There is extensive and unequivocal evidence that secondary metabolism in filamentous fungi and plants is associated with oxidative stress. In support of this idea, transcription factors related to oxidative stress response in yeast, plants, and fungi have been shown to participate in controlling secondary metabolism. Aflatoxin biosynthesis, one model of secondary metabolism, has been demonstrated to be triggered and intensified by reactive oxygen species buildup. An oxidative stress-related bZIP transcription factor AtfB is a key player in coordinate expression of antioxidant genes and genes involved in aflatoxin biosynthesis. Recent findings from our laboratory provide strong support for a regulatory network comprised of at least four transcription factors that bind in a highly coordinated and timely manner to promoters of the target genes and regulate their expression. In this review, we will focus on transcription factors involved in co-regulation of aflatoxin biosynthesis with oxidative stress response in aspergilli, and we will discuss the relationship of known oxidative stress-associated transcription factors and secondary metabolism in other organisms. We will also talk about transcription factors that are involved in oxidative stress response, but have not yet been demonstrated to be affiliated with secondary metabolism. The data support the notion that secondary metabolism provides a secondary line of defense in cellular response to oxidative stress.

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.

Aflatoxins are among the principal mycotoxins that contaminate economically important food and feed crops. Aflatoxin B₁ is the most potent naturally occurring carcinogen known and is also an immunosuppressant. Occurrence of aflatoxins in crops has vast economic and human health impacts worldwide. Thus, the study of aflatoxin biosynthesis has become a focal point in attempts to reduce human exposure to aflatoxins. This review highlights recent advances in the field of aflatoxin biosynthesis and explores the functional connection between aflatoxin biosynthesis, endomembrane trafficking, and response to oxidative stress. Dissection of the regulatory mechanisms involves a complete comprehension of the aflatoxin biosynthetic process and the dynamic network of transcription factors that orchestrates coordinated expression of the target genes. Despite advancements in the field, development of a safe and effective multifaceted approach to solve the aflatoxin food contamination problem is still required.

In filamentous fungi, several lines of experimental evidence indicate that secondary metabolism is triggered by oxidative stress; however, the functional and molecular mechanisms that mediate this association are unclear. The basic leucine zipper (bZIP) transcription factor AtfB, a member of the bZIP/CREB family, helps regulate conidial tolerance to oxidative stress. In this work, we investigated the role of AtfB in the connection between oxidative stress response and secondary metabolism in the filamentous fungus Aspergillus parasiticus. This well characterized model organism synthesizes the secondary metabolite and carcinogen aflatoxin. Chromatin immunoprecipitation with specific anti-AtfB demonstrated AtfB binding at promoters of seven genes in the aflatoxin gene cluster that carry CREs. Promoters lacking CREs did not show AtfB binding. The binding of AtfB to the promoters occurred under aflatoxin-inducing but not under aflatoxin-noninducing conditions and correlated with activation of transcription of the aflatoxin genes. Deletion of veA, a global regulator of secondary metabolism and development, nearly eliminated this binding. Electrophoretic mobility shift analysis demonstrated that AtfB binds to the nor-1 (an early aflatoxin gene) promoter at a composite regulatory element that consists of highly similar, adjacent CRE1 and AP-1-like binding sites. The five nucleotides immediately upstream from CRE1, AGCC(G/C), are highly conserved in five aflatoxin promoters that demonstrate AtfB binding. We propose that AtfB is a key player in the regulatory circuit that integrates secondary metabolism and cellular response to oxidative stress.

The mycotoxin aflatoxin is a secondary metabolite and potent human carcinogen. We investigated one mechanism that links stress response with coordinate activation of genes involved in aflatoxin biosynthesis in Aspergillus parasiticus. Electrophoretic mobility shift assays demonstrated that AtfB , a basic leucine zipper (bZIP) transcription factor, is a master co‐regulator that binds promoters of early (fas‐1), middle (ver‐1), and late (omtA) aflatoxin biosynthetic genes as well as stress‐response genes (mycelia‐specific cat1 and mitochondria‐specific Mn sod) at cAMP response element motifs. A novel conserved motif 5′‐T/GNT/CAAG CCNNG/AA/GC/ANT/C‐3′ was identified in promoters of the aflatoxin biosynthetic and stress‐response genes. A search for transcription factors identified SrrA as a transcription factor that could bind to the motif. Moreover, we also identified a STRE motif (5′‐CCCCT‐3′) in promoters of aflatoxin biosynthetic and stress‐response genes, and competition EMSA suggested that MsnA binds to this motif. Our study for the first time provides strong evidence to suggest that at least four transcription factors ( AtfB , SrrA , AP‐1 , and MsnA ) participate in a regulatory network that induces aflatoxin biosynthesis as part of the cellular response to oxidative stress in A. parasiticus.

Summary: The 27 genes involved in aflatoxin biosynthesis are clustered within a 70 kb region in the Aspergillus parasiticus genome. Using chromatin immunoprecipitation, we demonstrated a positive correlation between the initiation and spread of histone H4 acetylation in aflatoxin promoters and the onset of accumulation of aflatoxin proteins and aflatoxin. Histone H4 acetylation in the pksA (encodes an ‘early’ biosynthetic pathway enzyme) promoter peaked at 30 h, prior to the increased acetylation in the omtA and ordA (encode ‘late’ enzymes) promoters detected at 40 h. The specific order in which pksA, ver‐1 (encodes a ‘middle’ enzyme) and omtA transcripts accumulated in cells paralleled the pattern of spread of histone H4 acetylation. Binding of AflR, a positive regulator of aflatoxin biosynthesis, to the ordA promoter showed a positive correlation with the spread of histone H4 acetylation. The data suggest that the order of genes within the aflatoxin cluster determines the timing and order of transcriptional activation, and that the site of initiation and spread of histone H4 acetylation mediate this process. Our data indicate that the aflatoxin and adjacent sugar utilization clusters are part of a larger ‘regulatory unit’.