Kojic Acid Gene Clusters and the Transcriptional Activation Mechanism of Aspergillus flavus KojR on Expression of Clustered Genes.

Kojic acid (KA) is a fungal metabolite and has a variety of applications in the cosmetics and food industries. Aspergillus oryzae is a well-known producer of KA, and its KA biosynthesis gene cluster has been identified. In this study, we showed that nearly all section Flavi aspergilli except for A. avenaceus had complete KA gene clusters, and only one Penicillium species, P. nordicum, contained a partial KA gene cluster. Phylogenetic inference based on KA gene cluster sequences consistently grouped section Flavi aspergilli into clades as prior studies. The Zn(II)2Cys6 zinc cluster regulator KojR transcriptionally activated clustered genes of kojA and kojT in Aspergillus flavus. This was evidenced by the time-course expression of both genes in kojR-overexpressing strains whose kojR expression was driven by a heterologous Aspergillus nidulans gpdA promoter or a homologous A. flavus gpiA promoter. Using sequences from the kojA and kojT promoter regions of section Flavi aspergilli for motif analyses, we identified a consensus KojR-binding motif to be an 11-bp palindromic sequence of 5'-CGRCTWAGYCG-3' (R = A/G, W = A/T, Y = C/T). A CRISPR/Cas9-mediated gene-targeting technique showed that the motif sequence, 5'-CGACTTTGCCG-3', in the kojA promoter was critical for KA biosynthesis in A. flavus. Our findings may facilitate strain improvement and benefit future kojic acid production.

Genome Sequence of an Aspergillus flavus CA14 Strain That Is Widely Used in Gene Function Studies.

Aspergillus flavus produces aflatoxins that adversely impact human health and the economy. We report the genome sequence of A. flavus CA14 that has been widely used in gene function studies. The information will benefit A. flavus functional genomics studies on fungal development, secondary metabolite production, and fungus-host plant interactions.

Identification of a copper-transporting ATPase involved in biosynthesis of A. flavus conidial pigment.

Conidia are asexual spores and play a crucial role in fungal dissemination. Conidial pigmentation is important for tolerance against UV radiation and contributes to survival of fungi. The molecular basis of conidial pigmentation has been studied in several fungal species. In spite of sharing the initial common step of polyketide formation, other steps for pigment biosynthesis appear to be species-dependent. In this study, we isolated an Aspergillus flavus spontaneous mutant that produced yellow conidia. The underlying genetic defect, a three-nucleotide in-frame deletion in the gene, AFLA_051390, that encodes a copper-transporting ATPase, was identified by a comparative genomics approach. This genetic association was confirmed by disruption of the wild-type gene. When yellow mutants were grown on medium supplemented with copper ions or chloride ions, green conidial color was partially and nearly completely restored, respectively. Further disruption of AFLA_045660, an orthologue of Aspergillus nidulans yA (yellow pigment) that encodes a multicopper oxidase, in wild type and a derived strain producing dark green conidia showed that it yielded mutants that produced gold conidia. The results placed formation of the gold pigment after that of the yellow pigment and before that of the dark green pigment. Using reported inhibitors of DHN-melanin (tricyclazole and phthalide) and DOPA-melanin (tropolone and kojic acid) pathways on a set of conidial color mutants, we investigated the involvement of melanin biosynthesis in A. flavus conidial pigment formation. Results imply that both pathways have no bearing on conidial pigment biosynthesis of A. flavus.

The Transcriptional Regulator Hbx1 Affects the Expression of Thousands of Genes in the Aflatoxin-Producing Fungus Aspergillus flavus.

In filamentous fungi, homeobox proteins are conserved transcriptional regulators described to control conidiogenesis and fruiting body formation. Eight homeobox (hbx) genes are found in the genome of the aflatoxin-producing ascomycete, Aspergillus flavus While loss-of-function of seven of the eight genes had little to no effect on fungal growth and development, disruption of hbx1, resulted in aconidial colonies and lack of sclerotial production. Furthermore, the hbx1 mutant was unable to produce aflatoxins B1 and B2, cyclopiazonic acid and aflatrem. In the present study, hbx1 transcriptome analysis revealed that hbx1 has a broad effect on A. flavus gene expression, and the effect of hbx1 increases overtime, impacting more than five thousand protein-coding genes. Among the affected genes, those in the category of secondary metabolism (SM), followed by that of cellular transport, were the most affected. Specifically, regarding the effect of hbx1 on SM, we found that genes in 44 SM gene clusters where upregulated while 49 were downregulated in the absence of hbx1, including genes in the SM clusters responsible for the synthesis of asparasone, piperazine and aflavarin, all known to be associated with sclerotia. In addition, our study revealed that hbx1 affects the expression of other transcription factor genes involved in development, including the conidiation central regulatory pathway and flb genes.

Aspergillus flavus GPI-anchored protein-encoding ecm33 has a role in growth, development, aflatoxin biosynthesis, and maize infection.

Many glycosylphosphatidylinositol-anchored proteins (GPI-APs) of fungi are membrane enzymes, organization components, and extracellular matrix adhesins. We analyzed eight Aspergillus flavus transcriptome sets for the GPI-AP gene family and identified AFLA_040110, AFLA_063860, and AFLA_113120 to be among the top 5 highly expressed genes of the 36 family genes analyzed. Disruption of the former two genes did not drastically affect A. flavus growth and development. In contrast, disruption of AFLA_113120, an orthologue of Saccharomyces cerevisiae ECM33, caused a significant decrease in vegetative growth and conidiation, promoted sclerotial production, and altered conidial pigmentation. The A. flavus ecm33 null mutant, compared with the wild type and the complemented strain, produced predominantly aflatoxin B2 but accumulated comparable amounts of cyclopiazonic acid. It showed decreased sensitivity to Congo red at low concentrations (25-50 µg/mL) but had increased sensitivity to calcofluor white at high concentrations (250-500 µg/mL). Analyses of cell wall carbohydrates indicated that the α-glucan content was decreased significantly (p < 0.05), but the contents of chitin and ß-glucan were increased in the mutant strain. In a maize colonization study, the mutant was shown to be impaired in its infectivity and produced 3- to 4-fold lower amounts of conidia than the wild type and the complemented strain. A. flavus Ecm33 is required for proper cell wall composition and plays an important role in normal fungal growth and development, aflatoxin biosynthesis, and seed colonization.

The Aspergillus flavus Homeobox Gene, hbx1, is Required for Development and Aflatoxin Production.

Homeobox proteins, a class of well conserved transcription factors, regulate the expression of targeted genes, especially those involved in development. In filamentous fungi, homeobox genes are required for normal conidiogenesis and fruiting body formation. In the present study, we identified eight homeobox (hbx) genes in the aflatoxin-producing ascomycete, Aspergillus flavus, and determined their respective role in growth, conidiation and sclerotial production. Disruption of seven of the eight genes had little to no effect on fungal growth and development. However, disruption of the homeobox gene AFLA_069100, designated as hbx1, in two morphologically different A. flavus strains, CA14 and AF70, resulted in complete loss of production of conidia and sclerotia as well as aflatoxins B1 and B2, cyclopiazonic acid and aflatrem. Microscopic examination showed that the Δhbx1 mutants did not produce conidiophores. The inability of Δhbx1 mutants to produce conidia was related to downregulation of brlA (bristle) and abaA (abacus), regulatory genes for conidiophore development. These mutants also had significant downregulation of the aflatoxin pathway biosynthetic genes aflC, aflD, aflM and the cluster-specific regulatory gene, aflR. Our results demonstrate that hbx1 not only plays a significant role in controlling A. flavus development but is also critical for the production of secondary metabolites, such as aflatoxins.

Aspergillus flavus aswA, a gene homolog of Aspergillus nidulans oefC, regulates sclerotial development and biosynthesis of sclerotium-associated secondary metabolites.

Aspergillus flavus aswA (AFLA_085170) is a gene encoding a Zn(II)2Cys6 DNA-binding domain and a transcriptional activation domain, DUF3468. Disruption of aswA yielded strains that made a truncated gene transcript and generated a fungus that produced a greatly increased number of sclerotia. These sclerotia were odd-shaped and non-pigmented (white) and different from oval and pigmented (dark brown to black) mature sclerotia. Transcriptomic analysis of the ΔaswA strain grown on potato dextrose agar plates and Wickerham agar plates showed that expression of clustering genes involved in the biosynthesis of three sclerotium-associated secondary metabolites was down-regulated. These included gene clusters of asparasone, aflatrem, and aflavarin. In contrast, those of aflatoxin, cyclopiazonic acid and kojic acid were not affected. Metabolite analyses confirmed that the non-pigmented sclerotia contained aflatoxin and cyclopiazonic acid but not other aforementioned metabolites, three asparasone analogs and dihydroxyaflavinine commonly present in mature sclerotia. Impairment in aswA gene function stalls normal sclerotial development, which in turn prevents biosynthesis and accumulation of sclerotium-specific metabolites.

The Aspergillus flavus fluP-associated metabolite promotes sclerotial production.

Aspergillus flavus is able to synthesize a variety of polyketide-derived secondary metabolites including the hepatocarcinogen, aflatoxin B1. The fungus reproduces and disseminates predominantly by production of conidia. It also produces hardened mycelial aggregates called sclerotia that are used to cope with unfavourable growth environments. In the present study, we examined the role of A. flavus fluP, the backbone polyketide synthase gene of secondary metabolite gene cluster 41, on fungal development. The A. flavus CA14 fluP deletion mutant (AfΔfluP) grew and accumulated aflatoxin normally but produced a lower amount of sclerotia than the parental strain. This was also true for the Aspergillus parasiticus BN9 fluP deletion mutant (ApΔfluP). The A. flavus fluP gene was positively regulated by developmental regulators of VeA and VelB but not by the global regulator of secondary metabolism, LaeA. Overexpression of fluP in AfΔfluP (OEfluP) elevated its ability to produce sclerotia compared to that of the parental strain. Coculture of OEfluP with CA14, AfΔfluP, ApΔfluP, or an A. flavus pptA deletion mutant incapable of producing functional polyketide synthases also allowed increased sclerotial production of the respective strains at edges where colonies made contact. Acetone extracts of OEfluP but not of AfΔfluP exhibited the same effect in promoting sclerotial production of AfΔfluP. These results suggest that FluP polyketide synthase is involved in the synthesis of a diffusible metabolite that could serve as a signal molecule to regulate sclerotiogenesis.

High sequence variations in the region containing genes encoding a cellular morphogenesis protein and the repressor of sexual development help to reveal origins of Aspergillus oryzae.

Aspergillus oryzae and Aspergillus flavus are closely related fungal species. The A. flavus morphotype that produces numerous small sclerotia (S strain) and aflatoxin has a unique 1.5 kb deletion in the norB-cypA region of the aflatoxin gene cluster (i.e. the S genotype). Phylogenetic studies have indicated that an isolate of the nonaflatoxigenic A. flavus with the S genotype is the ancestor of A. oryzae. Genome sequence comparison between A. flavus NRRL3357, which produces large sclerotia (L strain), and S-strain A. flavus 70S identified a region (samA-rosA) that was highly variable in the two morphotypes. A third type of samA-rosA region was found in A. oryzae RIB40. The three samA-rosA types were later revealed to be commonly present in A. flavus L-strain populations. Of the 182 L-strain A. flavus field isolates examined, 46%, 15% and 39% had the samA-rosA type of NRRL3357, 70S and RIB40, respectively. The three types also were found in 18 S-strain A. flavus isolates with different proportions. For A. oryzae, however, the majority (80%) of the 16 strains examined had the RIB40 type and none had the NRRL3357 type. The results suggested that A. oryzae strains in the current culture collections were mostly derived from the samA-rosA/RIB40 lineage of the nonaflatoxigenic A. flavus with the S genotype.

Transcriptomic profiles of Aspergillus flavus CA42, a strain that produces small sclerotia, by decanal treatment and after recovery.

Aspergillus flavus is a ubiquitous saprophyte and is capable of producing many secondary metabolites including the carcinogenic aflatoxins. The A. flavus population that produces small sclerotia (S strain) has been implicated as the culprit for persistent aflatoxin contamination in field crops. We investigated how the plant volatile decanal, a C10 fatty aldehyde, affected the growth and development of the S strain A. flavus. Decanal treatment yielded fluffy variants lacking sclerotia and conidia and exhibiting a dosage-dependent radial colony growth. We used RNA-Seq analysis to examine transcriptomic changes caused by decanal and after removal of decanal. Mature sclerotia contained only 80% of the total transcripts detected in all samples in comparison to 94% for the decanal treated culture. Gene ontology (GO) analysis showed that decanal treatment increased expression of genes involved in oxidoreductase activity, cellular carbohydrate metabolism, alcohol metabolism and aflatoxin biosynthesis. The treatment affected cellular components associated with cell wall, and gene expression of glucanases, α-amylases, pectinesterase and peptidase required for its biosynthesis was increased. After decanal was removed, the culture resumed sclerotial production. Moreover, its GO terms significantly overlapped with those of the untreated culture; five of the enriched molecular functions, oxidoreductase activity, monooxygenase activity, electron carrier activity, heme binding, and iron binding were found in the untreated culture. The GO term of cellular component enriched was mainly integral protein constituents of the membrane. The results suggested that decanal halted development at the vegetative state rendering the fungus unable to produce conidia and sclerotia. The induced fluffy phenotype could be related to lower transcript abundance of flbB, flbD, and flbE but not to veA expression. Increased abundance of the laeA transcript in the treated culture correlated with early transcriptional activation of aflatoxin and kojic acid biosynthesis gene clusters. Expression profiles revealed subtle differences in timing of activation of the respective 55 secondary metabolite gene clusters.

Aspergillus flavus VelB acts distinctly from VeA in conidiation and may coordinate with FluG to modulate sclerotial production.

The proteins VeA, VelB and LaeA of Aspergillus nidulans form a heterotrimeric complex (the velvet complex) in the dark to coordinate sexual development and production of some secondary metabolites. VeA and VelB of A. nidulans and Aspergillus fumigatus also are repressors of conidiation, but VeA of Aspergillus flavus in studied strains acts positively on conidiation. In the present study, we show via yeast-two hybrid assays that interactions among A. flavus VeA, VelB, and LaeA are conserved as in the A. nidulans velvet complex. We found that FluG, which is required for conidiophore formation in A. nidulans but whose deletion in A. flavus delays onset of conidiation, was probably an interacting partner of VelB. Deletion of velB in A. flavus CA14 severely impaired conidiation in the dark although to a lesser extent than deletion of veA. In both mutants fluG deletion resulted in further decreased conidiation even in the light. Deletion of fluG in the ΔlaeA strain, however, did not affect conidiation. All mutant types were unable to produce aflatoxin and sclerotia. Cross-complementation of the ΔvelB strain with gpdA::veA restored conidiation but not aflatoxin production although aflR, the aflatoxin pathway regulatory gene, was expressed at a normal level. Cross-complementation of the ΔveA strain with gpdA::velB failed to restore conidiation and aflatoxin production. The ΔvelB strain complemented with or a wild type transformed by gpdA::velB had elevated sclerotial production as the ΔfluG strain. Concerted interactions of A. flavus VeA and VelB with LaeA are critical for conidiation and aflatoxin biosynthesis. VelB may have a dual role and likely coordinates with FluG to modulate sclerotial production.

Deletion of the Aspergillus flavus orthologue of A. nidulans fluG reduces conidiation and promotes production of sclerotia but does not abolish aflatoxin biosynthesis.

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.

Effects of laeA deletion on Aspergillus flavus conidial development and hydrophobicity may contribute to loss of aflatoxin production.

LaeA of Aspergillus nidulans is a putative methyltransferase and a component of the velvet complex; it is thought to mainly affect expression of genes required for the production of secondary metabolites. We found that although Aspergillus flavus CA14 laeA deletion mutants showed no aflatoxin production, expression of some of the early genes involved in aflatoxin formation, but not the later genes, could still be detected. The mutants grown in minimal medium supplemented with simple sugars and on some complex media exhibited altered conidial development. On potato dextrose agar (PDA) medium the deletion mutants showed reduced conidial chain elongation, increased production of conidiophores, and decreased colony hydrophobicity when compared to the parental strain. The loss of hydrophobicity and the other developmental changes in the laeA deletion mutants could affect the ability of the fungus to produce aflatoxins.

Identification of genetic defects in the atoxigenic biocontrol strain Aspergillus flavus K49 reveals the presence of a competitive recombinant group in field populations.

Contamination of corn, cotton, peanuts and tree nuts by aflatoxins is a severe economic burden for growers. A current biocontrol strategy is to use non-aflatoxigenic Aspergillus flavus strains to competitively exclude field toxigenic Aspergillus species. A. flavus K49 does not produce aflatoxins and cyclopiazonic acid (CPA) and is currently being tested in corn-growing fields in Mississippi. We found that its lack of production of aflatoxins and CPA resulted from single nucleotide mutations in the polyketide synthase gene and hybrid polyketide-nonribosomal peptide synthase gene, respectively. Furthermore, based on single nucleotide polymorphisms of the aflatoxin biosynthesis omtA gene and the CPA biosynthesis dmaT gene, we conclude that K49, AF36 and previously characterized TX9-8 form a biocontrol group. These isolates appear to be derived from recombinants of typical large and small sclerotial morphotype strains. This finding provides an easy way to select future biocontrol strains from the reservoir of non-aflatoxigenic populations in agricultural fields.

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.

HypC, the anthrone oxidase involved in aflatoxin biosynthesis.

On the basis of gene disruption and enzyme activity, hypC, an open reading frame in the region between the pksA (aflC) and nor-1 (aflD) genes in the aflatoxin biosynthesis gene cluster, encodes a 17-kDa oxidase that converts norsolorinic acid anthrone to norsolorinic acid.

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.

Absence of the aflatoxin biosynthesis gene, norA, allows accumulation of deoxyaflatoxin B1 in Aspergillus flavus cultures.

Biosynthesis of the highly toxic and carcinogenic aflatoxins in select Aspergillus species from the common intermediate O-methylsterigmatocystin has been postulated to require only the cytochrome P450 monooxygenase, OrdA (AflQ). We now provide evidence that the aryl alcohol dehydrogenase NorA (AflE) encoded by the aflatoxin biosynthetic gene cluster in Aspergillus flavus affects the accumulation of aflatoxins in the final steps of aflatoxin biosynthesis. Mutants with inactive norA produced reduced quantities of aflatoxin B(1) (AFB(1)), but elevated quantities of a new metabolite, deoxyAFB(1). To explain this result, we suggest that, in the absence of NorA, the AFB(1) reduction product, aflatoxicol, is produced and is readily dehydrated to deoxyAFB(1) in the acidic medium, enabling us to observe this otherwise minor toxin produced in wild-type A. flavus.

Are the genes nadA and norB involved in formation of aflatoxin G(1)?

Aflatoxins, the most toxic and carcinogenic family of fungal secondary metabolites, are frequent contaminants of foods intended for human consumption. Previous studies showed that formation of G-group aflatoxins (AFs) from O-methylsterigmatocystin (OMST) by certain Aspergillus species involves oxidation by the cytochrome P450 monooxygenases, OrdA (AflQ) and CypA (AflU). However, some of the steps in the conversion have not yet been fully defined. Extracts of Aspergillus parasiticus disruption mutants of the OYE-FMN binding domain reductase-encoding gene nadA (aflY) contained a 386 Da AFG(1) precursor. A compound with this mass was predicted as the product of sequential OrdA and CypA oxidation of OMST. Increased amounts of a 362 Da alcohol, the presumptive product of NadA reduction, accumulate in extracts of fungi with disrupted aryl alcohol dehydrogenase-encoding gene norB. These results show that biosynthesis of AFG(1) involves NadA reduction and NorB oxidation.