Functional expression and sub-cellular localization of the early aflatoxin pathway enzyme Nor-1 in Aspergillus parasiticus

Hong, Sung Yong; Linz, John E.

doi:10.1016/j.mycres.2009.01.013

Cited by 35 publications

(41 citation statements)

References 34 publications

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“…There is evidence to suggest that many secondary metabolite biosynthetic pathways in fungi are restricted to specific organelles, as shown for cyclosporine, penicillin, and aflatoxin biosynthesis (51)(52)(53)(54)(55)(56). Recently specific synthesis vesicles, aflatoxisomes (57), were discovered in Aspergillus parasiticus.…”

Section: Discussionmentioning

confidence: 99%

Two Novel Classes of Enzymes Are Required for the Biosynthesis of Aurofusarin in Fusarium graminearum

Frandsen

Schütt

Lund

et al. 2011

Journal of Biological Chemistry

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Previous studies have reported the functional characterization of 9 out of 11 genes found in the gene cluster responsible for biosynthesis of the polyketide pigment aurofusarin in Fusarium graminearum. Here we reanalyze the function of a putative aurofusarin pump (AurT) and the two remaining orphan genes, aurZ and aurS. Targeted gene replacement of aurZ resulted in the discovery that the compound YWA1, rather than nor-rubrofusarin, is the primary product of F. graminearum polyketide synthase 12 (FgPKS12). AurZ is the first representative of a novel class of dehydratases that act on hydroxylated ␥-pyrones. Replacement of the aurS gene resulted in accumulation of rubrofusarin, an intermediate that also accumulates when the GIP1, aurF, or aurO genes in the aurofusarin cluster are deleted. Based on the shared phenotype and predicted subcellular localization, we propose that AurS is a member of an extracellular enzyme complex (GIP1-AurF-AurO-AurS) responsible for converting rubrofusarin into aurofusarin. This implies that rubrofusarin, rather than aurofusarin, is pumped across the plasma membrane. Replacement of the putative aurofusarin pump aurT increased the rubrofusarin-to-aurofusarin ratio, supporting that rubrofusarin is normally pumped across the plasma membrane. These results provide functional information on two novel classes of proteins and their contribution to polyketide pigment biosynthesis.The plant pathogenic fungus Fusarium graminearum (teleomorph Gibberella zeae) is capable of producing a plethora of secondary metabolites, many of which belong to the polyketide class of compounds, such as the mycotoxins zearalenone, fusarin C, and aurofusarin (1). The F. graminearum genome encodes 15 polyketide synthases (PKS) 2 (2, 3), of which FgPKS12 is the progenitor of nor-rubrofusarin/rubrofusarin/aurofusarin (4, 5), FgPKS4 and FgPKS13 are the progenitors of zearalenone (1, 6), FgPKS10 is the progenitor of fusarin C (7, 8), and FgPKS3 is the progenitor of an uncharacterized black/purple perithecial pigment (8). PKS genes are typically found in gene clusters that comprise genes encoding transcription factor, transporters, and tailoring enzymes required for biosynthesis of the final product (1, 4 -6). In addition to genes encoding well characterized classes of enzymes, the clusters typically also include orphan genes that encode proteins annotated as "hypothetical" or "conserved hypothetical proteins," signifying that no similarity to any previously characterized protein has been found. In the case of F. graminearum, more than half of all gene models found in the Fusarium graminearum Genome Database from the Munich Information Center for Protein Sequences (MIPS) fall into either of these two categories (9).Functional characterization of hypothetical proteins is challenging, but those belonging to secondary metabolite gene clusters provide a unique opportunity because their location suggests a function in the respective biosynthetic pathways. The PKS12 gene cluster in F. graminearum, which is responsible for biosyn...

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Section: Discussionmentioning

confidence: 99%

Two Novel Classes of Enzymes Are Required for the Biosynthesis of Aurofusarin in Fusarium graminearum

Frandsen

Schütt

Lund

et al. 2011

Journal of Biological Chemistry

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“…A. parasiticus strain AFS10 (aflR), derived from SU-1, was used as a non-aflatoxinproducing control strain. A. parasiticus strain B62 (nor-1 brn-1; ATCC 24690) was derived from SU-1 and carries a genetic block in the aflatoxin biosynthetic pathway (9). Strain B62 accumulates large quantities of the bright red aflatoxin pathway intermediate norsolorinic acid (NA) as well as significantly reduced quantities of aflatoxin compared to SU-1 (9).…”

Section: Methodsmentioning

confidence: 99%

“…A. parasiticus strain B62 (nor-1 brn-1; ATCC 24690) was derived from SU-1 and carries a genetic block in the aflatoxin biosynthetic pathway (9). Strain B62 accumulates large quantities of the bright red aflatoxin pathway intermediate norsolorinic acid (NA) as well as significantly reduced quantities of aflatoxin compared to SU-1 (9). YES liquid medium (2% yeast extract and 6% sucrose, pH 5.8) was used as an aflatoxin-inducing growth medium.…”

Section: Methodsmentioning

confidence: 99%

A Possible Role for Exocytosis in Aflatoxin Export in Aspergillus parasiticus

Chanda¹,

Roze²,

Linz³

2010

Eukaryot Cell

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Filamentous fungi synthesize bioactive secondary metabolites with major human health and economic impacts. Little is known about the mechanisms that mediate the export of these metabolites to the cell exterior. Aspergillus parasiticus synthesizes aflatoxin, a secondary metabolite that is one of the most potent naturally occurring carcinogens known. We previously demonstrated that aflatoxin is synthesized and compartmentalized in specialized vesicles called aflatoxisomes and that these subcellular organelles also play a role in the export process. In the current study, we tested the hypothesis that aflatoxisomes fuse with the cytoplasmic membrane to facilitate the release of aflatoxin into the growth environment. Microscopic analysis of A. parasiticus grown under aflatoxin-inducing and non-aflatoxin-inducing conditions generated several lines of experimental evidence that supported the hypothesis. On the basis of the evidence, we propose that export of the mycotoxin aflatoxin in Aspergillus parasiticus occurs by exocytosis, and we present a model to illustrate this export mechanism.Secondary metabolites are chemically diverse natural products synthesized by plants, fungi, bacteria, algae, and animals. Secondary metabolites have an enormous impact on humans. Antibiotics, for example, are essential elements of the multibillion-dollar pharmaceutical industry, whereas mycotoxins cause hundreds of millions of dollars in damage to agriculture annually (11,15). These chemicals help the producing organism to survive nutrient limitation (16). They also contribute to cellular defense mechanisms and development (11, 12), reduce cellular oxidative stress (10), and help maintain cellular homeostasis by regulating carbon flow in the cell (17).Many fungal secondary metabolites are exported outside the cell; examples include antibiotics and mycotoxins (3,14). We and others conducted extensive studies on the regulation of fungal secondary metabolism at the molecular (11, 15) and cellular (3, 7) levels. However, little is known about the mechanisms that mediate secondary metabolite export or why export occurs.The filamentous fungus Aspergillus parasiticus produces aflatoxin, a secondary metabolite and the most potent naturally occurring carcinogen known. More than 90% of aflatoxin is exported to the cell exterior (3), making A. parasiticus an excellent model for studying secondary metabolite export. We recently demonstrated that specialized trafficking vesicles called aflatoxisomes play a key role in aflatoxin synthesis and export (3). As synthesis initiates, vesicle-vacuole fusion is downregulated by the global regulator Velvet, resulting in the accumulation of aflatoxisomes which contain at least the last two functional enzymes in the aflatoxin pathway and sequester aflatoxin (3). Treatments that block vesicle-vacuole fusion increase the number of aflatoxisomes, increase the quantity of aflatoxin accumulated in aflatoxisomes, and increase aflatoxin export to the cell exterior (3). On the basis of these previous observations, we ...

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“…We previously observed that early (Nor-1), middle (Ver-1), and late (OmtA) aflatoxin enzymes localize in vesicles and vacuoles in A. parasiticus (6)(7)(8). To determine the functional role of these organelles in aflatoxin synthesis, a vesicle-vacuole fraction (V) was first isolated from A. parasiticus grown for 36 h in aflatoxin-inducing medium (YES) using a high-density sucrose cushion method.…”

Section: Vesicles and Vacuoles Are One Primary Site For The Late Stepmentioning

confidence: 99%

“…1 and 2) and flavonoids (e.g., aurone) (reviewed in refs. 1 and 3) in plants and the nonribosomal peptide cyclosporin (4), the ␤-lactam antibiotic penicillin (5) (localization of ACVS is still controversial), and the polyketide aflatoxin (6)(7)(8) in fungi. However, the functional role of these compartments in secondary metabolism was unclear because these organelles potentially could be involved in synthesis, storage, protein turnover, transport, or export of the end product or biosynthetic enzymes.…”

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confidence: 99%

A key role for vesicles in fungal secondary metabolism

Chanda

Roze

Kang

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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197

244

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Eukaryotes have evolved highly conserved vesicle transport machinery to deliver proteins to the vacuole. In this study we show that the filamentous fungus Aspergillus parasiticus employs this delivery system to perform new cellular functions, the synthesis, compartmentalization, and export of aflatoxin; this secondary metabolite is one of the most potent naturally occurring carcinogens known. Here we show that a highly pure vesicle-vacuole fraction isolated from A. parasiticus under aflatoxin-inducing conditions converts sterigmatocystin, a late intermediate in aflatoxin synthesis, to aflatoxin B 1 ; these organelles also compartmentalize aflatoxin. The role of vesicles in aflatoxin biosynthesis and export was confirmed by blocking vesicle-vacuole fusion using 2 independent approaches. Disruption of A. parasiticus vb1 (encodes a protein homolog of AvaA, a small GTPase known to regulate vesicle fusion in A. nidulans) or treatment with Sortin3 (blocks Vps16 function, one protein in the class C tethering complex) increased aflatoxin synthesis and export but did not affect aflatoxin gene expression, demonstrating that vesicles and not vacuoles are primarily involved in toxin synthesis and export. We also observed that development of aflatoxigenic vesicles (aflatoxisomes) is strongly enhanced under aflatoxin-inducing growth conditions. Coordination of aflatoxisome development with aflatoxin gene expression is at least in part mediated by Velvet (VeA), a global regulator of Aspergillus secondary metabolism. We propose a unique 2-branch model to illustrate the proposed role for VeA in regulation of aflatoxisome development and aflatoxin gene expression.S econdary metabolites, natural products generated by filamentous fungi, plants, bacteria, algae, and animals, have an enormous impact on humans due to their application in health, medicine, and agriculture. Many secondary metabolites are beneficial (antibiotics, statins, morphine, etc.), though phytotoxins (e.g., ricin, crotin, amygdalin) and fungal poisons called mycotoxins (e.g., aflatoxin, sterigmatocystin, fumonisin) are detrimental to humans and animals. To control or customize biosynthesis of these natural products we must understand how and where secondary metabolism is orchestrated within the cell.Vacuoles and vesicles are known to sequester secondary metabolites to protect host cells from self-toxicity (1). Enzymes involved in secondary metabolism are often found in vesicles and vacuoles, including those for biosynthesis of alkaloids (e.g., berberine, sanguinarine, camptotecin, and morphine; reviewed in refs. 1 and 2) and flavonoids (e.g., aurone) (reviewed in refs. 1 and 3) in plants and the nonribosomal peptide cyclosporin (4), the ␤-lactam antibiotic penicillin (5) (localization of ACVS is still controversial), and the polyketide aflatoxin (6-8) in fungi. However, the functional role of these compartments in secondary metabolism was unclear because these organelles potentially could be involved in synthesis, storage, protein turnover, transport, or export of...

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Functional expression and sub-cellular localization of the early aflatoxin pathway enzyme Nor-1 in Aspergillus parasiticus

Cited by 35 publications

References 34 publications

Two Novel Classes of Enzymes Are Required for the Biosynthesis of Aurofusarin in Fusarium graminearum

Two Novel Classes of Enzymes Are Required for the Biosynthesis of Aurofusarin in Fusarium graminearum

A Possible Role for Exocytosis in Aflatoxin Export in Aspergillus parasiticus

A key role for vesicles in fungal secondary metabolism

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