Genetic regulation of aflatoxin biosynthesis: From gene to genome

Aflatoxins are notorious toxic secondary metabolites known for their impacts on human and animal health, and their effects on the marketability of key grain and nut crops. Understanding aflatoxin biosynthesis is the focus of a large and diverse research community. Concerted efforts by this community have led not only to a well-characterized biosynthetic pathway, but also to the discovery of novel regulatory mechanisms. Common to secondary metabolism is the clustering of biosynthetic genes and their regulation by pathway specific as well as global regulators. Recent data show that arrangement of secondary metabolite genes in clusters may allow for an important global regulation of secondary metabolism based on physical location along the chromosome. Available genomic and proteomic tools are now allowing us to examine aflatoxin biosynthesis more broadly and to put its regulation in context with fungal development and fungal ecology. This review covers our current understanding of the biosynthesis and regulation of aflatoxin and highlights new and emerging information garnered from structural and functional genomics. The focus of this review will be on studies in Aspergillus flavus and Aspergillus parasiticus, the two agronomically important species that produce aflatoxin. Also covered will be the important contributions gained by studies on production of the aflatoxin precursor sterigmatocystin in Aspergillus nidulans.     

Cloning of the afl-2 gene involved in aflatoxin biosynthesis from Aspergillus flavus.

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.     

Molecular genetic analysis and regulation of aflatoxin biosynthesis

Aflatoxins, produced by some Aspergillus species, are toxic and extremely carcinogenic furanocoumarins. Recent investigations of the molecular mechanism of AFB biosynthesis showed that the genes required for biosynthesis are in a 70 kb gene cluster. They encode a DNA-binding protein functioning in aflatoxin pathway gene regulation, and other enzymes such as cytochrome p450-type monooxygenases, dehydrogenases, methyltransferases, and polyketide and fatty acid synthases. Information gained from these studies has led to a better understanding of aflatoxin biosynthesis by these fungi. The characterization of genes involved in aflatoxin formation affords the opportunity to examine the mechanism of molecular regulation of the aflatoxin biosynthetic pathway, particularly during the interaction between aflatoxin-producing fungi and plants.     

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.     

Understanding the genetics of regulation of aflatoxin production and Aspergillus flavus development

Aflatoxins are polyketide-derived, toxic, and carcinogenic secondary metabolites produced primarily by two fungal species, Aspergillus flavus and A. parasiticus, on crops such as corn, peanuts, cottonseed, and treenuts. Regulatory guidelines issued by the U.S. Food and Drug Administration (FDA) prevent sale of commodities if contamination by these toxins exceeds certain levels. The biosynthesis of these toxins has been extensively studied. About 15 stable precursors have been identified. The genes involved in encoding the proteins required for the oxidative and regulatory steps in the biosynthesis are clustered in a 70 kb portion of chromosome 3 in the A. flavus genome. With the characterization of the gene cluster, new insights into the cellular processes that govern the genes involved in aflatoxin biosynthesis have been revealed, but the signaling processes that turn on aflatoxin biosynthesis during fungal contamination of crops are still not well understood. New molecular technologies, such as gene microarray analyses, quantitative polymerase chain reaction (PCR), and chromatin immunoprecipitation are being used to understand how physiological stress, environmental and soil conditions, receptivity of the plant, and fungal virulence lead to episodic outbreaks of aflatoxin contamination in certain commercially important crops. With this fundamental understanding, we will be better able to design improved non-aflatoxigenic biocompetitive Aspergillus strains and develop inhibitors of aflatoxin production (native to affected crops or otherwise) amenable to agricultural application for enhancing host-resistance against fungal invasion or toxin production. Comparisons of aflatoxin-producing species with other fungal species that retain some of the genes required for aflatoxin formation is expected to provide insight into the evolution of the aflatoxin gene cluster, and its role in fungal physiology. Therefore, information on how and why the fungus makes the toxin will be valuable for developing an effective and lasting strategy for control of aflatoxin contamination.     

Aflatoxins: Detection,toxicity, and biosynthesis

Aflatoxins are toxic and carcinogenic secondary metabolites produced mainly byAspergillus flavus andAspergillus parasiticus. The aflatoxins present in food and feed are hazardous to both human and animal health. A number of studies have been conducted on the detection, toxicity, biosynthesis, and regulation of aflatoxins due to the discovery of serious aflatoxicosis in farm animals, and the presence of aflatoxins in many food products. There are many reviews that focus on the biosynthesis of aflatoxin, yet there are few examinations of the overall aspects of aflatoxins, including detection, toxicity, and the regulation on biosynthesis. Thus, the goal of this article is to give an overview of the overall aspects of aflatoxins. This review consists of four parts; i) detection methods for aflatoxins, ii) the toxicity mechanism of aflatoxin B1, iii) gene cluster for aflatoxin biosynthesis, and iv) the regulation of aflatoxin biosynthesis.     

Cloning of a sugar utilization gene cluster in Aspergillus parasiticus

At one end of the 70 kb aflatoxin biosynthetic pathway gene cluster in Aspergillus parasiticus and Aspergillus flavus reported earlier, we have cloned a group of four genes that constitute a well-defined gene cluster related to sugar utilization in A. parasiticus: (1) sugR, (2) hxtA, (3) glcA and (4) nadA. No similar well-defined sugar gene cluster has been reported so far in any other related Aspergillus species such as A. flavus, A. nidulans, A. sojae, A. niger, A. oryzae and A. fumigatus. The expression of the hxtA gene, encoding a hexose transporter protein, was found to be concurrent with the aflatoxin pathway cluster genes, in aflatoxin-conducive medium. This is significant since a close linkage between the two gene clusters could potentially explain the induction of aflatoxin biosynthesis by simple sugars such as glucose or sucrose.     

Amino acid supplementation reveals differential regulation of aflatoxin biosynthesis in <Emphasis Type="Italic">Aspergillus flavus</Emphasis> NRRL 3357 and <Emphasis Type="Italic">Aspergillus parasiticus</Emphasis> SRRC 143

Aflatoxins are toxic and carcinogenic secondary metabolites produced by the fungi Aspergillus flavus and Aspergillus parasiticus. To better understand the molecular mechanisms that regulate aflatoxin production, the biosynthesis of the toxin in A. flavus and A. parasticus grown in yeast extract sucrose media supplemented with 50 mM tryptophan (Trp) were examined. Aspergillus flavus grown in the presence of 50 mM tryptophan was found to have significantly reduced aflatoxin B₁ and B₂ biosynthesis, while A. parasiticus cultures had significantly increased B₁ and G₁ biosynthesis. Microarray analysis of RNA extracted from fungi grown under these conditions revealed 77 genes that are expressed significantly different between A. flavus and A. parasiticus, including the aflatoxin biosynthetic genes aflD (nor-1), aflE (norA), and aflO (omtB). It is clear that the regulatory mechanisms of aflatoxin biosynthesis in response to Trp in A. flavus and A. parasiticus are different. These candidate genes may serve as regulatory factors of aflatoxin biosynthesis.     

Aspergillus flavus expressed sequence tags and microarray as tools in understanding aflatoxin biosynthesis

Aflatoxins are the most toxic and carcinogenic naturally occurring mycotoxins. They are produced primarily byAspergillus flavus andA. parasiticus. In order to better understand the molecular mechanisms that control aflatoxin production, identification of genes usingA. flavus expressed sequence tags (ESTs) and microarrays is currently being performed. Sequencing and annotation ofA. flavus ESTs from a normalizedA. flavus cDNA library identified 7,218 unique EST sequences. Genes that are putatively involved in aflatoxin biosynthesis, regulation and signal transduction, fungal virulence or pathogenicity, stress response or antioxidation, and fungal development were identified from these ESTs. Microarrays containing over 5,000 uniqueA. flavus gene amplicons were constructed at The Institute for Genomic Research. Gene expression profiling under aflatoxin-producing and non-producing conditions using this microarray has identified hundreds of genes that are potentially involved in aflatoxin production. Further investigations on the functions of these genes by gene knockout experiments are underway. This research is expected to provide information for developing new strategies for controlling aflatoxin contamination of agricultural commodities.     

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.     

A fragmented aflatoxin-like gene cluster in the forest pathogen Dothistroma septosporum

The polyketide toxin dothistromin is very similar in structure to the aflatoxin precursor, versicolorin B. Dothistromin is made by a pine needle pathogen, Dothistroma septosporum, both in culture and in planta. Orthologs of aflatoxin biosynthetic genes have been identified that are required for dothistromin biosynthesis in D. septosporum. In contrast to the situation in aflatoxin-producing fungi where 25 aflatoxin biosynthetic and regulatory genes are tightly clustered in one region of the genome, the dothistromin gene cluster is fragmented. Three mini-clusters of dothistromin genes have been identified, each located on a 1.3-Mb chromosome and each grouped with non-dothistromin genes. There are no obvious patterns of repeated sequences or transposon relics to suggest recent recombination events. Most dothistromin genes within the mini-clusters are co-regulated, suggesting that coordinate control of gene expression is achieved despite this unusual arrangement of secondary metabolite biosynthetic genes.     

Clustered genes involved in cyclopiazonic acid production are next to the aflatoxin biosynthesis gene cluster in Aspergillus flavus

Cyclopiazonic acid (CPA), an indole-tetramic acid mycotoxin, is produced by many species of Aspergillus and Penicillium. In addition to CPA Aspergillus flavus produces polyketide-derived carcinogenic aflatoxins. Aflatoxin biosynthesis genes form a gene cluster in a subtelomeric region. Isolates of A. flavus lacking aflatoxin production due to the loss of the entire aflatoxin gene cluster and portions of the subtelomeric region are often unable to produce CPA, which suggests a physical link of genes involved in CPA biosynthesis to the aflatoxin gene cluster. Examining the subtelomeric region in A. flavus isolates of different chemotypes revealed a region possibly associated with CPA production. Disruption of three of the four genes present in this region predicted to encode a monoamine oxidase, a dimethylallyl tryptophan synthase, and a hybrid polyketide non-ribosomal peptide synthase abolished CPA production in an aflatoxigenic A. flavus strain. Therefore, some of the CPA biosynthesis genes are organized in a mini-gene cluster that is next to the aflatoxin gene cluster in A. flavus.     

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 veA gene of the pine needle pathogen Dothistroma septosporum regulates sporulation and secondary metabolism

Fungi possess genetic systems to regulate the expression of genes involved in complex processes such as development and secondary metabolite biosynthesis. The product of the velvet gene veA, first identified and characterized in Aspergillus nidulans, is a key player in the regulation of both of these processes. Since its discovery and characterization in many Aspergillus species, VeA has been found to have similar functions in other fungi, including the Dothideomycete Mycosphaerella graminicola. Another Dothideomycete, Dothistroma septosporum, is a pine needle pathogen that produces dothistromin, a polyketide toxin very closely related to aflatoxin (AF) and sterigmatocystin (ST) synthesized by Aspergillus spp. Dothistromin is unusual in that, unlike most other secondary metabolites, it is produced mainly during the early exponential growth phase in culture. It was therefore of interest to determine whether the regulation of dothistromin production in D. septosporum differs from the regulation of AF/ST in Aspergillus spp. To begin to address this question, a veA ortholog was identified and its function analyzed in D. septosporum. Inactivation of the veA gene resulted in reduced dothistromin production and a corresponding decrease in expression of dothistromin biosynthetic genes. Expression of other putative secondary metabolite genes in D. septosporum such as polyketide synthases and non-ribosomal peptide synthases showed a range of different responses to loss of Ds-veA. Asexual sporulation was also significantly reduced in the mutants, accompanied by a reduction in the expression of a putative stuA regulatory gene. The mutants were, however, able to infect Pinus radiata seedlings and complete their life cycle under laboratory conditions. Overall this work suggests that D. septosporum has a veA ortholog that is involved in the control of both developmental and secondary metabolite biosynthetic pathways.     

veA is required for toxin and sclerotial production in Aspergillus parasiticus

It was long been noted that secondary metabolism is associated with fungal development. In Aspergillus nidulans, conidiation and mycotoxin production are linked by a G protein signaling pathway. Also in A. nidulans, cleistothecial development and mycotoxin production are controlled by a gene called veA. Here we report the characterization of a veA ortholog in the aflatoxin-producing fungus A. parasiticus. Cleistothecia are not produced by Aspergillus parasiticus; instead, this fungus produces spherical structures called sclerotia that allow for survival under adverse conditions. Deletion of veA from A. parasiticus resulted in the blockage of sclerotial formation as well as a blockage in the production of aflatoxin intermediates. Our results indicate that A. parasiticus veA is required for the expression of aflR and aflJ, which regulate the activation of the aflatoxin gene cluster. In addition to these findings, we observed that deletion of veA reduced conidiation both on the culture medium and on peanut seed. The fact that veA is necessary for conidiation, production of resistant structures, and aflatoxin biosynthesis makes veA a good candidate gene to control aflatoxin biosynthesis or fungal development and in this way to greatly decrease its devastating impact on health and the economy.