Gary A. Payne

Clustered Pathway Genes in Aflatoxin Biosynthesis

Aflatoxins, a group of polyketide-derived furanocoumarins (Fig. [1][1]), are the most toxic and carcinogenic compounds among the known mycotoxins. Among the at least 16 structurally related aflatoxins characterized, however, there are only four major aflatoxins, B1, B2, G1, and G2 (AFB1, AFG1, AFB2

Whole genome comparison of Aspergillus flavus and A. oryzae

Aspergillus flavus is a plant and animal pathogen that also produces the potent carcinogen aflatoxin. Aspergillus oryzae is a closely related species that has been used for centuries in the food fermentation industry and is Generally Regarded As Safe (GRAS). Whole genome sequences for these two fungi are now complete, providing us with the opportunity to examine any genomic differences that may explain the different ecological niches of these two fungi, and perhaps to identify pathogenicity factors in A. flavus. These two fungi are very similar in genome size and number of predicted genes. The estimated genome size (36·8 Mb) and predicted number of genes (12 197) for A. flavus is similar to that of A. oryzae (36·7 Mb and 12 079, respectively). These two fungi have significantly larger genomes than Aspergillus nidulans (30·1) and Aspergillus fumigatus (29·4). The A. flavus and A. oryzae genomes are enriched in genes for secondary metabolism, but do not differ greatly from one another in the predicted number of polyketide synthases, nonribosomal peptide synthases or the number of genes coding for cytochrome P450 enzymes. A micro-scale analysis of the two fungi did show differences in DNA correspondence between the two species and in the number of transposable elements. Each species has approximately 350 unique genes. The high degree of sequence similarity between the two fungi suggests that they may be ecotypes of the same species and that A. oryzae has resulted from the domestication of A. flavus.

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.

Diverse inhibitors of aflatoxin biosynthesis

Pre-harvest and post-harvest contamination of maize, peanuts, cotton, and tree nuts by members of the genus Aspergillus and subsequent contamination with the mycotoxin aflatoxin pose a widespread food safety problem for which effective and inexpensive control strategies are lacking. Since the discovery of aflatoxin as a potently carcinogenic food contaminant, extensive research has been focused on identifying compounds that inhibit its biosynthesis. Numerous diverse compounds and extracts containing activity inhibitory to aflatoxin biosynthesis have been reported. Only recently, however, have tools been available to investigate the molecular mechanisms by which these inhibitors affect aflatoxin biosynthesis. Many inhibitors are plant-derived and a few may be amenable to pathway engineering for tissue-specific expression in susceptible host plants as a defense against aflatoxin contamination. Other compounds show promise as protectants during crop storage. Finally, inhibitors with different modes of action could be used in comparative transcriptional and metabolomic profiling experiments to identify regulatory networks controlling aflatoxin biosynthesis.

Beyond aflatoxin: four distinct expression patterns and functional roles associated with Aspergillus flavus secondary metabolism gene clusters

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.

Aspergillus niger absorbs copper and zinc from swine wastewater

Wastewater from swine confined-housing operations contains elevated levels of copper and zinc due to their abundance in feed. These metals may accumulate to phytotoxic levels in some agricultural soils of North Carolina due to land application of treated swine effluent. We evaluated fungi for their ability to remove these metals from wastewater and found Aspergillus niger best suited for this purpose. A. niger was able to grow on plates amended with copper at a level five times that inhibitory to the growth of Saccharomyes cerevisiae. We also found evidence for internal absorption as the mechanism used by A. niger to detoxify its environment of copper, a property of the fungus that has not been previously exploited for metal bioremediation. In this report, we show that A. niger is capable of removing 91% of the copper and 70% of the zinc from treated swine effluent.

Unlocking the secrets behind secondary metabolism: a review of Aspergillus flavus from pathogenicity to functional genomics.

Aspergillus flavus has received a considerable amount of attention due to its ability to produce aflatoxin, a secondary metabolite that is both immunosuppressive and carcinogenic to animals and humans. Research on aflatoxin over the last 40 years has made it one of the best studied fungal secondary metabolites. In spite of the large volume of research in this area, many unanswered questions remain concerning the genetic regulation of aflatoxin production and the molecular signals that intimately associate the synthesis of aflatoxin with specific environmental and nutritional conditions. It is anticipated that the tools now available in the field of genomics will build upon our existing knowledge and provide answers to some of these questions. Complete genome sequences are now available for a number of fungal species that are closely related to A. flavus. This information can be used along with current genomic analyses in A. flavus to more closely examine the biosynthesis and regulation of secondary metabolism. The intent of this review is to summarize the large body of knowledge that exists from many years of research on A. flavus, with the hope that this information in the light of new genomic studies may bring scientists closer to unraveling the web of regulatory circuits that govern aflatoxin biosynthesis. Specifically, scientific findings in the following areas will be presented: classification and phylogenetic analyses of A. flavus, population biology, ecology and pathogenicity in agricultural environments, classical genetics including linkage group and mutant analyses, gene clusters, regulation of aflatoxin biosynthesis, and genomics.

Infection and Fumonisin Production by Fusarium verticillioides in Developing Maize Kernels

ABSTRACT Fusarium ear rot and fumonisin contamination are serious problems for maize growers, particularly in the southeastern United States. The lack of maize genotypes highly resistant to infection by Fusarium verticillioides or to fumonisin contamination emphasizes the need for management strategies to prevent contamination by this mycotoxin. Information on the initial appearance of infection and fumonisin contamination of kernels and their increase over time is needed to determine if early harvest may be an appropriate control strategy. Maize ears from replicated studies at two locations in eastern North Carolina were harvested weekly, starting 2 weeks after pollination and continuing for 14 weeks. The percentage of kernels infected with F. verticillioides and the fumonisin contamination in the harvested samples were determined. Kernel infection by F. verticillioides and fumonisin contamination appeared as kernels neared physiological maturity and increased up to the average harvest date for maize in North Carolina. Beyond this date, the concentrations of fumonisin fluctuated. Under years conducive for fumonisin contamination, early harvest (greater than 25% grain moisture) may help reduce the level of contamination.

Identification of two aflatrem biosynthesis gene loci in Aspergillus flavus and metabolic engineering of Penicillium paxilli to elucidate their function.

ABSTRACT Aflatrem is a potent tremorgenic toxin produced by the soil fungus Aspergillus flavus, and a member of a structurally diverse group of fungal secondary metabolites known as indole-diterpenes. Gene clusters for indole-diterpene biosynthesis have recently been described in several species of filamentous fungi. A search of Aspergillus complete genome sequence data identified putative aflatrem gene clusters in the genomes of A. flavus and Aspergillus oryzae. In both species the genes for aflatrem biosynthesis cluster at two discrete loci; the first, ATM1, is telomere proximal on chromosome 5 and contains a cluster of three genes, atmG, atmC, and atmM, and the second, ATM2, is telomere distal on chromosome 7 and contains five genes, atmD, atmQ, atmB, atmA, and atmP. Reverse transcriptase PCR in A. flavus demonstrated that aflatrem biosynthesis transcript levels increased with the onset of aflatrem production. Transfer of atmP and atmQ into Penicillium paxilli paxP and paxQ deletion mutants, known to accumulate paxilline intermediates paspaline and 13-desoxypaxilline, respectively, showed that AtmP is a functional homolog of PaxP and that AtmQ utilizes 13-desoxypaxilline as a substrate to synthesize aflatrem pathway-specific intermediates, paspalicine and paspalinine. We propose a scheme for aflatrem biosynthesis in A. flavus based on these reconstitution experiments in P. paxilli and identification of putative intermediates in wild-type cultures of A. flavus.

Heritabilities and Correlations of Fusarium Ear Rot Resistance and Fumonisin Contamination Resistance in Two Maize Populations

In volume 46, issue 1, p. 353–361, the estimates of heritability for fumonisin concentration and Fusarium ear rot in the NC3003 B104 recombinant inbred population were computed incorrectly. The corrected estimates of heritability on an entry mean basis are 0.88 (SE 5 0.03) for fumonisin concentration and 0.86 (SE 5 0.03) for ear rot. The corrected estimates of heritability on a plot basis are 0.58 (SE 5 0.06) for fumonisin concentration and 0.55 (SE 5 0.07) for ear rot. The corrected estimate of heritability on an individual plant basis for ear rot is 0.22 (SE 5 0.03). The corrected estimate of the relative efficiency of indirect selection is 0.85. All of the corrected parameter estimates are higher than the original estimates reported, and the changes do not affect our conclusions.