Identification of averantin as an aflatoxin B1 precursor: placement in the biosynthetic pathway.

A new blocked mutant of Aspergillus parasiticus produces no detectable aflatoxin B1, but accumulates several polyhydroxyanthraquinones. One of these pigments was identified as averantin. This is the first report of its formation by A. parasiticus. Radiotracer studies with [14C]averantin showed that 15.3% of label from averantin was incorporated into aflatoxin B1. This incorporation was blocked by dichlorvos. With radiotracers and other mutants, averantin was placed after norsolorinic acid and before averufin in the biosynthetic pathway in which the general steps are norsolorinic acid leads to averantin leads to averufin leads to versiconal hemiacetal acetate leads to versicolorin A leads to sterigmatocystin leads to aflatoxin B1.     

Enzymatic function of the nor-1 protein in aflatoxin biosynthesis in Aspergillus parasiticus

The nor-1 gene is involved in aflatoxin biosynthesis in Aspergillus parasiticus and was predicted to encode a norsolorinic acid ketoreductase. Recombinant Nor-1 expressed in Escherichia coli converted the 1' keto group of norsolorinic acid to the 1' hydroxyl group of averantin in crude E. coli cell extracts in the presence of NADPH. The results confirm that Nor-1 functions as a ketoreductase in vitro.     

Inhibition of aflatoxin biosynthesis by phenolic compounds

The phenolic compounds acetosyringone, syringaldehyde and sinapinic acid inhibited the biosynthesis of aflatoxin B1 (AFB1) by A. flavus. Acetosyringone was the most active among the three compounds, inhibiting aflatoxin level by 82% at 2 m moll-1. The synthesis and accumulation of norsolorinic acid, an aflatoxin biosynthetic intermediate, was also inhibited. These results suggest that at least one step early in the AFB1 biosynthetic pathway is inhibited by the phenolics.     

Cloning of the Aspergillus parasiticus apa-2 gene associated with the regulation of aflatoxin biosynthesis.

An Aspergillus parasiticus gene, designated apa-2, was identified as a regulatory gene associated with aflatoxin biosynthesis. The apa-2 gene was cloned on the basis of overproduction of pathway intermediates following transformation of fungal strains with cosmid DNA containing the aflatoxin biosynthetic genes nor-1 and ver-1. Transformation of an O-methylsterigmatocystin-accumulating strain, A. parasiticus SRRC 2043, with a 5.5-kb HindIII-XbaI DNA fragment containing apa-2 resulted in overproduction of all aflatoxin pathway intermediates analyzed. Specific enzyme activities associated with the conversion of norsolorinic acid and sterigmatocystin were increased approximately twofold. The apa-2 gene was found to complement an A. flavus afl-2 mutant strain for aflatoxin production, suggesting that apa-2 is functionally homologous to afl-2. Comparison of the A. parasiticus apa-2 gene DNA sequence with that of the A. flavus afl-2 gene (G. A. Payne, G. J. Nystorm, D. Bhatnagar, T. E. Cleveland, and C. P. Woloshuk, Appl. Environ. Microbiol. 59:156-162, 1993) showed that they shared > 95% DNA homology. Physical mapping of cosmid subclones placed apa-2 approximately 8 kb from ver-1.     

Conversion of a new metabolite to aflatoxin B2 by Aspergillus parasiticus

A new metabolite which could be converted to aflatoxin (AF) B2 was detected during cofermentation analysis of two nonaflatoxigenic strains (SRRC 2043 and SRRC 163) of Aspergillus parasiticus. SRRC 2043, which accumulates the xanthone O-methylsterigmatocystin (OMST), a late precursor in the AFB1 pathway, was observed to accumulate another chemically related compound (HOMST; molecular weight, 356); SRRC 163 is blocked early in the pathway and accumulates averantin. During cofermentation of the two strains, levels of OMST and HOMST were observed to be greatly reduced in the culture, with simultaneous production of AFB1, AFB2, and AFG1. Intact cells of SRRC 163 were able to convert pure OMST or its precursor, sterigmatocystin, to AFB1 and AFG1 without AFB2 accumulation; the same cells converted isolated HOMST to AFB2 with no AFB1 or AFG1 production. The results indicate that AFB2 is produced from a separate branch in the AF biosynthetic pathway than are AFB1 and AFG1; AFB2 arises from HOMST, and AFB1 and AFG1 arise from sterigmatocystin and OMST.     

Conversion of a new metabolite to aflatoxin B2 by Aspergillus parasiticus.

A new metabolite which could be converted to aflatoxin (AF) B2 was detected during cofermentation analysis of two nonaflatoxigenic strains (SRRC 2043 and SRRC 163) of Aspergillus parasiticus. SRRC 2043, which accumulates the xanthone O-methylsterigmatocystin (OMST), a late precursor in the AFB1 pathway, was observed to accumulate another chemically related compound (HOMST; molecular weight, 356); SRRC 163 is blocked early in the pathway and accumulates averantin. During cofermentation of the two strains, levels of OMST and HOMST were observed to be greatly reduced in the culture, with simultaneous production of AFB1, AFB2, and AFG1. Intact cells of SRRC 163 were able to convert pure OMST or its precursor, sterigmatocystin, to AFB1 and AFG1 without AFB2 accumulation; the same cells converted isolated HOMST to AFB2 with no AFB1 or AFG1 production. The results indicate that AFB2 is produced from a separate branch in the AF biosynthetic pathway than are AFB1 and AFG1; AFB2 arises from HOMST, and AFB1 and AFG1 arise from sterigmatocystin and OMST.     

Characterization of the function of the ver-1A and ver-1B genes, involved in aflatoxin biosynthesis in Aspergillus parasiticus.

The ver-1A gene was cloned and its nucleotide sequence was determined as part of a previous study on aflatoxin B1 (AFB1) biosynthesis in the filamentous fungus Aspergillus parasiticus SU-1. A second copy of this gene, ver-1B, was tentatively identified in this fungal strain. In this study, ver-1B was cloned by screening an A. parasiticus cosmid library with a ver-1A probe. The nucleotide sequence of ver-1B was determined. The predicted amino acid sequence of ver-1B had 95% identity with ver-1A. A translational stop codon, found in the ver-1B gene coding region, indicated that it encodes a truncated polypeptide. To confirm the function of the ver-1 genes in AFB1 synthesis, a plasmid (pDV-VA) was designed to disrupt ver-1A and/or ver-1B by transformation of the AFB1 producer A. parasiticus NR-1. One disruptant, VAD-102, which accumulated the pathway intermediate versicolorin A was obtained. Southern hybridization analysis of VAD-102 revealed that ver-1A but not ver-1B was disrupted. A functional ver-1A gene was transformed back into strain VAD-102. Transformants which received ver-1A produced AFB1, confirming that ver-1A is the only functional ver-1 gene in A. parasiticus SU-1 and that its gene product is involved in the conversion of versicolorin A to sterigmatocystin in AFB1 biosynthesis. A duplicated chromosomal region (approximately 12 kb) was identified upstream from ver-1A and ver-1B by Southern hybridization analysis. This duplicated region contained the aflR gene, which is proposed to be one regulator of AFB1, synthesis. A similar gene duplication was also identified in several other strains of A. parasiticus.     

Interactions of saprophytic yeasts with a nor mutant of Aspergillus flavus.

The nor mutant of Aspergillus flavus has a defective norsolorinic acid reductase, and thus the aflatoxin biosynthetic pathway is blocked, resulting in the accumulation of norsolorinic acid, a bright red-orange pigment. We developed a visual agar plate assay to monitor yeast strains for their ability to inhibit aflatoxin production by visually scoring the accumulation of this pigment of the nor mutant. We identified yeast strains that reduced the red-orange pigment accumulation in the nor mutant. These yeasts also reduced aflatoxin accumulation by a toxigenic strain of A. flavus. These yeasts may be useful for reducing aflatoxin contamination of food commodities.     

Biosynthetic origin of aflatoxin G1: confirmation of sterigmatocystin and lack of confirmation of aflatoxin B1 as precursors

The origin of aflatoxin G1 was studied using mutant strains of Aspergillus parasiticus blocked early in the pathway and by tracing 14C-labelled aflatoxin B1 (AFB1) in wild-type A. flavus and A. parasiticus strains. Sterigmatocystin (ST) was a precursor of AFB1, AFG1 and AFG2 in the four mutants examined. The identity of AFG1 was confirmed by mass spectrometry. No evidence for conversion of AFB1 to AFG1 was found. A rigorously controlled study of conversions of radioactivity based on preparative thin-layer chromatography of aflatoxins demonstrated that low levels of aflatoxin interconversions previously reported in the literature might actually be artifacts.     

Interactions of Saprophytic Yeasts with a nor Mutant of Aspergillus flavus

The nor mutant of Aspergillus flavus has a defective norsolorinic acid reductase, and thus the aflatoxin biosynthetic pathway is blocked, resulting in the accumulation of norsolorinic acid, a bright red-orange pigment. We developed a visual agar plate assay to monitor yeast strains for their ability to inhibit aflatoxin production by visually scoring the accumulation of this pigment of the nor mutant. We identified yeast strains that reduced the red-orange pigment accumulation in the nor mutant. These yeasts also reduced aflatoxin accumulation by a toxigenic strain of A. flavus. These yeasts may be useful for reducing aflatoxin contamination of food commodities.     

Chromosomal location plays a role in regulation of aflatoxin gene expression in Aspergillus parasiticus.

The nor-1 gene in the filamentous fungus Aspergillus parasiticus encodes a ketoreductase involved in aflatoxin biosynthesis. To study environmental influences on nor-1 expression, we generated plasmid pAPGUSNNB containing a nor-1 promoter-beta-glucuronidase (GUS) (encoded by uidA) reporter fusion with niaD (encodes nitrate reductase) as a selectable marker. niaD transformants of A. parasiticus strain NR-1 (niaD) carried pAPGUSNNB integrated predominantly at the nor-1 or niaD locus. Expression of the native nor-1 and nor-1::GUS reporter was compared in transformants grown under aflatoxin-inducing conditions by Northern and Western analyses and by qualitative and quantitative GUS activity assays. The timing and level of nor-1 promoter function with pAPGUSNNB integrated at nor-1 was similar to that observed for the native nor-1 gene. In contrast, nor-1 promoter activity in pAPGUSNNB and a second nor-1::GUS reporter construct, pBNG3.0, was not detectable when integration occurred at niaD. Because niaD-dependent regulation could account for the absence of expression at niaD, a third chromosomal location was analyzed using pAPGUSNP, which contained nor-1::GUS plus pyrG (encodes OMP decarboxylase) as a selectable marker. GUS expression was detectable only when pAPGUSNP integrated at nor-1 and was not detectable at pyrG, even under growth conditions that required pyrG expression. nor-1::GUS is regulated similarly to the native nor-1 gene when it is integrated at its homologous site within the aflatoxin gene cluster but is not expressed at native nor-1 levels at two locations outside of the aflatoxin gene cluster. We conclude that the GUS reporter system can be used effectively to measure nor-1 promoter activity and that nor-1 is subject to position-dependent regulation in the A. parasiticus chromosome.     

The affinity purification and characterization of a dehydrogenase from Aspergillus parasiticus involved in aflatoxin B1 biosynthesis.

A two step scheme has been developed for the purification of a dehydrogenase from mycelia of 84 hours old Aspergillus parasiticus (1-11-105 Wh 1), which catalyzes the conversion of norsolorinic acid (NA) to averantin (AVN). The dehydrogenase was purified from cell-free extracts using reactive green 19-agarose and norsolorinic acid-agarose affinity chromatography. The latter affinity matrix was synthesised by attaching norsolorinic acid to omega-aminohexylagarose. The purified protein was shown to be homogenous on non-denaturing polyacrylamide gel electrophoresis. A final purification of 215-fold was achieved. Results of gel filtration chromatography indicated the approximate molecular mass of the native protein to be 140,000 daltons. The isoelectric point of the protein was about 5.5 as determined by chromatofocusing. The reaction catalyzed by the dehydrogenase was optimum at pH 8.5 and between 25 degrees to 35 degrees C. The Km of the enzyme for NA and NADPH was determined to be 3.45 microM and 103 microM respectively.     

Regulation of aflatoxin synthesis by FadA/cAMP/protein kinase A signaling in Aspergillus parasiticus

Analysis of fadA and pkaA mutants in the filamentous fungus Aspergillus nidulans demonstrated that FadA (Galpha) stimulates cyclic AMP (cAMP)-dependent protein kinase A (PKA) activity resulting, at least in part, in inhibition of conidiation and sterigmatocystin (ST) biosynthesis. In contrast, cAMP added to the growth medium stimulates aflatoxin (AF) synthesis in Aspergillus parasiticus. Our goal was to explain these conflicting reports and to provide mechanistic detail on the role of FadA, cAMP, and PKA in regulation of AF synthesis and conidiation in A. parasiticus. cAMP or dibutyryl-cAMP (DcAMP) were added to a solid growth medium and intracellular cyclic nucleotide levels, PKA activity, and nor-1 promoter activity were measured in A. parasiticus D8D3 (nor1::GUS reporter) and TJYP1-22 (fadAGA2R, activated allele). Similar to Tice and Buchanan [34], cAMP or DcAMP stimulated AF synthesis (and conidiation) associated with an AflR-dependent increase in nor-1 promoter activity. However, treatment resulted in a 100-fold increase in intracellular cAMP/DcAMP accompanied by a 40 to 80 fold decrease in total PKA activity. ThefadAG42R allele in TJYP1-22 decreased AF synthesis and conidiation, increased basal PKA activity 10 fold, and decreased total PKA activity 2 fold. In TJYP1-22, intracellular cAMP increased 2 fold without cAMP or DcAMP treatment; treatment did not stimulate conidiation or AF synthesis. Based on these data, we conclude that: (1) FadA/PKA regulate toxin synthesis and conidiation via similar mechanisms in Aspergillus spp.; and (2) intracellular cAMP levels, at least in part, mediate a PKA-dependent regulatory influence on conidiation and AF synthesis.     

Pathways for the incorporation of exogenous fatty acids into phosphatidylethanolamine in Escherichia coli

Two distinct pathways for the incorporation of exogenous fatty acids into phospholipids were identified in Escherichia coli. The predominant route originates with the activation of fatty acids by acyl-CoA synthetase followed by the distribution of the acyl moieties into all phospholipid classes via the sn-glycerol-3-phosphate acyltransferase reaction. This pathway was blocked in mutants (fadD) lacking acyl-CoA synthetase activity. In fadD strains, exogenous fatty acids were introduced exclusively into the 1-position of phosphatidylethanolamine. This secondary route is related to 1-position fatty acid turnover in phosphatidylethanolamine and proceeds via the acyl-acyl carrier protein synthetase/2-acylglycerophosphoethanolamine acyltransferase system. The turnover pathway exhibited a preference for saturated fatty acids, whereas the acyl-CoA synthetase-dependent pathway was less discriminating. Both pathways were inhibited in mutants (fadL) lacking the fatty acid permease, demonstrating that the fadL gene product translocates exogenous fatty acids to an intracellular pool accessible to both synthetases. These data demonstrate that acyl-CoA synthetase is not required for fatty acid transport in E. coli and that the metabolism of exogenous fatty acids is segregated from the metabolism of acyl-acyl carrier proteins derived from fatty acid biosynthesis.     

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.