Perng-Kuang Chang

Aflatoxin Biosynthesis Cluster Gene cypA Is Required for G Aflatoxin Formation

ABSTRACT Aspergillus flavus isolates produce only aflatoxins B1 and B2, while Aspergillus parasiticus and Aspergillus nomius produce aflatoxins B1, B2, G1, and G2. Sequence comparison of the aflatoxin biosynthesis pathway gene cluster upstream from the polyketide synthase gene, pksA, revealed that A. flavus isolates are missing portions of genes (cypA and norB) predicted to encode, respectively, a cytochrome P450 monooxygenase and an aryl alcohol dehydrogenase. Insertional disruption of cypA in A. parasiticus yielded transformants that lack the ability to produce G aflatoxins but not B aflatoxins. The enzyme encoded by cypA has highest amino acid identity to Gibberella zeae Tri4 (38%), a P450 monooxygenase previously shown to be involved in trichodiene epoxidation. The substrate for CypA may be an intermediate formed by oxidative cleavage of the A ring of O-methylsterigmatocystin by OrdA, the P450 monooxygenase required for formation of aflatoxins B1 and B2.

Association of aflatoxin biosynthesis and sclerotial development 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.

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.

Nonfunctionality of Aspergillus sojae aflR in a strain of Aspergillus parasiticus with a disrupted aflR gene

ABSTRACT Aspergillus sojae belongs to the Aspergillus section Flavi but does not produce aflatoxins. The functionality of the A. sojae aflR gene (aflRs) was examined by transforming it into an ΔaflR strain of A. parasiticus, derived from a nitrate-nonutilizing, versicolorin A (VERA)-accumulating strain. The A. parasiticus aflR gene (aflRp) transformants produced VERA, but the aflRs transformants did not. Even when aflRs was placed under the control of the amylase gene (amyB) promoter of Aspergillus oryzae, the amy(p)::aflRs transformants did not produce VERA. A chimeric construct containing the aflRs promoter plus the aflRs N- and aflRp C-terminal coding regions could restore VERA production, but a construct containing the aflRp promoter plus the aflRp N- and aflRs C-terminal coding regions could not. These results show that the A. sojae aflR promoter is functional in A. parasiticus and that the HAHA motif does not affect the function of the resulting hybrid AflR. We conclude that the lack of aflatoxin production by A. sojae can be attributed, at least partially, to the premature termination defect in aflRs, which deletes the C-terminal transcription activation domain that is critical for the expression of aflatoxin biosynthetic genes.

Characterization of the Aspergillus parasiticus major nitrogen regulatory gene, areA

The major nitrogen regulatory gene, areA, was cloned from Aspergillus parasiticus. It encoded a polypeptide of 864 amino acids which contained a nuclear localization signal (NLS), a highly acidic region from positions 497 to 542, a Cys-X(2)-Cys-X(17)-Cys-X(2)-Cys DNA-binding motif and a conserved carboxy-terminus. Electrophoretic mobility shift assays suggested that the A. parasiticus AREA DNA-binding domain fusion protein bound cooperatively to single GATA elements in the A. parasiticus niaD-niiA intergenic region. AREA also bound to the aflR-aflJ intergenic region of the aflatoxin biosynthesis gene cluster. Regions of areA were fused to a yeast GAL4 DNA-binding domain coding region to localize putative transcription activation domain(s) of AREA based on activation of the GAL1(p)::lacZ reporter gene expression. The portion between NLS and the acidic domain demonstrated 16-20-fold higher activation activities than other portions of AREA, which suggests that the transcription activation domain is located in this region.

Repressor-AFLR interaction modulates aflatoxin biosynthesis in Aspergillus parasiticus.

Regulation of aflatoxin (AF) biosynthesis likely involves a complex interplay of positive- and negative-acting factors that are affected by physiological cues responsive to internal and external stimuli. These factors, presumably, modulate the expression of the AF pathway-specific regulatory gene, aflR, whose product, AFLR, a zinc cluster transcription factor, then turns on or off the transcription of other AF genes. To determine if the AFLR carboxyl region (AFLRC) interacts with positive-or negative-acting proteins, we fused the Aspergillus parasiticus aflR carboxyl coding region(aflRC) to the promoter of A. parasiticusnitrite reductase gene (niiA(p)::aflRC), and transformed it into A. parasiticus SRRC 2043. Transformants that contained two copies of niiA(p)::aflRC, one at the niaD locus and another at the aflR locus, over produced AF precursors independent of the nitrogen source. The higher copy number of the integrated niiA(p)::aflRC correlated with increased production of AF precursors by the transformants as well as increased expression of both aflRC and native aflR in potato dextrose broth and A & M medium. Since aflRC does not encode a DNA-binding domain, the expressed AFLRC should not bind to the promoters of AF pathway genes and affect transcription directly. The results are consistent with AFLRC titrating out a putative repressor that interacts with AFLR under different growth conditions and modulates AF biosynthesis. This interaction also indirectly affects sclerotial development.

Development and refinement of a high-efficiency gene-targeting system for Aspergillus flavus

An efficient gene-targeting system based on impairment of the nonhomologous end-joining pathway and the orotidine monophosphate decarboxylase gene (pyrG) in Aspergillus flavus was established. It was achieved by replacing the ku70 gene with the Aspergillus oryzae pyrithiamine resistance (ptr) gene and by inserting the Aspergillus parasiticus cypA gene into the pyrG locus. The utility of this system was demonstrated by disruption of nine candidate genes for conidial pigment biosynthesis. The gene-targeting frequencies ranged from 80 to 100%. Two linked genes on chromosome 4, wA and olgA, were confirmed to be involved in pigment formation. In contrast to the parental strain which produced yellowish-green conidia, the knockout mutants produced white and olive-green conidia, respectively. The system was further refined by restoring the pyrithiamine sensitivity and uracil auxotrophy in the A. flavus transformation recipient with an engineered pyrG marker. The improvement allowed gene manipulation using the reusable pyrG marker as shown by the restoration of laeA-mediated aflatoxin production in an A. flavus laeA-deleted mutant.

What does genetic diversity of Aspergillus flavus tell us about Aspergillus oryzae

Aspergillus flavus and Aspergillus oryzae belong to Aspergillus section Flavi. They are closely related and are of significant economic importance. The former species has the ability to produce harmful aflatoxins while the latter is widely used in food fermentation and industrial enzyme production. This review summarizes the current understanding of the similarity of the A. flavus and A. oryzae genomes, the genetic diversity in A. flavus and A. oryzae populations, the causes of this diversity, and the relatedness of nonaflatoxigenic A. flavus strains to A. oryzae.

Loss of msnA, a putative stress regulatory gene, in Aspergillus parasiticus and Aspergillus flavus increased production of conidia, aflatoxins and kojic acid.

Production of the harmful carcinogenic aflatoxins by Aspergillus parasiticus and Aspergillus flavus has been postulated to be a mechanism to relieve oxidative stress. The msnA gene of A. parasiticus and A. flavus is the ortholog of Saccharomyces cerevisiae MSN2 that is associated with multi-stress response. Compared to wild type strains, the msnA deletion (∆msnA) strains of A. parasiticus and A. flavus exhibited retarded colony growth with increased conidiation. The ∆msnA strains also produced slightly higher amounts of aflatoxins and elevated amounts of kojic acid on mixed cereal medium. Microarray assays showed that expression of genes encoding oxidative stress defense enzymes, i.e., superoxide dismutase, catalase, and cytochrome c peroxidase in A. parasiticus ∆msnA, and the catalase A gene in A. flavus ∆msnA, was up-regulated. Both A. parasiticus and A. flavus ∆msnA strains produced higher levels of reactive oxygen species (ROS), and ROS production of A. flavus msnA addback strains was decreased to levels comparable to that of the wild type A. flavus. The msnA gene appears to be required for the maintenance of the normal oxidative state. The impairment of msnA resulted in the aforementioned changes, which might be used to combat the increased oxidative stress in the cells.

Deletion of the Aspergillus flavus Orthologue of A. nidulans fluG Reduces Conidiation and Promotes Production of Sclerotia but Does Not Abolish Aflatoxin Biosynthesis

ABSTRACT The fluG gene is a member of a family of genes required for conidiation and sterigmatocystin production in Aspergillus nidulans. We examined the role of the Aspergillus flavus fluG orthologue in asexual development and aflatoxin biosynthesis. Deletion of fluG in A. flavus yielded strains with an approximately 3-fold reduction in conidiation but a 30-fold increase in sclerotial formation when grown on potato dextrose agar in the dark. The concurrent developmental changes suggest that A. flavus FluG exerts opposite effects on a mutual signaling pathway for both processes. The altered conidial development was in part attributable to delayed expression of brlA, a gene controlling conidiophore formation. Unlike the loss of sterigmatocystin production by A. nidulans fluG deletion strains, aflatoxin biosynthesis was not affected by the fluG deletion in A. flavus. In A. nidulans, FluG was recently found to be involved in the formation of dehydroaustinol, a component of a diffusible signal of conidiation. Coculturing experiments did not show a similar diffusible meroterpenoid secondary metabolite produced by A. flavus. These results suggest that the function of fluG and the signaling pathways related to conidiation are different in the two related aspergilli.