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

Kenneth C. Ehrlich , Perng-Kuang Chang , Leslie L. Scharfenstein , Jeffrey W. Cary , Jason M. Crawford , Craig A. Townsend
DOI: http://dx.doi.org/10.1111/j.1574-6968.2010.01914.x 65-70 First published online: 1 April 2010

Abstract

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 B1 (AFB1), but elevated quantities of a new metabolite, deoxyAFB1. To explain this result, we suggest that, in the absence of NorA, the AFB1 reduction product, aflatoxicol, is produced and is readily dehydrated to deoxyAFB1 in the acidic medium, enabling us to observe this otherwise minor toxin produced in wild-type A. flavus.

Keywords
  • Aspergillus flavus
  • aflatoxin biosynthesis
  • gene disruption
  • MS
  • aryl alcohol dehydrogenase
  • aflatoxicol

Introduction

Many species of Aspergillus produce the xanthone metabolite sterigmatocystin (Fig. 1), but only a few are capable of converting sterigmatocystin into the far more toxic and carcinogenic aflatoxins (AFs: AFB1, AFB2, AFG1, AFG2) (Frisvad et al., 2007). Because Aspergillus species are common agricultural contaminants and because ingestion of aflatoxins can lead to hepatocellular carcinoma, a better understanding of the final steps of aflatoxin biosynthesis is needed. For aflatoxin B1 (AFB1) biosynthesis, sterigmatocystin must first be methylated by an O-methyltransferase unique to aflatoxin biosynthesis (Bhatnagar et al., 1987a, b). The resulting methylated intermediate, O-methylsterigmatocystin (OMST) (Yu et al., 1998), is then oxidized by the cytochrome P450 monooxygenase, OrdA (AflQ). Because AFB1 was produced when either OMST or its presumptive initial oxidation product, 11-hydroxy-OMST (HOMST), was fed to yeast cells expressing the Aspergillus parasiticus cytochrome P450 monooxygenase OrdA (Prieto et al., 1996; Udwary et al., 2002), it was proposed that OrdA is the only enzyme required for the conversion of OMST to AFB1. To be consistent with the yeast-feeding experiment, OrdA must also introduce an oxygen atom into HOMST (Fig. 1). The subsequent conversion steps require hydration, ring-opening, cyclization, decarboxylation, and demethylation to produce AFB1.

1

Known or previously postulated intermediates in the conversion of sterigmatocystin (ST) to AFB1. Molecular weights are shown below each compound. OmtA, O-methyltransferase.

The oxidative ring cleavage and rearrangement necessary for the formation of the coumarin ring system in AFB1 must be consistent with the following observations: (a) NADPH is utilized in the conversion (Singh & Hsieh, 1976); (b) an ‘NIH hydride shift’ occurs so that the C-11 hydrogen is retained (Simpson et al., 1983); (c) an oxygen atom and carbon-11 in the A-ring of OMST are lost as carbon dioxide (Chatterjee & Townsend, 1994); and (d) an oxygen atom incorporated into the B-ring (Scheme 1) is retained (Watanabe & Townsend, 1996).

The role of the putative aryl alcohol dehydrogenase NorA (AflE) in aflatoxin biosynthesis has not been definitively ascribed, although it was originally thought to function in the reduction of norsolorinic acid to averantin (hence the name ‘Nor’) (Cary et al., 1996; Yu et al., 2004). NorA shares >60% amino acid identity with NorB (AflF), an aryl alcohol dehydrogenase shown to be involved in the formation of AFG1 (Ehrlich et al., 2008). Genes encoding both enzymes are part of the aflatoxin biosynthesis gene cluster. The sterigmatocystin gene cluster of Aspergillus nidulans possesses only one of these genes: stcV (Brown et al., 1996). Based on blast searches of genome sequence databases, genes encoding aryl alcohol dehydrogenases are common in many filamentous fungi and yeast. In the Aspergillus flavus aflatoxin gene cluster, the promoter and translation start codon of norB are missing due to a large DNA deletion in this region (Ehrlich et al., 2004). Therefore, the role of norA is most easily examined in A. flavus. We now provide evidence that A. flavus, lacking a functional copy of norA, accumulates a new metabolite, deoxyAFB1, a shunt metabolite that most likely is formed by dehydration of aflatoxicol (AFOH) in the acidic culture medium.

Materials and methods

Preparation of norA mutants in A. flavus AF13

A vector for insertional inactivation of norA in A. flavus was constructed by PCR with the oligonucleotide primers P1, 5′-acgactacaagaatagcggtgacat and P2, 5′-tattctagagacgcagactcttggtatgg (GenBank accession #AY510451; 47574–48188) and P3, 5′-tattctagagtactgggccgcggtcagtt and P4, 5′-aatggtacctcgagtccgcgacaactaggctcattttg (48516–49107) to amplify 5′- and 3′-portions of AF13ΔnorA, respectively (Fig. 2a). The resulting 614- and 591-bp PCR fragments were cloned into the SphI/XbaI and XbaI/KpnI sites of pUC18, respectively. An XbaI fragment from the niaD-containing plasmid, pSL82 (Chang et al., 1996), was then inserted into the internal XbaI site of the norA fragments in pUC18 to create the knockout vector.

2

Disruption of norA in Aspergillus flavus AF13. (a) Schematic showing the design of the knockout vector. Numbers denote the position of the region in AF13 DNA (GenBank accession number AY51051). P1–P4 are the oligonucleotide primers used for amplification of the 5′- and 3′-ends of the gene. The neighboring genes estA and ver-1 are shown. niaD was obtained from an XbaI digest of pSL82. The construct was prepared in pUC18. (b) PCR with oligonucleotide primers P1 and P4 of AF13 DNA from different transformant clones. Lane 1, 1 kb+ marker (Invitrogen); lane 2, AF13 (pSL82) control; lanes 3–5, AF13ΔnorA-clones 3, 15, 21, respectively.

Transformation of A. flavus AF13ΔniaD protoplasts was performed as described previously using the PEG procedure (Ehrlich et al., 2004) with 10 μg of the XhoI/SphI-linearized plasmid. Confirmation that norA was insertionally inactivated (double crossover event) in the resulting transformants was carried out by PCR using the outer oligonucleotide primers (P1 and P4, Fig. 2b) with DNA from the putative transformants or from pSL82-transformed AF13 as the control.

Thin-layer chromatography (TLC) and liquid chromatography/mass spectrometry (LC/MS)

Fungal cultures grown from spores at 30 °C for 3 days on potato dextrose agar (PDA, Difco, Voigt Global Distribution, Lawrence, KS) were extracted with acetone and chloroform as described previously (Ehrlich et al., 2004). Aliquots of the extract were analyzed by TLC on 250-μm silica gel plates (J.T. Baker, Phillipsburg, NJ) developed with toluene : ethyl acetate : acetic acid (8 : 1 : 1). A prominent blue-fluorescent compound from ΔnorA cultures was partially purified by preparative TLC. The unpurified extract, the TLC-purified metabolite, and authentic standards (AFB1, synthetic aflatoxicol, synthetic deoxyAFB1, OMST, and synthetic HOMST) were analyzed in the positive ion mode by LC/MS. The materials were dissolved in methanol, injected on a Luna C18 100 × 4.6 mm column (5 μm, 100 Å, Phenomenex) equilibrated in 10% acetonitrile/0.1% formic acid and 90% aqueous formic acid (0.1%), and eluted with a gradient to 100% acetonitrile/0.1% formic acid over 30 min. Metabolites were monitored by both diode array UV-visible spectrophotometry and quadrupole MS (Agilent 6130).

Feeding studies

Aflatoxicol (AFOH) and deoxyAFB1 were prepared by zinc borohydride reduction of AFB1 (Sigma, St. Louis, MO) (Hsia & Chu, 1977). Aflatoxicol (AFOH) was partially purified from the reaction mix by preparative TLC. Synthetic AFOH was dissolved in 200 μL dimethyl sulfoxide and added to 3-day mycelial cultures of A. parasiticus SRRC2043 (accumulates OMST only) in low-sugar replacement medium (Bhatnagar et al., 1987a, b) or to Saccharomyces cerevisiae expressing norA or ordA after induction with galactose (Yu et al., 1998). Following a 4-h incubation, metabolites were extracted into methylene chloride and aliquots were examined by TLC.

blast searches

blast searches (tblastx and blastp) were performed against the sequenced fungal genome datasets in Pubmed (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi), the Broad Institute fungal database (http://www.broadinstitute.org/annotation/genome/aspergillus_group/MultiHome.html), and the A. flavus genomic sequence (http://www.aspergillusflavus.org/genomics). The cut-off for matches was E −30.

Results

Transformation of A. flavus AF13ΔniaD with the linearized norA knockout vector (Fig. 2a) yielded approximately 60 colonies, three of which had slightly darker orange mycelia when regrown on PDA plates. The three darker orange transformants were confirmed to be double crossover norA disruptants by PCR (Fig. 2b). A 1.5-kb PCR band was obtained for intact norA in the AF13 control strain and an 8 kb product for the positive ΔnorA transformants (Fig. 2b). The latter product is consistent with the size expected with the 7 kb niaD selection marker inserted into the norA gene.

Only acetone extracts of the norA knockout cultures and cultures transformed with the selection marker were examined by liquid chromatography combined with mass spectrometry (LC/MS; Fig. 3 and Table 1). A metabolite eluted after AFB1 (14.1 min compared with 13.7 min) and exhibited a blue-shifted (λmax=332 nm) chromophore compared with that of AFB1max=362 nm). This less polar compound was identified as deoxyAFB1 by its positive ion mass spectrum (M+H=297; deoxyAFB1, M=296 Da) and having a retention time and UV-visible chromophore identical to that of deoxyAFB1 prepared by established synthetic methods (Hsia & Chu, 1977). The LC data showed that deoxyAFB1 accumulated in at least 20-fold greater amounts in the norA knockout strain than in the selection marker-only transformed strain (Fig. 3).

3

LC profile of the Aspergillus flavus AF13ΔnorA extract and the AF13 control extract obtained during the LC/MS analysis.

View this table:
1

LC/MS data from extracts of AF13ΔnorA and control cultures

Comparison of other metabolites in the acetone extracts of an AF13ΔnorA clone (#15) and the AF13 control with natural or synthetic standards by UV-visible spectrophotometry and positive ion LC/MS confirmed the presence of OMST (15.9 min), HOMST (12.4 min, M+H=355, M=354), and AFB1 (13.8 min) (Table 1). The metabolites shared identical LC retention times, UV-visible chromophores, and mass spectra with their respective standard. Several unknown compounds were also observed in extracts of fungi with both mutant and intact norA. One exhibited a chromophore (λmax=318 nm, shoulder at 360 nm; M+H=371, M=370) similar to those of OMST and HOMST, suggesting that it could be a related intermediate in the pathway. Two unknown compounds eluting at 10.9 and 13.0 min with the same mass (M+H=329, M=328) were found in extracts from control and norA mutant fungi. One of them eluted at 10.9 and 13.0 min, and exhibited a chromophore similar to that of AFB1max=360 nm). This metabolite was found mainly in the AF13ΔnorA extract. Another compound with M+H=371, identified only in the AF13ΔnorA extract, eluted at 15.6 min. Taken together, the observed alteration in the metabolic flux between the control and knockout transformants suggests the presence of other minor natural products and intermediates in the biosynthetic pathway to AFB1.

An ion with the expected mass, elution time, and chromophore for AFOH (314 Da, 10.3 min) was detected in extracts of a 2-day A. flavus norA knockout culture, but not in the control culture extract. AFOH, after feeding to a strain of A. parasiticus with defective ordA, but intact norA, was readily oxidized to AFB1 (Fig. 4, lane 3); deoxyAFB1 was not detected. Similarly, AFOH was oxidized to AFB1 by yeast cells whether or not they expressed norA or ordA (Fig. 4, lanes 7–9).

4

Feeding of AFOH to Aspergillus parasiticus SRRC2043 or Saccharomyces cerevisiae: Lane 1, A. parasiticus SRRC2043 (accumulates OMST only) extract; lanes 2 and 6, AFB1 standard; lane 3, A. parasiticus SRRC2043 incubated with partially purified AFOH (AFOH is converted to AFB1); lanes 4 and 10, partially purified synthetic AFOH; lane 5, OMST standard; lanes 7–9, AFOH fed to S. cerevisiae expressing norA, ordA, and nontransformed control, respectively. The dotted line indicates the TLC origin. Fluorescent materials at the top of the TLC plates are not shown.

Discussion

Orthologs of the aryl alcohol dehydrogenase-encoding gene norA are found in the gene clusters of all aflatoxin- and sterigmatocystin-producing Aspergillus species (Ehrlich et al., 2005). The role of NorA in aflatoxin biosynthesis has not yet been defined. In previous studies, mutants of norA in A. parasiticus failed to show a detectable phenotype (J.W. Cary and K.C. Ehrlich; P.-K. Chang and K.C. Ehrlich, unpublished data). Our results show that A. flavus lacking norA accumulate deoxyAFB1. This is the first time that deoxyAFB1 has been shown to be a natural metabolite of aflatoxin-producing Aspergillus cultures. DeoxyAFB1 most likely results from dehydration of aflatoxicol (AFOH) as had been demonstrated previously in synthetic studies and confirmed here (Lau & Chu, 1983). AFOH is a natural enzymatic reduction product of AFB1. Therefore, we suggest that A. flavus norA mutants lacking the aryl alcohol dehydrogenase accumulate an increased amount of the presumed NorA substrate AFOH, compared with cultures with intact norA, and that AFOH undergoes acid-catalyzed dehydration in the acidic growth medium to yield deoxyAFB1 (Fig. 5).

5

Scheme showing possible intermediates in the conversion of the putative 370 Da lactone intermediate to AFB1 and deoxyAFB1. Molecular weights are shown below the compound.

The presence of AFB1 in AF13ΔnorA mutant extracts indicates that only a portion of AFB1 is reduced to AFOH in the absence of NorA, suggesting an oxidative role for NorA that minimizes accumulation of AFOH. This provides an insight into the previously reported phenomenon that aflatoxin producers and nonproducers are capable of interconverting AFB1 and AFOH (Nakazato et al., 1990). The counterpart reductive enzymes involved in this oxidation-state balance as well as the underlying ecological rationale for the activity remain undefined. A blastp search of the translated A. flavus genomic DNA database with the A. flavus NorA sequence revealed the presence of six genes predicted to encode proteins (AFLA_134080, E=0; AFLA_077060, E=0; AFLA_124600, E=−175; AFLA_096620, E=−107; AFLA_027250, E=−42; AFLA_093600, NorB, E=−44) with a high degree of homology (E value<−40). It is possible that these homologs could complement the function of NorA to some extent, even in the absence of NorB.

The LC/MS results show that the presumptive first OrdA oxidation product HOMST is present in the extracts of both AF13ΔnorA mutants and the AF13 control strain. HOMST was implicated as a potential intermediate in synthetic feeding studies with either A. parasiticus cultures or with yeast expressing ordA (Udwary et al., 2002), and this intermediate was confirmed here in our product analysis. Our results indicate that NorA is involved in a catalytic step after OrdA oxidation and are consistent with the route proposed in Fig. 5, where OrdA is predicted to catalyze oxidation of HOMST to a putative 370 Da lactone.

The subsequent rearrangement steps of the presumptive 370 Da lactone are less clear. Ultimately, these are likely to result in the formation of the 326 Da methyl enolether shown in Fig. 5, which is likely to be the immediate AFB1 precursor. Recent results suggest that the aflatoxin biosynthesis gene, hypE, encodes a protein with an EthD domain that may be involved in the oxidative demethylation of this methyl enolether (Holmes, 2008). Proteins with an EthD domain, previously only reported in bacteria, are required for oxidative ethyl-tert-butyl ether degradation in the presence of a cytochrome P450 monooxygenase (Chauvaux et al., 2001). Disruption of hypE in A. flavus led to accumulation of a compound with the intense blue fluorescence characteristic of deoxyAFB1 and aflatoxins, but that migrated faster than AFB1 on TLC. This new metabolite exhibited a mass of 328 Da, which is consistent with the methyl ether shown in Fig. 5. Oxidation of the methyl ether in either the 326 or the 328 Da intermediates may occur with HypE and an unknown cytochrome P450 enzyme [possibly OrdA or CypX (AflV)] to cause loss of the methyl as formaldehyde and directly yield AFB1 or AFOH, respectively. AFOH resulting from demethylation of the 328 Da ether would require NorA-catalyzed oxidation to AFB1.

In the absence of NorA, the 326 Da methyl enolether or AFB1 may be partially reduced to the 328 Da methyl ether in the reductive metabolic environment of the cell as shown in Fig. 5. As suggested previously, the formation of increased quantities of deoxyAFB1 rather than AFOH in the absence of NorA could be a consequence of the precursor metabolites being produced and isolated under acidic culture conditions. In our studies, synthetic AFOH was found to dehydrate readily under mild acidic conditions. In the fungal cell, the pH is likely to be significantly higher and therefore, if AFOH is formed, it is unlikely that it would be subjected to acid-catalyzed dehydration. The balance in the cellular environment between oxidation and reduction as well as the availability of active transport out of the cell of AFB1 would be expected to play critical roles in determining the levels of the individual precursors and in maintaining the oxidation state of AFB1.

Because synthetic HOMST fed to yeast expressing ordA was converted to AFB1 (5, 6), NorA is not required for the formation of AFB1 by yeast or by the norA knockout mutants (Fig. 4). Conversion of AFB1 to AFOH was found by Nakazato (1990) when AFB1 was fed to strains of fungi incapable of toxin production. NorA, therefore, may serve as a maintenance alcohol dehydrogenase to prevent derailment of AFB1 production. Our study suggests that, while conversion of OMST to AFB1 may only require a single cytochrome P450 monooxygenase, other enzymes are important to minimize derailment of AFB1 production.

Acknowledgements

We wish to thank Beverly Montalbano for early contributions to this work. The work at Southern Regional Research Center was supported by CRIS 6435-41420-004-P and at Johns Hopkins by US National Institutes of Health grant ES001670 awarded to C.A.T. J.M.C. is currently a Damon Runyon Cancer Research Foundation Fellow (DRG-2002-09) in the Department of Biological Chemistry & Molecular Pharmacology, Harvard Medical School.

Authors' contribution

K.C.E. and P.-K.C. contributed equally to this work.

Footnotes

  • Editor: Richard Staples

References

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