An aflatoxin biosynthesis cluster gene encodes a novel oxidase required for conversion of versicolorin a to sterigmatocystin

Disruption of the aflatoxin biosynthesis cluster gene aflY (hypA) gave Aspergillus parasiticus transformants that accumulated versicolorin A. This gene is predicted to encode the Baeyer-Villiger oxidase necessary for formation of the xanthone ring of the aflatoxin precursor demethylsterigmatocystin.     

Cloning and characterization of the O-methyltransferase I gene (dmtA) from Aspergillus parasiticus associated with the conversions of demethylsterigmatocystin to sterigmatocystin and dihydrodemethylsterigmatocystin to dihydrosterigmatocystin in aflatoxin biosynthesis.

O-Methyltransferase I catalyzes both the conversion of demethylsterigmatocystin to sterigmatocystin and the conversion of dihydrodemethylsterigmatocystin to dihydrosterigmatocystin during aflatoxin biosynthesis. In this study, both genomic cloning and cDNA cloning of the gene encoding O-methyltransferase I were accomplished by using PCR strategies, such as conventional PCR based on the N-terminal amino acid sequence of the purified enzyme, 5' and 3' rapid amplification of cDNA ends PCR, and thermal asymmetric interlaced PCR (TAIL-PCR), and genes were sequenced by using Aspergillus parasiticus NIAH-26. A comparison of the genomic sequences with the cDNA of the dmtA region revealed that the coding region is interrupted by three short introns. The cDNA of the dmtA gene is 1,373 bp long and encodes a 386-amino-acid protein with a deduced molecular weight of 43,023, which is consistent with the molecular weight of the protein determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The C-terminal half of the deduced protein exhibits 76.3% identity with the coding region of the Aspergillus nidulans StcP protein, whereas the N-terminal half of dmtA exhibits 73.0% identity with the 5' flanking region of the stcP gene, suggesting that translation of the stcP gene may start at a site upstream from methionine that is different from the site that has been suggested previously. Also, an examination of the 5' and 3' flanking regions of the dmtA gene in which TAIL-PCR was used demonstrated that the dmtA gene is located in the aflatoxin biosynthesis cluster between (and in the same orientation as) the omtA and ord-2 genes. Northern blotting revealed that expression of the dmtA gene is influenced by both medium composition and culture temperature and that the pattern correlates with the patterns observed for other genes in the aflatoxin gene cluster. Furthermore, Southern blotting and PCR analyses of the dmtA gene showed that a dmtA homolog is present in Aspergillus oryzae SYS-2.     

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.     

Enzymatic conversion of sterigmatocystin into aflatoxin B1 by cell-free extracts of Aspergillus parasiticus.

A cell-free extract, prepared from Aspergillus parasiticus ATCC 15517 grown in synthetic medium, was active in converting [14C]sterigmatocystin into aflatoxin B1 in the presence of reduced nicotinamide adenine dinucleotide phosphate. The activity was demonstrated by the time course of conversion and the linear dependence of the yield of product on enzyme concentrations. Optimum activity was obtained at pH 7.5 to 7.8 at 27 C. The results confirm sterigmatocystin as a biogenetic precursor of aflatoxin B1. Techniques were developed for enzymatic studies on aflatoxin biosynthesis.     

Conversion of sterigmatocystin to aflatoxin B 1 by Aspergillus parasiticus

¹⁴C-Sterigmatocystin isolated from cultures of $\text{Aspergillus}$$\text{versicolor}$ supplemented with (1-¹⁴C)acetate was shown to be efficiently converted to aflatoxin B₁ by the resting mycelium of $\text{A. parasiticus}$. The experimental results may indicate a biosynthetic pathway leading from 5-hydroxysterigmatocystin to sterigmatocystin and then to aflatoxin B₁.     

Enzymatic conversion of sterigmatocystin into aflatoxin B1 by cell-free extracts of Aspergillus parasiticus.

A cell-free extract, prepared from Aspergillus parasiticus ATCC 15517 grown in synthetic medium, was active in converting [14C]sterigmatocystin into aflatoxin B1 in the presence of reduced nicotinamide adenine dinucleotide phosphate. The activity was demonstrated by the time course of conversion and the linear dependence of the yield of product on enzyme concentrations. Optimum activity was obtained at pH 7.5 to 7.8 at 27 C. The results confirm sterigmatocystin as a biogenetic precursor of aflatoxin B1. Techniques were developed for enzymatic studies on aflatoxin biosynthesis.     

Appearance of enzyme activities catalyzing conversion of sterigmatocystin to aflatoxin B1 in late-growth-phase Aspergillus parasiticus cultures.

Two activities involved in terminal pathway conversion of sterigmatocystin to aflatoxin B1 were isolated from an aflatoxin-nonproducing mutant of Aspergillus parasiticus (avn-1), and the time course of appearance of the activities in culture was determined. Subcellular fractionation of fungal mycelia resolved the two activities into a postmicrosomal activity which catalyzed conversion of sterigmatocystin to O-methylsterigmatocystin and a microsomal activity which converted O-methylsterigmatocystin to aflatoxin B1. The two activities were absent in 24-h-old cells, increased to optimum levels during the stationary phase, and then declined.     

Appearance of enzyme activities catalyzing conversion of sterigmatocystin to aflatoxin B1 in late-growth-phase Aspergillus parasiticus cultures

Two activities involved in terminal pathway conversion of sterigmatocystin to aflatoxin B1 were isolated from an aflatoxin-nonproducing mutant of Aspergillus parasiticus (avn-1), and the time course of appearance of the activities in culture was determined. Subcellular fractionation of fungal mycelia resolved the two activities into a postmicrosomal activity which catalyzed conversion of sterigmatocystin to O-methylsterigmatocystin and a microsomal activity which converted O-methylsterigmatocystin to aflatoxin B1. The two activities were absent in 24-h-old cells, increased to optimum levels during the stationary phase, and then declined.     

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)     

The affinity purification and characterization of a dehydrogenase from Aspergillus parasiticus involved in aflatoxin B1 biosynthesis.

A two step scheme has been developed for the purification of a dehydrogenase from mycelia of 84 hours old Aspergillus parasiticus (1-11-105 Wh 1), which catalyzes the conversion of norsolorinic acid (NA) to averantin (AVN). The dehydrogenase was purified from cell-free extracts using reactive green 19-agarose and norsolorinic acid-agarose affinity chromatography. The latter affinity matrix was synthesised by attaching norsolorinic acid to omega-aminohexylagarose. The purified protein was shown to be homogenous on non-denaturing polyacrylamide gel electrophoresis. A final purification of 215-fold was achieved. Results of gel filtration chromatography indicated the approximate molecular mass of the native protein to be 140,000 daltons. The isoelectric point of the protein was about 5.5 as determined by chromatofocusing. The reaction catalyzed by the dehydrogenase was optimum at pH 8.5 and between 25 degrees to 35 degrees C. The Km of the enzyme for NA and NADPH was determined to be 3.45 microM and 103 microM respectively.     

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.     

stcS, a putative P-450 monooxygenase, is required for the conversion of versicolorin A to sterigmatocystin in Aspergillus nidulans.

Sterigmatocystin (ST) and aflatoxin are carcinogenic end point metabolites derived from the same biochemical pathway, which is found in several Aspergillus spp. Recently, an ST gene cluster, containing approximately 25 distinct genes that are each proposed to function specifically in ST biosynthesis, has been identified in Aspergillus nidulans. Each of these structural genes is named stc (sterigmatocystin) followed by a consecutive letter of the alphabet. We have previously described stcU (formerly verA) as encoding a keto-reductase required for the conversion of versicolorin A to ST. We now describe a second A. nidulans gene, stcS (formerly verB), that is located within 2 kb of stcU in the ST gene cluster. An stcS-disrupted strain of A. nidulans, TSS17, was unable to produce ST and converted ST/aflatoxin precursors to versicolorin A rather than ST, indicating that stcS functions at the same point in the pathway as stcU. Genomic sequence analysis of stcS shows that it encodes a cytochrome P-450 monooxygenase and constitutes a novel P-450 family, CYP59. Assuming that StcU activity mimics that of similar P-450s, it is likely that StcU catalyzes one of the proposed oxidation steps necessary to convert versicolorin A to ST. These results constitute the first genetic proof that the conversion of versicolorin A to ST requires more than one enzymatic activity.     

The aflatoxin biosynthesis cluster gene, aflX, encodes an oxidoreductase involved in conversion of versicolorin A to demethylsterigmatocystin

Biosynthesis of the toxic and carcinogenic aflatoxins by the fungus Aspergillus flavus is a complicated process involving more that 27 enzymes and regulatory factors encoded by a clustered group of genes. Previous studies found that three enzymes, encoded by verA, ver-1, and aflY, are required for conversion of versicolorin A (VA), to demethylsterigmatocystin. We now show that a fourth enzyme, encoded by the previously uncharacterized gene, aflX (ordB), is also required for this conversion. A homolog of this gene, stcQ, is present in the A. nidulans sterigmatocystin (ST) biosynthesis cluster. Disruption of aflX in Aspergillus flavus gave transformants that accumulated approximately 4-fold more VA and fourfold less aflatoxin than the untransformed strain. Southern and Northern blot analyses confirmed that aflX was the only gene disrupted in these transformants. Feeding ST or O-methylsterigmatocystin, but not VA or earlier precursor metabolites, restored normal levels of AF production. The protein encoded by aflX is predicted to have domains typical of an NADH-dependent oxidoreductase. It has 27% amino acid identity to a protein encoded by the aflatoxin cluster gene, aflO (avfA). Some of domains in the protein are similar to those of epoxide hydrolases.     

Isolation and characterization of Aspergillus parasiticus mutants with impaired aflatoxin production by a novel tip culture method.

A convenient procedure consisting of UV photography (K. Yabe, Y. Ando, M. Ito, and N. Terakado, Appl. Environ. Microbiol. 53:230-234, 1987) and a tip culture method has been devised for the isolation and characterization of Aspergillus parasiticus mutants relating to aflatoxin production. With the latter procedure, the production of aflatoxins excreted into the culture medium and precursors in the mycelium were easily measured quantitatively or semiquantitatively. A total of 38 mutants in which the aflatoxigenicity was decreased or lost were obtained by UV radiation; 3 were found to be blocked mutants, which accumulated the aflatoxin precursors versicolorin A or averantin.