin biosynthetic genes nor-1 and ver-1. Transformation of an O-methylsterigmatocystin-accumulating strain, A. parasiticus SRRC 2043, with a 5.5-kb HindIII-XbaI DNA fragment containing apa-2 resulted in overproduction of all aflatoxin pathway intermediates analyzed. Specific enzyme activities associated with the conversion of norsolorinic acid and sterigmatocystin were increased approximately twofold. The apa-2 gene was found to complement an A. flavus afl-2 mutant strain for aflatoxin production, suggesting that apa-2 is functionally homologous to afl-2. Comparison of the A. parasiticus apa-2 gene DNA sequence with that of the A. flavus afl-2 gene (G. A. Payne, G. J. Nystorm, D. Bhatnagar, T. E. Cleveland, and C. P. Woloshuk, Appl. Environ. Microbiol. 59:156-162, 1993) showed that they shared > 95% DNA homology. Physical mapping of cosmid subclones placed apa-2 approximately 8 kb from ver-1.     

Cloning and characterization of a cDNA from Aspergillus parasiticus encoding an O-methyltransferase involved in aflatoxin biosynthesis.

Aflatoxins are polyketide-derived secondary metabolites produced by the fungi Aspergillus flavus and Aspergillus parasiticus. Among the catalytic steps in the aflatoxin biosynthetic pathway, the conversion of sterigmatocystin to O-methylsterigmatocystin and the conversion of dihydrosterigmatocystin to dihydro-O-methylsterigmatocystin are catalyzed by an S-adenosylmethionine-dependent O-methyltransferase. A cDNA library was constructed by using RNA isolated from a 24-h-old culture of wild-type A. parasiticus SRRC 143 and was screened by using polyclonal antiserum raised against a purified 40-kDa O-methyltransferase protein. A clone that harbored a full-length cDNA insert (1,460 bp) containing the 1,254-bp coding region of the gene omt-1 was identified by the antiserum and isolated. The complete cDNA sequence was determined, and the corresponding 418-amino-acid sequence of the native enzyme with a molecular weight of 46,000 was deduced. This 46-kDa native enzyme has a leader sequence of 41 amino acids, and the mature form of the enzyme apparently consists of 377 amino acids and has a molecular weight of 42,000. Direct sequencing of the purified mature enzyme from A. parasiticus SRRC 163 showed that 19 of 22 amino acid residues were identical to the amino acid residues in an internal region of the deduced amino acid sequence of the mature protein. The 1,460-bp omt-1 cDNA was cloned into an Escherichia coli expression system; a Western blot (immunoblot) analysis of crude extracts from this expression system revealed a 51-kDa fusion protein (fused with a 5-kDa beta-galactosidase N-terminal fragment).(ABSTRACT TRUNCATED AT 250 WORDS)