Increased conidiation associated with progression along the sterigmatocystin biosynthetic pathway

The Aspergillus nidulans sterigmatocystin (ST) gene cluster contains both regulatory (aflR) and biosynthetic genes (stc genes) required for ST production. A total of 26 genes are in the cluster, 13 of which have been assigned a known function in the biosynthetic pathway. This complex secondary pathway represents a physiological cost to the fungus. We tested the amount of asexual spore production using a series of isogenic lines of A. nidulans, differing only in a mutation in aflR (resulting in a strain containing no ST intermediates) or a mutation in three stc genes that produced either no ST intermediates (ΔstcJ), an early ST intermediate, norsoloroinic acid (ΔstcE) or a late ST intermediate, versicolorin A (ΔstcU). In two independently replicated experiments we compared the numbers of conidia produced by each of these mutant strains and a wild type ST producer in a neutral (growth media) and a host (corn seed) environment. A stepwise increase in asexual spore production was observed with each progressive step in the ST pathway. Thus, the data suggest that recruitment or loss of these secondary metabolite pathway genes has a selective advantage apart from the physiological activity of the metabolite.     

Functional and phylogenetic analysis of the Aspergillus ochraceoroseus aflQ (ordA) gene ortholog

Within the Aspergillus parasiticus and A. flavus aflatoxin (AF) biosynthetic gene cluster the aflQ (ordA) and aflP (omtA) genes encode respectively an oxidoreductase and methyltransferase. These genes are required for the final steps in the conversion of sterigmatocystin (ST) to aflatoxin B(1) (AFB(1)). Aspergillus nidulans harbors a gene cluster that produces ST, as the aflQ and aflP orthologs are either non-functional or absent in the genome. Aspergillus ochraceoroseus produces both AF and ST, and it harbors an AF/ST biosynthetic gene cluster that is organized much like the A. nidulans ST cluster. The A. ochraceoroseus cluster also does not contain aflQ or aflP orthologs. However the ability of A. ochraceoroseus to produce AF would indicate that functional aflQ and aflP orthologs are present within the genome. Utilizing degenerate primers based on conserved regions of the A. flavus aflQ gene and an A. nidulans gene demonstrating the highest level of homology to aflQ, a putative aflQ ortholog was PCR amplified from A. ochraceoroseus genomic DNA. The A. ochraceoroseus aflQ ortholog demonstrated 57% amino acid identity to A. flavus AflQ. Transformation of an O-methylsterigmatocystin (OMST)-accumulating A. parasiticus aflQ mutant with the putative A. ochraceoroseus aflQ gene restored AF production. Although the aflQ gene does not reside in the AF/ST cluster it appears to be regulated in a manner similar to other A. ochraceoroseus AF/ST cluster genes. Phylogenetic analysis of AflQ and AflQ-like proteins from a number of ST- and AF-producing Aspergilli indicates that A. ochraceoroseus might be ancestral to A. nidulans and A. flavus.     

stcS, a putative P-450 monooxygenase, is required for the conversion of versicolorin A to sterigmatocystin in Aspergillus nidulans.

Sterigmatocystin (ST) and aflatoxin are carcinogenic end point metabolites derived from the same biochemical pathway, which is found in several Aspergillus spp. Recently, an ST gene cluster, containing approximately 25 distinct genes that are each proposed to function specifically in ST biosynthesis, has been identified in Aspergillus nidulans. Each of these structural genes is named stc (sterigmatocystin) followed by a consecutive letter of the alphabet. We have previously described stcU (formerly verA) as encoding a keto-reductase required for the conversion of versicolorin A to ST. We now describe a second A. nidulans gene, stcS (formerly verB), that is located within 2 kb of stcU in the ST gene cluster. An stcS-disrupted strain of A. nidulans, TSS17, was unable to produce ST and converted ST/aflatoxin precursors to versicolorin A rather than ST, indicating that stcS functions at the same point in the pathway as stcU. Genomic sequence analysis of stcS shows that it encodes a cytochrome P-450 monooxygenase and constitutes a novel P-450 family, CYP59. Assuming that StcU activity mimics that of similar P-450s, it is likely that StcU catalyzes one of the proposed oxidation steps necessary to convert versicolorin A to ST. These results constitute the first genetic proof that the conversion of versicolorin A to ST requires more than one enzymatic activity.     

Aspergillus nidulans verA is required for production of the mycotoxin sterigmatocystin.

Aspergillus nidulans produces the carcinogenic mycotoxin sterigmatocystin (ST), the next-to-last precursor in the aflatoxin (AF) biosynthetic pathway found in the closely related fungi Aspergillus flavus and Aspergillus parasiticus. We identified and characterized an A. nidulans gene, verA, that is required for converting the AF precursor versicolorin A to ST. verA is closely related to several polyketide biosynthetic genes involved in polyketide production in Streptomyces spp. and exhibits extended sequence similarity to A. parasiticus ver-1, a gene proposed to encode an enzyme involved in converting versicolorin A to ST. By performing a sequence analysis of the region 3' to verA, we identified two additional open reading frames, designated ORF1 and ORF2. ORF2 is closely related to a number of cytochrome P-450 monooxygenases, while ORF1 shares identity with the gamma subunit of translation elongation factor 1. Given that several steps in the ST-AF pathway may require monooxygenase activity and that AF biosynthetic genes are clustered in A. flavus and A. parasiticus, we suggest that verA may be part of a cluster of genes required for ST biosynthesis. We disrupted the verA coding region by inserting the A. nidulans argB gene into the center of the coding region and transformed an A. nidulans argB2 mutant to arginine prototrophy. Seven transformants that produced DNA patterns indicative of a verA disruption event were grown under ST-inducing conditions, and all of the transformants produced versicolorin A but negligible amounts of ST (200-fold to almost 1,000-fold less than the wild type), confirming the hypothesis that verA encodes an enzyme necessary for converting versicolorin A to ST.     

RcoA has pleiotropic effects on Aspergillus nidulans cellular development

Aspergillus nidulans rcoA encodes a member of the WD repeat family of proteins. The RcoA protein shares sequence similarity with other members of this protein family, including the Saccharomyces cerevisiae Tup1p and Neurospora crassa RCO1. Tup1p is involved in negative regulation of an array of functions including carbon catabolite repression. RCO1 functions in regulating pleiotropic developmental processes, but not carbon catabolite repression. In A. nidulans, deletion of rcoA (DeltarcoA), a recessive mutation, resulted in gross defects in vegetative growth, asexual spore production and sterigmatocystin (ST) biosynthesis. Expression of the asexual and ST pathway-specific regulatory genes, brlA and aflR, respectively, but not the signal transduction genes (i.e. flbA, fluG or fadA) regulating brlA and aflR expression was delayed (brlA) or eliminated (aflR) in a DeltarcoA strain. Overexpression of aflR in a DeltarcoA strain could not rescue normal expression of downstream targets of AflR. CreA-dependent carbon catabolite repression of starch and ethanol utilization was only weakly affected in a DeltarcoA strain. The strong role of RcoA in development, vegetative growth and ST production, compared with a relatively weak role in carbon catabolite repression, is similar to the role of RCO1 in N. crassa.     

Reciprocal oxylipin-mediated cross-talk in the Aspergillus–seed pathosystem

In Aspergilli, mycotoxin production and sporulation are governed, in part, by endogenous oxylipins (oxygenated, polyunsaturated fatty acids and metabolites derived therefrom). In Aspergillus nidulans , oxylipins are synthesized by the dioxygenase enzymes PpoA, PpoB and PpoC. Structurally similar oxylipins are synthesized in seeds via the action of lipoxygenase (LOX) enzymes. Previous reports have shown that exogenous application of seed oxylipins to Aspergillus cultures alters sporulation and mycotoxin production. Herein, we explored whether a plant oxylipin biosynthetic gene ( ZmLOX3 ) could substitute functionally for A. nidulans ppo genes. We engineered ZmLOX3 into wild-type A. nidulans , and into a Δ ppoAC strain that was reduced in production of oxylipins, conidia and the mycotoxin sterigmatocystin. ZmLOX3 expression increased production of conidia and sterigmatocystin in both backgrounds. We additionally explored whether A. nidulans oxylipins affect seed LOX gene expression during Aspergillus colonization. We observed that peanut seed pnlox2–3 expression was decreased when infected by A. nidulans Δ ppo mutants compared with infection by wild type. This result provides genetic evidence that fungal oxylipins are involved in plant LOX gene expression changes, leading to possible alterations in the fungal/host interaction. This report provides the first genetic evidence for reciprocal oxylipin cross-talk in the Aspergillus –seed pathosystem.     

Reciprocal oxylipin-mediated cross-talk in the Aspergillus-seed pathosystem.

In Aspergilli, mycotoxin production and sporulation are governed, in part, by endogenous oxylipins (oxygenated, polyunsaturated fatty acids and metabolites derived therefrom). In Aspergillus nidulans, oxylipins are synthesized by the dioxygenase enzymes PpoA, PpoB and PpoC. Structurally similar oxylipins are synthesized in seeds via the action of lipoxygenase (LOX) enzymes. Previous reports have shown that exogenous application of seed oxylipins to Aspergillus cultures alters sporulation and mycotoxin production. Herein, we explored whether a plant oxylipin biosynthetic gene (ZmLOX3) could substitute functionally for A. nidulans ppo genes. We engineered ZmLOX3 into wild-type A. nidulans, and into a DeltappoAC strain that was reduced in production of oxylipins, conidia and the mycotoxin sterigmatocystin. ZmLOX3 expression increased production of conidia and sterigmatocystin in both backgrounds. We additionally explored whether A. nidulans oxylipins affect seed LOX gene expression during Aspergillus colonization. We observed that peanut seed pnlox2-3 expression was decreased when infected by A. nidulansDeltappo mutants compared with infection by wild type. This result provides genetic evidence that fungal oxylipins are involved in plant LOX gene expression changes, leading to possible alterations in the fungal/host interaction. This report provides the first genetic evidence for reciprocal oxylipin cross-talk in the Aspergillus-seed pathosystem.     

Requirement of monooxygenase-mediated steps for sterigmatocystin biosynthesis by Aspergillus nidulans

Sterigmatocystin (ST) and aflatoxin B(1) (AFB(1)) are two polyketide-derived Aspergillus mycotoxins synthesized by functionally identical sets of enzymes. ST, the compound produced by Aspergillus nidulans, is a late intermediate in the AFB(1) pathway of A. parasiticus and A. flavus. Previous biochemical studies predicted that five oxygenase steps are required for the formation of ST. A 60-kb ST gene cluster in A. nidulans contains five genes, stcB, stcF, stcL, stcS, and stcW, encoding putative monooxygenase activities. Prior research showed that stcL and stcS mutants accumulated versicolorins B and A, respectively. We now show that strains disrupted at stcF, encoding a P-450 monooxygenase similar to A. parasiticus avnA, accumulate averantin. Disruption of either StcB (a putative P-450 monooxygenase) or StcW (a putative flavin-requiring monooxygenase) led to the accumulation of averufin as determined by radiolabeled feeding and extraction studies.     

Some genetical aspects of ornithine metabolism in Aspergillus nidulans.

A possible minor route of ornithine catabolism in Aspergillus nidulans might begin with the ornithine decarboxylase reaction and end with the succinic semialdehyde dehydrogenase reaction. It is therefore of interest that the putative structural genes for these two enzymes, puA and ssuA, respectively, are tightly linked group II. However, this linkage is unlikely to have regulatory significance because ileA, the structural gene for threonine dehydratase, separates them. The gene order in this region is ssuA-ileA-puA-mauB-anB. (mauB- mutations result in loss of monoamine oxidase whilst anB- mutations lead to aneurin auxotrophy.) 2. An auxotrophy for ornithine or putrescine in A. nidulans occurs in double mutants lacking arginase and blocked before ornithine in the arginine biosynthetic pathway. Some residual ornithine synthesis in such double mutants can be catalysed by ornithine delta-transaminase, especially if it is synthesised constitutively.