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Species of Aspergillus produce a diverse array of secondary metabolites, and recent genomic analysis has predicted that these species have the capacity to synthesize many more compounds. It has been possible to infer the presence of 55 gene clusters associated with secondary metabolism in Aspergillus flavus; however, only three metabolic pathways-aflatoxin, cyclopiazonic acid (CPA) and aflatrem-have been assigned to these clusters. To gain an insight into the regulation of and to infer the ecological significance of the 55 secondary metabolite gene clusters predicted in A. flavus, we examined their expression over 28 diverse conditions. Variables included culture medium and temperature, fungal development, colonization of developing maize seeds and misexpression of laeA, a global regulator of secondary metabolism. Hierarchical clustering analysis of expression profiles allowed us to categorize the gene clusters into four distinct clades. Gene clusters for the production of aflatoxins, CPA and seven other unknown compound(s) were identified as belonging to one clade. To further explore the relationships found by gene expression analysis, aflatoxin and CPA production were quantified under five different cell culture environments known to be conducive or nonconducive for aflatoxin biosynthesis and during the colonization of developing maize seeds. Results from these studies showed that secondary metabolism gene clusters have distinctive gene expression profiles. Aflatoxin and CPA were found to have unique regulation, but are sufficiently similar that they would be expected to co-occur in substrates colonized with A. flavus.

aflJ resides within the aflatoxin biosynthetic gene cluster adjacent to the pathway regulatory gene aflR and is involved in aflatoxin production, but its function is unknown. Over-expression of aflJ in the aflatoxin-producing strain 86-10 resulted in increased aflatoxin. In an effort to study the function and regulation of aflJ, strain 649-1 lacking the entire biosynthetic cluster was transformed with either reporter constructs, expression constructs, or cosmid clones and analysed for gene expression or metabolite accumulation. Over-expression of aflJ did not result in elevated transcription of ver-1, omtA or aflR. To determine if over-expression of aflJ leads to an increase in early pathway intermediates, strain 649-1 was transformed with cosmid 5E6 and either gpdA::aflJ alone, gpdA::aflR alone, or aflJ and aflR together. Cosmid 5E6 contains the genes pksA, nor-1, fas-1, and fas-2, which are required for the biosynthesis of the early pathway intermediate averantin. 649-1 transformants containing 5E6 alone produced no detectable averantin. In contrast, 5E6 transformants with gpdA::aflR produced averantin, but only half as much as those transformants containing both aflR and aflJ. Northern blot analysis showed that 5E6 transformants containing both aflR and aflJ had five times more pksA transcripts and four times more nor-1 transcripts than 5E6 transformants containing gpdA::aflR alone. Further, aflJ transcription was regulated by aflR. Over-expression of aflR resulted in elevated aflJ transcription. aflJ appears to modulate the regulation of early genes in aflatoxin biosynthesis.

Aflatoxins are toxic and carcinogenic polyketide metabolites produced by certain fungal species, including Aspergillus flavus and A. parasiticus. Many internal and external factors, such as nutrition and environment affect aflatoxin biosynthesis; therefore, we analyzed the transcriptome of A. flavus using expressed sequence tags (ESTs) from a normalized cDNA expression library constructed from mycelia harvested under several conditions. A total of 7218 unique ESTs were identified from 26,110 sequenced cDNA clones. Functional classifications were assigned to these ESTs and genes, potentially involved in the aflatoxin contamination process, were identified. Based on this EST sequence information, a genomic DNA amplicon microarray was constructed at The Institute for Genomic Research (TIGR). To identify potential regulatory networks controlling aflatoxin contamination in food and feeds, gene expression profiles in aflatoxin-supportive media versus non-aflatoxin-supportive media were evaluated in A. flavus and A. parasiticus. Genes consistently expressed in several aflatoxin-supportive media are reported.

Aflatoxins are toxic secondary metabolites produced by a 70-kb cluster of genes in Aspergillus flavus. The cluster genes are coordinately regulated and reside as a single copy within the genome. Diploids between a wild-type strain and a mutant (649) lacking the aflatoxin gene cluster fail to produce aflatoxin or transcripts of the aflatoxin pathway genes. This dominant phenotype is rescued in diploids between a wild-type strain and a transformant of the mutant containing an ectopic copy of aflR, the transcriptional regulator of the aflatoxin biosynthetic gene cluster. Further characterization of the mutant showed that it is missing 317 kb of chromosome III, including the known genes for aflatoxin biosynthesis. In addition, 939 kb of chromosome II is present as a duplication on chromosome III in the region previously containing the aflatoxin gene cluster. The lack of aflatoxin production in the diploid was not due to a unique or a mis-expressed repressor of aflR. Instead a form of reversible silencing based on the position of aflR is likely preventing the aflatoxin genes from being expressed in 649 x wild-type diploids. Gene expression analysis revealed the silencing effect is specific to the aflatoxin gene cluster.

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.

The genes encoding the aflatoxin biosynthetic pathway enzymes have been localized as a cluster to a 75-kb DNA fragment. The enzymatic functions of the products of most of the genes in the cluster are known, but there are a few genes that have not yet been characterized. We report here the characterization of one of these genes, a gene designated aflJ. This gene resides in the cluster adjacent to the pathway regulatory gene, aflR, and the two genes are divergently transcribed. Disruption of aflJ in Aspergillus flavus results in a failure to produce aflatoxins and a failure to convert exogenously added pathway intermediates norsolorinic acid, sterigmatocystin, and O-methylsterigmatocystin to aflatoxin. The disrupted strain does, however, accumulate pksA, nor-1, ver-1, and omtA transcripts under conditions conducive to aflatoxin biosynthesis. Therefore, disruption of aflJ does not affect transcription of these genes, and aflJ does not appear to have a regulatory function similar to that of aflR. Sequence analysis of aflJ and its putative peptide, AflJ, did not reveal any enzymatic domains or significant similarities to proteins of known function. The putative peptide does contain three regions predicted to be membrane-spanning domains and a microbodies C-terminal targeting signal.

O-methyltransferase (OMT) is one of the key enzymes in aflatoxin (AF) biosynthesis in the fungi, Aspergillus flavus (Af) and A. parasiticus (Ap). Genomic DNA clones containing the omtA genes from Ap strain SRRC 143 and Af strain CRA01-2B were sequenced. Comparison of the genomic DNA sequences with the cDNA of this Ap gene revealed the presence of four introns ranging from 52 to 60 bp in length in both species; the region encoding the putative S-adenosylmethionine-binding motif was located between the third and fourth introns. The coding sequence of omtA from Ap strain SRRC 143 demonstrated a greater than 97% sequence identity with that from Af strain CRA01-2B, within the coding region.

Aflatoxins are toxic and carcinogenic secondary metabolites produced by the fungi Aspergillus flavus and A. parasiticus. Aflatoxins are synthesized by condensation of acetate units; their synthesis is estimated to involve at least 16 different enzymes. In this study we have shown that at least nine genes involved in the aflatoxin biosynthetic pathway are located within a 60-kb DNA fragment. Four of these genes, nor-1, aflR, ver-1, and omtA (previously named omt-1), have been cloned in A. flavus and A. parasiticus. In addition, five other genes, pksA, uvm8, aad, ord-1, and ord-2 have been recently cloned in A. parasiticus. The pksA, aad, and uvm8 genes exhibit sequence homologies to polyketide synthase, aryl-alcohol dehydrogenase, and fatty acid synthase genes, respectively. The cDNA sequences of ord-1 and ord-2 genes, which may be involved in later steps of aflatoxin biosynthesis, have been determined; the ord-1 gene product exhibits homology to cytochrome P-450-type enzymes. By characterizing the overlapping regions of the DNA inserts in different cosmid and lambda DNA clones, we have determined the order of these aflatoxin pathway genes within this 60-kb DNA region to be pksA, nor-1, uvm8, aflR, aad, ver-1, ord-1, ord-2, and omtA in A. parasiticus and nor-1, aflR, ver-1, ord-1, ord-2, and omtA in A. flavus. The order is related to the order in enzymatic steps required for aflatoxin biosynthesis. The physical distances (in kilobase pairs) and the directions of transcription of these genes have been determined for both aflatoxigenic species.

Aflatoxins belong to a family of decaketides that are produced as secondary metabolites by Aspergillus flavus and A. parasiticus. The aflatoxin biosynthetic pathway involves several enzymatic steps that appear to be regulated by the afl2 gene in A. flavus and the apa2 gene in A. parasiticus. Several lines of evidence indicate that these two genes are homologous. The DNA sequences of the two genes are highly similar, they both are involved in the regulation of aflatoxin biosynthesis, and apa2 can complement the afl2 mutation in A. flavus. Because of these similarities, we propose that these two genes are homologs, and because of the ability of these genes to regulate aflatoxin biosynthesis, we suggest that they be designated aflR. We report here the further characterization of aflR from A. flavus and show that aflR codes for a 2,078-bp transcript with an open reading frame of 1,311 nucleotides that codes for 437 amino acids and a putative protein of 46,679 daltons. Analysis of the predicted amino acid sequence indicated that the polypeptide contains a zinc cluster motif between amino acid positions 29 and 56. This region contains the consensus sequence Cys-Xaa2-Cys-Xaa6-Cys-Xaa6-Cys-Xaa2-Cys-Xaa6+ ++-Cys. This motif has been found in several fungal transcriptional regulatory proteins. DNA hybridization of the aflR gene with genomic digests of seven polyketide-producing fungi revealed similar sequences in three other species related to A. flavus: A. parasiticus, A. oryzae, and A. sojae. Finally, we present evidence for an antisense transcript (aflRas) derived from the opposite strand of aflR, suggesting that the aflR locus involves some form of antisense regulation.

An Aspergillus flavus cDNA library was screened by differential hybridization to isolate clones corresponding to genes that are actively transcribed under culture conditions conducive to aflatoxin biosynthesis. One clone with a 1.28-kb insert was isolated, and its nucleotide sequence was determined. The nucleotide sequence of this clone had 75% DNA identity to those of the alcohol dehydrogenase genes from Aspergillus nidulans, and the putative polypeptide translated from the cDNA sequence had 82% similarity with the amino acid sequences of the A. nidulans proteins. Thus, this gene has been designated adh1. Southern hybridization analysis of genomic DNA from A. flavus indicated that there was one copy of the adh1 gene. Northern (RNA) hybridization analysis indicated that the adh1 transcript accumulated in culture medium conducive to aflatoxin production and the timing of accumulation of adh1 transcripts was similar to that for aflatoxin. Fusion of the promoter region of adh1 to a beta-glucuronidase reporter gene indicated that accumulation of the adh1 transcript was the result of transcriptional activation. These molecular data support previous physiological evidence that showed the importance of carbohydrate metabolism during 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.

Aflatoxins are extremely potent carcinogens produced by Aspergillus flavus and Aspergillus parasiticus. Cloning of genes in the aflatoxin pathway provides a specific approach to understanding the regulation of aflatoxin biosynthesis and, subsequently, to the control of aflatoxin contamination of food and feed. This paper reports the isolation of a gene involved in aflatoxin biosynthesis by complementation of an aflatoxin-nonproducing mutant with a wild-type genomic cosmid library of A. flavus. Strain 650-33, blocked in aflatoxin biosynthesis at the afl-2 allele, was complemented by a 32-kb cosmid clone (B9), resulting in the production of aflatoxin. The onset and profile of aflatoxin accumulation was similar for the transformed strain and the wild-type strain (NRRL 3357) of the fungus, indicating that the integrated gene is under the same control as in wild-type strains. Complementation analyses with DNA fragments from B9 indicated that the gene resides within a 2.2-kb fragment. Because this gene complements the mutated afl-2 allele, it was designated afl-2. Genetic evidence obtained from a double mutant showed that afl-2 is involved in aflatoxin biosynthesis before the formation of norsolorinic acid, the first stable intermediate identified in the pathway. Further, metabolite feeding studies with the mutant, transformed, and wild-type cultures and enzymatic activity measurements in cell extracts of these cultures suggest that afl-2 regulates gene expression or the activity of other aflatoxin pathway enzymes. This is the first reported isolation of a gene for aflatoxin biosynthesis in A. flavus.