Fungal Genetics and Biology 45 (2008) 1081–1093

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Fungal Genetics and Biology

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Involvement of the nadA gene in formation of G-group aflatoxins in Aspergillus

parasiticus
Jingjing Cai a,1,4, Hongmei Zeng a,2,4, Yoko Shima a,d,4, Hidemi Hatabayashi a, Hiroyuki Nakagawa b,
Yasuhiro Ito a, Yoshikazu Adachi d, Hiromitsu Nakajima c, Kimiko Yabe a,*,3
a
Food Biotechnology Division, National Food Research Institute, Tsukuba, Ibaraki 305-8642, Japan
b
Food Safety Division, National Food Research Institute, Tsukuba, Ibaraki 305-8642, Japan
c
Faculty of Agriculture, Tottori University, Tottori 680-8553, Japan
d
Laboratory of Animal Health, School of Agriculture, Ibaraki University, Ibaraki 300-0393, Japan

a r t i c l e i n f o a b s t r a c t

Article history: The nadA gene is present at the end of the aflatoxin gene cluster in the genome of Aspergillus parasiticus as
Received 24 November 2007 well as in Aspergillus flavus. RT-PCR analyses showed that the nadA gene was expressed in an aflatoxin-
Accepted 10 March 2008 inducible YES medium, but not in an aflatoxin-non-inducible YEP medium. The nadA gene was not
Available online 16 March 2008
expressed in the aflR gene-deletion mutant, irrespective of the culture medium used. To clarify the nadA
gene’s function, we disrupted the gene in aflatoxigenic A. parasiticus. The four nadA-deletion mutants that
Keywords: were isolated commonly accumulated a novel yellow-fluorescent pigment (named NADA) in mycelia as
Aflatoxin biosynthesis
well as in culture medium. When the mutants and the wild-type strain were cultured for 3 days in YES
Aspergillus parasiticus
nadA gene
medium, the mutants each produced about 50% of the amounts of G-group aflatoxins that the wild-type
Aflatoxin G1 strain produced. In contrast, the amounts of B-group aflatoxins did not significantly differ between the
NADA mutants and the wild-type strain. The NADA pigment was so unstable that it could non-enzymatically
change to aflatoxin G1 (AFG1). LC–MS measurement showed that the molecular mass of NADA was
360, which is 32 higher than that of AFG1. We previously reported that at least one cytosol enzyme,
together with two other microsome enzymes, is necessary for the formation of AFG1 from O-methylste-
rigmatocystin (OMST) in the cell-free system of A. parasiticus. The present study confirmed that the cyto-
sol fraction of the wild-type A. parasiticus strain significantly enhanced the AFG1 formation from OMST,
whereas the cytosol fraction of the nadA-deletion mutant did not show the same activity. Furthermore,
the cytosol fraction of the wild-type strain showed the enzyme activity catalyzing the reaction from
NADA to AFG1, which required NADPH or NADH, indicating that NADA is a precursor of AFG1; in contrast,
the cytosol fraction of the nadA-deletion mutant did not show the same enzyme activity. These results
demonstrated that the NadA protein is the cytosol enzyme required for G-aflatoxin biosynthesis from
OMST, and that it catalyzes the reaction from NADA to AFG1, the last step in G-aflatoxin biosynthesis.
Ó 2008 Elsevier Inc. All rights reserved.

1. Introduction and Aspergillus ochraceoroseus have also been reported to produce

Aflatoxins are highly toxic and carcinogenic secondary metabo-
lites produced primarily by certain strains of Aspergillus flavus and
Aspergillus parasiticus (Bhatnagar et al., 1994; Payne and Brown,
1998; Bennett and Klich, 2003). Recently, some other strains of
Aspergillus nomius, Aspergillus pseudotamarii, Aspergillus bombycic,
* Corresponding author. Fax: +81 298 38 8122.
E-mail address: yabek@affrc.go.jp (K. Yabe).
1
Present address: Biotechnology Research Institute, Chinese Academy of Agricul-
tural Sciences, Beijing 100081, China.
2
Present address: Institute of Plant Protection, Chinese Academy of Agricultural
Sciences. Beijing 100094, China.
3
Present address: National Agriculture and Food Research Organization, Tsukuba,
Ibaraki 305-8517, Japan.
4
Equal contribution.
aflatoxins (Payne and Brown, 1998). The contamination of food
and feed crops such as wheat, corn, cotton, peanuts, and tree nuts
with aflatoxins is not only a very serious health hazard to both ani-
mals and humans but also causes economic problems all over the
world (Eaton and Groopman, 1994; Massey et al., 1995). Despite
this serious situation, there are currently no control approaches
to prevent aflatoxin contamination (Jelinek et al., 1989). To obtain
information useful for developing effective methods of preventing
aflatoxin contamination, the biosynthetic pathways of aflatoxin
have been extensively studied. Recently, the majority of the en-
zyme reactions in aflatoxin biosynthesis have been clarified (re-
viewed in Minto and Townsend, 1997; Yu et al., 2002; Yabe and
Nakajima, 2004).
The genes involved in aflatoxin biosynthesis have also been
studied. Most of the genes constitute a large gene cluster

1087-1845/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.fgb.2008.03.003
1082 J. Cai et al. / Fungal Genetics and Biology 45 (2008) 1081–1093

encompassing 70 kb in the fungal genome (Fig. 1A), and their
expression is positively regulated by the product of the regulatory
gene, aflR (Payne et al., 1993; Woloshuk and Prieto, 1998; Yu et al.,
2004a, 2004b; Price et al., 2006). The chromosomal location of the
genes in the cluster is also important in the regulation of gene
expression (Chiou et al., 2002). Although the cluster ends were first
suggested to be norB and hypA, recently one terminus of the hypA
gene was extended to the next gene, nadA, based on a microarray
study (Price et al., 2006). Four other genes, hxtA, glcA, sugR, and
orf, are adjacent to the nadA gene, and the first three of those genes
were suggested to constitute a sugar utilization gene cluster
(Fig. 1A) (Yu et al., 2000). No similar sugar gene cluster has been
reported in any other related Aspergillus species such as A. flavus
or A. oryzae (Yu et al., 2000). The function of the sugar gene cluster
has not been clarified.
More than 20 species, including A. nidulans, produce sterigmat-
ocystin, one of the latter intermediates in aflatoxin biosynthesis, as
their final product (Brown et al., 1996; Keller and Hohn, 1997). The
enzyme pathway for sterigmatocystin biosynthesis in A. nidulans is
the same as that for aflatoxin biosynthesis, and the genes in the
sterigmatocystin biosynthesis in A. nidulans are mostly found in
the aflatoxin gene cluster in the genomes of the aflatoxigenic fungi.
However, several genes in the aflatoxin gene cluster are not pres-
ent in the sterigmatocystin gene cluster and are thought to be
exclusively involved in aflatoxin formation reactions after steri-
matocystin formation (Yu et al., 2004a, 2004b). Among these genes
is nadA, whose deduced amino acid sequence showed significant
identity (30–40%) with other NADH oxidases, though the function
of this gene has not been clarified (Yu et al., 2000).
Among the naturally occurring aflatoxins, the four major ones
are afltoxin B1 (AFB1), AFB2, AFG1, and AFG2. The 1-group aflatoxins
(AFB1 and AFG1), each of which contains a dihydrobisfuran ring in
those molecules, are produced from O-methylsterigmatocystin
(OMST). The 2-group aflatoxins (AFB2 and AFG2), each containing
a tetrahydrobisfuran ring, are produced from dihydro-O-methyl-
sterigmatocystin (DHOMST) in the latter part of the biosynthetic
pathway (Yabe et al., 1988a). The branching step for these two
groups is the desaturation step from versicolorin B to versicolorin
A in aflatoxin biosynthesis (Yabe et al., 1991a). In biosynthesis of B-
group aflatoxins, either the reaction from OMST to AFB1 or that
from DHOMST to AFB2 is commonly catalyzed by a P450 cyto-
chrome monooxygenase enzyme, which is encoded by the ordA
gene in the aflatoxin gene cluster (Yu et al., 1998; Yabe et al.,
1999). The recombinant OrdA enzyme expressed in yeast showed
the conversion of OMST to AFB1 or that of DHOMST to AFB2, indi-
cating that OrdA may be the sole enzyme required for these path-
ways (Yu et al., 1998). The OrdA is thought to be a multi-functional
enzyme because either pathway from OMST to AFB1 and DHOMST
to AFB2 is composed of several types of complicated reactions, such
as oxidation, decarboxylation, and dehydration. Recently, 10-hy-
droxy-OMST (HOMST) was suggested to function as an intermedi-
ate between OMST and AFB1 (Udwary et al., 2002).
G-group aflatoxins, AFG1 and AFG2, are also produced from
OMST and DHOMST, respectively (Yabe et al., 1988a, 1999). We
previously reported that at least three enzymes, that are two mem-
brane enzymes and one cytosol enzyme, are required for G-group
aflatoxins production from these precursors (Yabe et al., 1999).
The activity of the OrdA monooxygenase enzyme was found in
the microsome fraction. Another monooxygenase gene, cypA, was
recently reported to be involved in the formation of G-group afla-
toxins by Ehrich et al. (Ehrlich et al., 2004), and the CypA enzyme
has some transmembrane regions on its deduced amino acid se-
quence, suggesting that it is a membrane enzyme. Therefore, the
OrdA and CypA enzymes likely correspond to the two membrane
enzymes required for G-group aflatoxins. In contrast, the gene
encoding the cytosol (soluble) enzyme has not been determined.
We recently found that the hypA gene, which is a presumptive
end of the aflatoxin gene cluster, was involved in the step from ver-
sicolorin A to demethylsterigmatocystin in aflatoxin biosynthesis,
and Ehrlich et al. independently reported the same result recently
(Ehrlich et al., 2005). The nadA gene exists next to and outside of
the hypA gene, and shares the promoter region in the 866 bp inter-
genic region with the hypA gene. A putative AflR-binding site was
recently reported in this region (Price et al., 2006). Therefore, we

Fig. 1. Genomic gene structures and expressions of the nadA gene. (A) The aflatoxin biosynthesis genes and assumptive sugar utilization genes are schematically shown. The
nadA gene was herein confirmed to be involved in the aflatoxin gene cluster. (B) Total RNA was prepared from the mycelia of the wild-type strain A. parasiticus SYS-4, which
has been cultured in YEP (aflatoxin-non-conductive) (b) or YES (aflatoxin-conductive) (c and d) medium for 48 h (b and c) or 72 h (d). Expression of the nadA (1), omtA (2) or
pkaC (3) gene was analyzed using the resulting RNA sample and each primer set of nadA-F/R, omtA-F/R, and pkaC-F/R (Table 1) in RT-PCR. PCR using the genomic DNA as the
template was also performed (a). (C) The RNA was prepared from the mycelia of the A. parasiticus SYS-4 (2, 5, and 8) or the aflR-gene deletion mutant (3, 6, and 9), which was
cultured in YES medium for 72 h. Expression of the nadA (2 and 3), omtA (5 and 6), or cmd (8 and 9) gene was analyzed using the RNA and each primer set of nadA-F-2/R-2,
omtA-F-2/R-2, and cmd-F/R (Table 1) in RT-PCR. The same PCR except using the A. parasiticus SYS-4 genomic DNA was also performed (1, 4, and 7).
 J. Cai et al. / Fungal Genetics and Biology 45 (2008) 1081–1093
supposed that the nadA gene is involved in the aflatoxin gene clus-
ter. In this work, RT-PCR revealed that the nadA gene expression
depended on the culture condition as well as on the presence of
the aflR gene, a regulatory gene that positively controls expressions
of the aflatoxin biosynthesis enzyme genes. We also disrupted the
nadA gene of A. parasiticus and then characterized the resulting dis-
ruptant to clarify the gene’s function. Finally, we confirmed that
the nadA gene encodes the cytosol enzyme required for the last
step in biosynthesis of G-group aflatoxins. A novel intermediate
of AFG1 was also found in this work.
2. Materials and methods
2.1. Fungal strains
Aspergillus parasiticus SYS-4 (=NRRL2999) was a wild-type afla-
toxin-producing strain, and the nadA-deletion mutants were newly
isolated in this work. A. parasiticus NIAH-26 was a UV-irradiated
mutant of A. parasiticus SYS-4 (Yabe et al., 1988b), and induced
all of the enzymes required to convert norsolorinic acid to aflatox-
ins, although it produced no aflatoxins or pigmented precursors
(Yabe et al., 1991b). This strain was suggested to be blocked in
the fas-2 gene (Suzuki R., personal communication).
Aspergillus parasiticus aflR-deletion mutant was also isolated
from A. parasiticus SYS-4 for RT-PCR analysis of nadA gene expres-
sion. The nadA gene of SYS-4 strain was replaced with the pyrithi-
amine (PT)-resistant marker (ptrA, PT-resistant gene, a selectable
marker (Kubodera et al., 2000) (Fig. 2A). The aflR gene disruption
cassettes were prepared by double-joint PCR (Yu et al., 2004a,
2004b). The 50 -flanking region (0.9 kb) and the 30 -flanking region
(0.9 kb) of coding region were, respectively, amplified using geno-
mic DNA of A. parasiticus SYS-4 with primer pairs of P1 [50 -CCGGC
TGGTTCGTGGAAGTC-30 ] and P2 [50 -GATGCAAGAGCGGCTCATCGT
CACCCCAGAAAAGCCCCACCGCCAGAGCA-30 ] or P3 [50 -CCAATGGGA
TCCCGTAATCAATTGCCCCGTGGAGGTGAGGAAGGAATTCA-30 ] and
P4 [50 -CATCGACCTTGTGGCCGACG-30 ], in which P2 and P3 primers
carried 24 bases of homologous sequence overlapping with the
ends of the ptrA gene (ptrA sequence is underlined). The ptrA gene
(2.0 kb) was also amplified with primer pairs P5 [50 -GGG
CAATTGATTACGGGATCCCATTG-30 ] and P6 [50 -GGGGTGACGATGAG
CCGCTCT-30 ]. After second round PCR using the all fragments with-
out any primers, third round PCR was performed with nested prim-
ers P7 [50 -CTGTGCAGGCCATGTGGGTG-30 ] and P8 [50 -CCTCCACATG
AGCCTTGAGCG-30 ]. The finally obtained DNA fragment was used as
a disruption cassette to transform A. parasiticus SYS-4. After trans-
formation of A. parasiticus SYS-4 wild strain, 25 PT-resistant trans-
formants were isolated. One among them was suggested to be the
aflR-deletion mutant when PCR analyses were done using the three
sets of gene-specific primer pairs: P9 [50 -GATCCATCGCGGATAGG-
30 ] and P11 [50 -GCAAGAGCGGCTCATCGTCA-30 ], P12 [50 -TGGGA TC
CCGTAATCAATTGCCC-30 ] and P10 [50 - TCTCGTATCTCGCCCATG-30 ],
or P13 [50 -TCATTCTCGATGCAGGTAATC-30 ] and P14 [50 -ATGG
TTGACCATATCTCCCC-30 ] (Fig. 2B). The deletion of the aflR gene in
the mutant was further confirmed by Southern blot analysis, in
which total genomic DNAs of the mutant and the wild strain
SYS-4 were, respectively, digested with BglII enzyme and then
hybridized with the 0.76 kb aflR probe, which had been amplified
from genomic DNA using primer pairs P15 [50 -CAATTTGAGGGTTTA
CAGGG-30 ] and P14, and then using nested primers, P13 and P16
[50 -TGCCGATTTCTTGGCTGA-30 ]. The SYS-4 DNA gave the expected
3.0-kb band together with another 6.0-kb band, in contrast, the
mutant DNA did not showed the 3.0-kb band, but showed only
the 6.0-kb band (Fig. 2C). A. parasiticus contains a partially dupli-
cated region of the aflatoxin gene cluster together with the whole
gene cluster in the genome, and the additional aflR, that is called
aflR-2, is a non-functional gene in aflatoxin biosynthesis (Cary
1083
et al., 2002; Chang and Yu, 2002). These results demonstrated that
the resulting mutant was the aflR-deletion mutant. The aflatoxin
productivity was in fact lost in the aflR-deletion mutant when
analyzed by thin-layer-chromatography (TLC) (Fig. 2D).
2.2. Media and growth conditions
GY (glucose 2%, yeast extract 0.5%), GY agar plates (GY supple-
mented with 2% agar) and YES medium (20% sucrose, 2% yeast ex-
tract) were used as aflatoxin-inducing media. YEP medium (20%
peptone, 2% yeast extract) was a non-aflatoxin-inducing
medium.
For standard culture and TLC or HPLC analysis, tip culture meth-
od was used (Yabe et al., 1988b). Spores (about 1  104 each) of
each of the wild-type strain SYS-4 or the nadA-deletion mutants
were inoculated into 250 ll of YES medium in a tip (Yabe et al.,
1988b). After stationary surface culture at 28 °C for 3 days, culture
media and the mycelia were separated by centrifugation and then
used for further analyses. To prepare larger amount of the nadA
pigment (NADA) or other intermediate for physic-chemical analy-
ses, the nadA-deletion mutant was cultured in 100 ml YES medium
in a bottle at 28 °C for 3–6 days (Wen et al., 2005).
2.3. Metabolites
The nadA pigment (NADA) was prepared as described below.
The NADA concentration was determined by UV absorption spec-
trum using the molecular coefficient of AFG1 (Cole and Cox,
1981). OMST was prepared by the methylation of ST with methyl
iodide and sodium carbonate in acetone (Yabe et al., 1988a).
2.4. RNA preparation and RT-PCR
The total RNA was prepared from the mycelia of A. parasiticus
SYS-4 or the aflR-deletion mutant, which had been cultured in
YES or YEP medium for 48 or 72 h using TRI reagent (200 ll, Sig-
ma–Aldrich, St. Louis, MO, USA) and FastPrep FP100A (Q-Biogene,
Santa Ana, CA, USA) as previously described (Yan et al., 2004). After
treatment of the RNA with DNase I to degrade the slight amount of
remaining DNA, RT-PCR was performed using an RT-PCR kit (Rev-
erTra Dash; Toyobo, Osaka, Japan) and the primers corresponding
to the nadA, omtA, pkaC, or calmodulin (cmd gene) (Table 1) accord-
ing to the manufacturer’s instructions. PCRs with the same primers
were also done using a genomic DNA of the wild-type SYS-4 strain
as the template.
2.5. Construction of nadA gene disruption vector
To disrupt the nadA gene in A. parasiticus SYS-4, the nadA gene
disruption plasmid, pNADA-L/R, was constructed in a two-step
procedure (Fig. 3A). A 1.3-kb PCR fragment containing 50 -flanking
region of the nadA gene in the genome of A. parasiticus SYS-4
(AY371490) was amplified with KOD-plus enzyme (Toyobo) using
the primers of nadA-L-F-SalI [50 -AACGCGTCGACTCGAAGTTGCCTAG
GCC-30 (SalI)] and nadA-L-R-PstI [50 -AAACTGCAGTCCAGCTCGA
GCCATTG-30 (PstI)]. The 1.4-kb PCR fragment containing 30 -flank-
ing region of the nadA gene was also amplified with the primers
of nadA-R-F-(KpnI) [50 -TGCAGGGGTACCGATCTC-30 ] and nadA-R-R
[50 -ATCCTAGCTGCTGCGGTG-30 ]. The PstI–SalI-digested PCR frag-
ment containing 50 -nadA and the KpnI-digested 30 -nadA fragment
were cloned sequentially into the PstI–XhoI site and then the
KpnI–EcoRV site of the pSP72-ptrA (Wen et al., 2005) to give pNA-
DA-L/R. The fragment containing the 50 -flanking region, ptrA, and
30 -flanking region was used for fungal transformation following
linearization of the plasmid pNADA-L/R by digestion with AlwNI
and XcmI enzymes.
1084 J. Cai et al. / Fungal Genetics and Biology 45 (2008) 1081–1093

Fig. 2. Disruption of the aflR gene via double-crossover recombination. (A) Strategy for the disruption of the aflR gene is shown. The aflR gene disruption cassette was
transformed into wild-type strain SYS-4. A. parasiticus has two copies of aflR gene in its genome (Cary et al., 2002; Chang and Yu, 2002), and the functional aflR gene and
another non-functional aflR (aflR-2) are shown. The double-crossover recombination events resulted in the replacement of the target gene aflR with the selectable marker ptrA
gene. Long arrows, gene direction; short arrows, positions of primers used for confirmation of gene disruptions; vertical arrow, gene replacement. (B) PCR analysis using
different combinations of primers was done to confirm that the aflR gene was deleted in the aflR disruptant. The expected lengths of the PCR products are shown as a table. (C)
Southern analysis of the aflR-deletion mutant. Genomic DNA of SYS-4 (1) or aflR-deleted mutant (2) were digested with BglII and analyzed by Southern hybridization using
aflR probe. kHindIII-digested markers were used as size standards. (D) Production of aflatoxin in the aflR disruptant. Aflatoxin produced by the aflR disruptant or SYS-4 were
analyzed by TLC. Lane 1, culture medium (10 ll) of SYS-4; lane 2, culture medium of the aflR disruptant; lane 3, medium only.

2.6. Fungal transformation 2005). The PT-resistant transformants were screened on a CD-
selective medium with 0.1 mg L1 PT (Kubodera et al., 2000) and
Protoplasts were prepared from A. parasiticus SYS-4. Transfor- then transferred to a GY agar plate to detect aflatoxin production
mation of fungi was done as previously described (Wen et al., and the accumulation of precursors.
 J. Cai et al. / Fungal Genetics and Biology 45 (2008) 1081–1093 1085

Table 1 medium was extracted with chloroform, and the resulting chloro-
The oligonucleotide primers used in RT-PCR form extract was analyzed using an HPLC apparatus (LC-10A, Shi-
Primera Sequence Positionb madzu, Kyoto, Japan) equipped with a silica gel HPLC column
nadA-F 0
5 -CAATGGCTCGAGCTGGAC-3 0
70446–70463 (0.46  15 cm), the mobile phase of toluene–ethyl acetate–formic
nadA-R 50 -GACTGATAAGGGAGCCGC-30 71817–71834 acid (99%)–methanol (89:7.5:2:1.5, vol/vol/vol/vol) at a flow rate
nadA-R-2 50 -AAGTCCAATGCCGTCAAC-30 71523–71540 of 1.0 ml min1 at 35 °C. The retention times of AFB1, AFB2, AFG1,
omtA-F 50 -CCTTCCTCGCCTTTGCG-30 54776–54792 and AFG2 were compared with those of standard samples (afla-
omtA-R 50 -GGTGAGACGAAGAGCCC-30 56125–56141
omtA-F-2 50 -CCTTCCTCGCCTTTGCG-30 54776–54792
toxin B–aflatoxin G mixture; Sigma).
omtA-R-2 50 -GGTGAGACGAAGAGCCC-30 56125–56141
pkaC-F 50 -AGGTGGTCAAGATGAAGCAG -30 563–582c 2.11. Purification and characterization of the NADA pigment
pkaC-R 50 -TGCTGCTATTTCTGTGGC-30 1708–1722c
cmd-F 50 -GGTGATGGCCAGATCACCAC-30
NADA was extracted with acetone from the mycelia of the
cmd-R 50 -CCGATGGAGGTCATGACGTG-30
nadA-deletion mutant, which had been cultured in YES medium
a
F, forward primer; R, reverse primer. for 3 days, and purified with silica gel TLC using the developing
b
Positions correspond to the genome sequence of A. parasiticus (AY371490).
c
solution. The spot corresponding to the novel yellow pigment
Sequence of A. parasiticus protein kinase A catalytic subunit (the pkaC, Cai J.,
personal communication), and the numbers for pkaC gene were based on its start
above AFB1 on the TLC plate was scraped off and then extracted
site (+1). with ethyl acetate or acetone. Water was then added to the extract
up to a final concentration of about 3% because the pigment ap-
peared to be relatively stable in an aqueous condition. (1) Stability.
2.7. Confirmation of nadA deletion by PCR The ethyl acetate solution containing NADA was spotted on a TLC
plate, dried in air in the dark for various times, and then developed
The genomic DNAs of the resulting PT-resistant transformants with the same developing solution. The products were observed
were prepared from the mycelia with the rapid extraction method under 365-nm UV light. (2) TFA assay. A trifluoroacetic acid (TFA)
(Wen et al., 2005), and then PCR analyses were performed using solution (1 ll) was mixed with 10 ll of the NADA ethyl acetate
the three sets of gene-specific primer pairs: P17 (nadA-F) [50 - solution or the methanol solution containing four aflatoxins
CAATGGCTCGAGCTGGAC-30 ] and P18 (nadA-R) [50 -GACTGATAAG (AFB1, AFB2, AFG1, and AFG2) at room temperature for 30 min. After
GGAGCCGC-30 ], P19 (hypA-R2) [50 -GCTAACAGATCCTCCGTCAA drying in the air, the residues were dissolved in 10 ll methanol and
CGT-30 ] and P11 (ptrA-F), P12 (ptrA-R) and P20 (hxtA-F) [50 -TCAT spotted onto a TLC plate, then developed. The product from NADA
CCGCGGCATCGAG-30 ] (Fig. 2A). was compared with those from aflatoxins. (3) LC–MS measurement.
To determine the retention time in chromatogram, the molecular
2.8. Southern analysis mass, and the absorption spectrum of the reaction products, the
substances were analyzed with a liquid chromatography–mass
Genomic DNAs of nadA mutant and the SYS-4 were purified by spectrum (LC–MS) apparatus (LCMS-2010, Shimadzu). The LC–MS
Nucleon PhytoPure (GE Healthcare) according to the manufac- system consisted of a LC-VP separation module equipped with a
turer’s instruction. Total genomic DNA of each strain was subjected SPD-M10Avp photodiode array detector and a LC–MS 2010A single
to restriction enzyme digestion of BglII or XhoI and separated by quadrupole mass spectrometer with an atmospheric pressure pho-
agarose gel electrophoresis, followed by vacuum blotting to a pos- toionization (APPI) source probe. The probe was operated in the
itively charged nylon membrane, Hybond-N+ (GE Healthcare), positive/negative mode; the nebulizer temperature was 200 °C or
using VacuGene XL Vacuum Blotting System (GE Healthcare). The 250 °C. The moving solvents were: solution A, 0.1% acetic acid in
filters were hybridized with the nadA and ptrA probes. The 1.1-kb water; and solution B, 0.1% acetic acid in methanol. The ratio of
nadA probe was amplified from genomic DNA using primer pairs solution B was changed according to the following linear gradient
nadA-F3 [50 -CGTATCTCAGTTATGCAATGTCTC-30 ] and nadA-R3 [50 - cycle: 0 min, 5%; 2 min, 5%; 17 min, 95%; 22 min, 95%; 23 min, 5%;
GTAACAGTATACCATGAAGGC-30 ], nadA-F [50 -CAATGGCTCGAGCTG- and 33 min, 5%. An Inertsil column (150 mm  2.1 mm, 5 lm,
GAC-30 ] and nadA-R2 [50 -AAGTCCAATGCCGTCAAC-30 ] for nested Superco, USA) was used, and the flow rate was 0.2 ml min1.
PCR. The 2.0-kb ptrA probe was amplified from pPTRI plasmid
(Takara) using primer pairs ptrA-F2 [50 -GGGCAATTGATTACGG- 2.12. Preparation of microsome and cytosol fractions
GATCCCA-30 ] and ptrA-R2 [50 -TGACGATGAGCCGCTCTTGC-30 ].
Hybridization and detection were performed using AlkPhos Direct The microsome fraction was prepared from the mycelia of A.
Labelling and Detection System (GE Healthcare) according to the parasiticus NIAH-26 cultured in YES medium at 28 °C for 4 days
suppliers’ manuals. (Yabe et al., 1999). The cytosol fractions were, respectively, pre-
pared from the mycelia of the SYS-4 strain and the nadA-deletion
2.9. TIP culture and TLC analysis mutant (Yabe et al., 1999). Further purification of the cytosol pro-
teins was performed by successive gel-filtration and ion-exchange
The resulting nadA mutants or the SYS-4 were cultured in 250 ll chromatography as follows. The cytosol fraction was desalted
of YES medium in the tip culture for 3 days (Yabe et al., 1988b). The through a Sephadex G-25M gel-filtration column (PD-10; GE Phar-
resulting culture medium or mycelia extract of each fungus was macia LKB Biotechnology, Uppsalla, Sweden), which had been
analyzed by thin-layer-chromatography (TLC) using a silica gel equilibrated with Solution 1 (20 mM Tris–HCl [pH 7.5], 10% [vol/
Kieselgel 60 TLC plate (No. 5721; Merck, Rahway, NJ, USA) and vol] glycerol, 5 mM MgCl2, 0.4 mM EDTA, and 1 mM 2-mercap-
the developing solution of chloroform–ethyl acetate–formic acid toethanol). The desalted cytosol fraction (0.4 ml) was then diluted
(90%) (6:3:1, vol/vol/vol). The substances on the plate were then by adding the same volume of Solution 1 supplemented with 0.5%
observed under 365-nm UV light. [weight/vol] Tween 80, and then applied onto the wet DEAE–Seph-
acryl resin (GE Pharmacia LKB Biotechnology) in a filtration cup
2.10. HPLC analysis (UFC30 LH00, pore size 0.65 lm; or UFC3 OSV00, pore size 5 lm;
Millipore, Bedford, MA, USA), which had been equilibrated with
The aflatoxin concentration was determined by high perfor- Solution 1 supplemented with 0.5% [weight/vol] Tween 80, and
mance chromatography (HPLC) (Yabe et al., 1991a). The tip culture then mixed. After the whole set containing the mixture was kept
1086 J. Cai et al. / Fungal Genetics and Biology 45 (2008) 1081–1093

Fig. 3. Disruption of the nadA gene via double-crossover recombination. (A) Strategy for the disruption of the nadA gene is shown. The gene disruption vector pNADA-L/R was
linearized and then transformed into wild-type strain SYS-4. The double-crossover recombination events resulted in the replacement of the target gene nadA with the
selectable marker ptrA gene. Long arrows, gene direction; short arrows, positions of primers used for confirmation of gene disruptions; vertical arrow, gene replacement. The
expected lengths of the PCR products are shown as a table. (B) PCR analysis using different combinations of primers was done to confirm that the nadA gene was deleted in the
nadA disruptant. (C) Southern analysis of the nadA-deletion mutant. Genomic DNA of SYS-4 (a) or nadA-deleted mutant (b) were digested with BglII or XhoI and analyzed by
Southern hybridization using nadA gene coding region or ptrA selectable marker gene as probes. kHindIII-digested markers were used as size standards.

on ice for 5 min, it was centrifuged at 2000g using a mini-centri-
fuge machine (PMC-060, AU Techno Services, Osaka, Japan). The re-
sin in the cup was washed five times with 0.15 ml of the same
solution, and then five times with the same solution without
Tween 80, in order to remove the detergent which, if it contami-
nated the mixture, would inhibit enzyme activity in the cell-free
systems. The proteins bound to the resin was eluted applying
0.15 ml of Solution 1 supplemented with 0.5 M KCl to the resin fol-
lowed by centrifugation at 2000g. The resulting cytosol fraction
was stored at 80 °C until use.
2.13. Enzyme assays
For the enzyme reaction from OMST to aflatoxins, the pigment-
removed cytosol of the SYS-4 or the nadA-deletion mutant (final
concentration: 0.44 mg ml1) was incubated in the reaction mix-
 J. Cai et al. / Fungal Genetics and Biology 45 (2008) 1081–1093
ture (total volume: 50 ll) containing 60 lM OMST and the micro-
some fraction (1.8 mg ml1) of NIAH-26 in solution A containing
90 mM potassium phosphate [pH 7.5], 10% glycerol, and 4 mM
NADPH as described previously (Yabe et al., 1999). The volume of
the reaction mixture was 50 ll in a 0.5-ml microtube. After incuba-
tion at 24 °C for 40 min, the reaction was terminated by adding
70 ll of chloroform and mixing it with a Vortex mixer followed
by centrifugation at 10,000g for 2 min. An aliquot (usually 20 ll)
of the lower layer was analyzed by HPLC.
To detect the enzyme activity catalyzing the reaction from
NADA to AFG1, the same reaction mixture, except that it contained
110 lM NADA instead of OMST was incubated in the presence or
absence of either 4 lM NAPDH or NADH as described above. After
incubation at 24 °C for 40 min, aflatoxins produced were measured
by HPLC.
3. Results
3.1. Expression of the nadA gene
The wild-type A. parasiticus SYS-4 strain was cultured in YES
medium or in YEP medium. Expression of the nadA gene and the
aflatoxin-related gene omtA was detected when the fungus was
cultured in YES for 48 or 72 h. The RT-PCR product of 1328 bp using
the primers encompassing the whole coding region of the nadA
gene was slightly smaller than the PCR product (1389 bp) obtained
when the genomic DNA was used as the template, because the
amplified region contained a splicing site (Fig. 1B, 1, lanes a, c,
and d). In contrast, RT-PCR products were not detected when the
fungus was cultured in YEP (Fig. 1B, 1, lane b). An aflatoxin-related
gene, omtA, was also expressed in the YES medium, but not in the
YEP medium (Fig. 1B, lane 2). Expression of the pkaC gene, a consti-
tutively expressed protein kinase A catalytic unit gene, occurred in
both media (0.9-kb band, Fig. 1B, lane 3).
Furthermore, when the RNA was prepared from the aflR-dele-
tion mutant (Fig. 2) for RT-PCR, expression of the nadA and omtA
genes was not detected even in the YES medium (Fig. 1C, lanes 3
and 6). In contrast, the cmd gene, a calmodulin gene that is not re-
lated to aflatoxin production, was expressed irrespective of the
medium used (Fig. 1C, lane 9). These results indicated that regula-
tion of nadA gene expression is similarly to that of other aflatoxin-
related genes.
3.2. Disruption of the nadA gene
To investigate nadA gene function, we disrupted the nadA gene
of the SYS-4 strain by gene replacement with a ptrA selection
marker gene (Fig. 3A). Five independent transformation experi-
ments yielded 470 pyrithiamine-resistant transformants on CD
selection agar plates. PCR amplifications with several combina-
tions of primer pairs were then carried out to select the transfor-
mants lacking the nadA gene using their DNA samples as the
templates. Among 90 randomly selected transformants, 4 showed
the predicted results in the PCR analysis. When we used P17 and
P18 primers encompassing the nadA coding region, the recipient
strain SYS-4 gave a PCR product with a predicted size of 1.4 kb.
However, the mutants did not show the band (Fig. 3B, lane 1).
Also, only the mutants generated 1.45 kb (Fig. 3B, 2, lane a) and
1.5 kb (Fig. 3B, 3, lane a) PCR products when the primer pairs
P19–P11 and P12–P20 were used, respectively. We also did
Southern analysis to confirm the deletion of nadA gene in the
resulting mutants. As the expected aflatoxin gene cluster restric-
tion enzyme mapping data, the nadA gene coding region was pre-
sented on an approximately 4.1-kb BglII fragment or 6.6-kb XhoI
fragment of the wild-type SYS-4 genome. While nadA-deletion
mutants DNA gave no hybridization band (Fig. 3C, left). When
1087
using ptrA gene as a probe which is a thiazole synthase (thiA)
gene with mutation point in A. oryzae (Kubodera et al., 2000), be-
sides the band supposed to correspond to the putative homolo-
gous nmt-1 gene in A. parasiticus genome (Kubodera et al.,
2003), specific ptrA hybridization signals of 4.7 kb for BglII diges-
tion or 7.2 kb for XhoI digestion were only detected in the geno-
mic DNA of nadA-deletion mutant (Fig. 3C, right). These results
indicated that the ptrA-selectable marker was replaced with the
nadA gene in the genomes of the mutants. No transformants
showed any apparent morphological differences from the wild-
type SYS-4 strain and all of them made dark figures on the UV
pictures (Yabe et al., 1987), suggesting that disruption of the nadA
gene did not show a remarkable decrease on aflatoxin production.
3.3. A novel pigment produced by the nadA-deletion mutant
The nadA-deletion mutants and the recipient strain SYS-4 were
cultured in YES medium by the tip culture method for 3 days.
When the culture medium of the nadA-deletion mutant was ana-
lyzed by TLC, a novel substance, which showed bright yellow fluo-
rescence under 365 nm UV light, was remarkably detected
together with four kinds of aflatoxins (Fig. 4A, lanes 2–5). The same
pigment was also found in the SYS-4 strain, but in much smaller
quantities tan in the mutants (Fig. 4A, lane 6). The same pigment
was also obviously observed in the mutants’ mycelia extracts (data
not shown). The pigment, which was named NADA, was not pro-
duced when the mutants were cultured in an aflatoxin-non-induc-
ible YEP medium instead of YES (data not shown), supporting the
intermediacy of NADA in aflatoxin biosynthesis.
The amounts of aflatoxins accumulated in the culture media of
the nadA-deletion mutants after 3-day tip culture were analyzed
by HPLC (Fig. 4B). The nadA-deletion mutants did not show any
remarkable difference in production of B-group aflatoxin (AFB1
and AFB2) compared to the wild-type SYS-4 strain. In contrast, they
showed significant decrease in production of G-group aflatoxins
(AFG1 and AFG2), to about 50% of that by the wild-type strain.
The nadA gene was revealed to be involved in G-group, but not
B-group, aflatoxin formation.
3.4. NADA characterization
Interestingly, when the mutants and the wild-type strain were
cultured in YES medium in tip culture for 6 days, there was much
less NADA in the medium than in that cultured for 3 days (Fig. 5A).
Similar results were obtained when GY medium was used instead
of YES, indicating that medium was not related to the results
(Fig. 5B). Since the fungus’ aflatoxin productivity peak at around
3–4 days and then decreased in tip culture (Yabe et al., 1988b),
NADA once produced appeared to gradually change to another sub-
stance while in the medium. In contrast, aflatoxins in the medium
tended to increase for at least 6 days, since they are relatively sta-
ble substances.
We first tried to purify NADA from the mycelia. However, repe-
tition of TLC purification followed by extraction and drying caused
a spontaneous change of NADA into another compound, whose Rf
value was the same as that of AFG1 (Fig. 5C). HPLC confirmed that
the resulting substance was AFG1 (data not shown). Remarkably,
when a fresh sample of the TLC-purified NADA was injected into
the HPLC apparatus, AFG1 also appeared with the remaining NADA.
Furthermore, TFA treatment of the resulting AFG1-like substance
drastically increased the hydrophilicity of the substance, whose
Rf value was the same as that of AFG2a produced from AFG1 stan-
dard with TFA (Fig. 5D) (Cole and Cox, 1981). This demonstrated
that NADA could non-enzymatically changed to AFG1 in vitro.
LC–MS analyses revealed that NADA had a peak whose reten-
tion time (18.0 min) was different from that of AFG1 (16.7 min)
1088 J. Cai et al. / Fungal Genetics and Biology 45 (2008) 1081–1093

Fig. 4. Production of a novel intermediate, NADA, and aflatoxins in the nadA disruptants. (A) Culture medium (10 ll) of each of the nadA-deletion mutants no.21 (lane 2), no.
53 (3), no. 67 (4), no. 85 (5), the recipient SYS-4 strain (6) (indicated with D) and no fungus (7) after 3-day tip culture were analyzed by TLC. Lane 1, standard of aflatoxins B1,
B2, G1 and G2 mixture. (B) Aflatoxins in the media of the nadA-deletion mutants (2–5) and the SYS-4 (1) (indicated with D) were, respectively, analyzed by HPLC after 3-day tip
culture. Experiments were done in tetra-plicate. The total of B-group aflatoxins (AFB1 and AFB2) (left) and that of G-group aflatoxins (AFG1 and AFG2) (right) produced were
shown. Less than 5% of the total B- and G-group aflatoxins were AFB2 and AFG2, respectively. There were no significant differences among the mycelia weights of the nadA-
deletion mutants (2–5) and the SYS-4 (1) (23.4 ± 3.0 mg 250 ll1).

in LC chromatogram (Fig. 6A). NADA’s molecular mass peak was
measured using various nebulizer temperatures. NADA ionization
was as follows ([nebulizer temperature] m/z (relative intensity)):
temperature 200 °C, 361 [M+H]+ (100%), 329 (91.3%) (Fig. 6B,
upper), 359 [MH] (100%), 327 (0%) (Fig. 6B, lower); tempera-
ture 250 °C, 361 [M+H] + (20.9%), 329 (100%) (Fig. 6C, upper),
359 [MH] (100%), 327 (91.3%) (Fig. 6C, lower). These results
indicated that the NADA molecular mass was 360 and that this
substance was partially changed to AFG1 even under the mild
conditions for LC–MS measurement. The absorption spectra of
NADA and AFG1 by photodiode array (PDA) detection also dem-
onstrated that NADA was a different substance from AFG1
(Fig. 6D).
3.5. Preparation of the cytosol fraction
We first fed the NIAH-26 with purified NADA to confirm the NA-
DA’s intermediacy for aflatoxin biosynthesis. Although AFG1, but
no other aflatoxins, was apparently produced from NADA after 4
days’ culture (data not shown), the possibility of NADA spontane-
ously changing to AFG1 in the culture medium could not be
avoided. Therefore, we then tried to do cell-free experiments to de-
tect the enzyme activity relating to NADA.
However, since the SYS-4 and nadA-deletion strains endoge-
nously produced large amounts of aflatoxins together with small
amounts of their precursors, the cytosol fractions prepared from
those fungi contained aflatoxins and the precursors, which ham-
pering the sensitive detection of reaction products in the cell-free
systems. Therefore, we had to purify the cytosol proteins to remove
them before the cell-free experiments, using the following two-
step gel-filtration procedure using a PD10 column and then an-
ion-exchange chromatography using DEAE–Sepharose resin. The
addition of a non-ionic detergent, Tween 80, in the washing solu-
tion for the DEAE resin was useful for removing the hydrophobic
contaminants such as aflatoxins and their precursors, because the
non-ionic detergent effectively captured them from the proteins,
but did not bind to the ionic resin. A fraction containing soluble
proteins without any aflatoxins or precursors was obtained and
then used for cell-free assays.
3.6. Involvement of the nadA gene in the pathway from OMST to AFG1
When the microsome fraction of A. parasiticus NIAH-26 was
incubated with OMST in the presence of NADPH, a small amount
of AFG1 was produced, depending on the extent of cytosol con-
tamination in the microsome fraction (Table 2). When the puri-
 J. Cai et al. / Fungal Genetics and Biology 45 (2008) 1081–1093 1089

Fig. 5. Conversion of NADA to AFG1. The SYS-4 (lanes 2 and 3) or nadA-deletion mutant (4 and 5) was cultured for 3 days (2 and 4) or 6 days (3 and 5) in YES (A) or GY (B)
medium by the tip culture, and then the media were analyzed by TLC. Either medium without fungus was also analyzed by TLC (A1 and B1), in which a spot in YES medium
was an unknown substance different from NADA (A1). NADA is indicated with a closed triangle. (C) After analyzing the culture medium of the nadA-deletion mutant by TLC,
the spot corresponding to the NADA pigment was extracted from the TLC plate and then spotted onto the TLC plate again (lanes 2–5). Each spot was air dried for 1 h (2), 2 h
(3), 4 h (4) or overnight (5) and then developed by TLC. Lane 1, the culture medium of the nadA-deletion mutant. (D) NADA pigment and mixture of aflatoxins (AFB1, AFB2,
AFG1, and AFG2) were, respectively, treated with trifluoroacetic acid (TFA), and analyzed by TLC. Lane 1, mixture of aflatoxins: AFB1, AFB2, AFG1, and AFG2 from the top down;
2, the culture medium of the nadA-deletion mutant; 3, TLC-purified NADA pigment which was air dried on TLC plate for 1 h; 4, TFA-treated NADA, in which small amounts of
remaining AFG1 and newly formed AFG2a were detected; 5, TFA-treated aflatoxin mixture (5), in which AFB2, AFG2, AFB2a, and AFG2a were detected from the top down.

fied cytosol fraction of the SYS-4 recipient strain was further
added to the same reaction mixture, AFG1 production increased
remarkably, supporting the previous observation that the cytosol
fraction was required for AFG1 formation from OMST (Yabe et al.,
1999). The requirement of NADPH for the reaction was also con-
firmed. However, when the cytosol fraction of the nadA-deletion
mutant was used instead of that of the SYS-4 strain, the same
enhancement of AFG1 production was not detected. These results
indicated that the cytosol fraction required for the pathway from
OMST to AFG1 corresponded to the protein encoded by the nadA
gene.
Since OMST serves as the common precursor of AFB1 and AFG1
(Yabe et al., 1988a, 1999), AFB1 was also produced from OMST
depending on the presence of NADPH although the AFB1 formation
from OMST does not require the cytosol enzyme. In fact, the
amount of AFB1 produced in the presence of the cytosol of the
nadA-deletion mutant was almost same as that in the presence of
the cytosol of the wild strain (Table 2).
3.7. AFG1 formation from NADA
Since NADA seemed to be the last precursor just before AFG1,
we tried to determine the enzyme activity using NADA as the sub-
strate. When the cytosol fraction of the SYS-4 strain was incubated
with NADA in the presence of NADPH, AFG1 was significantly pro-
duced, although a small amount of AFG1 was formed from NADA in
the absence of the cytosol because of spontaneous conversion of
NADA to AFG1 (Table 3). Almost the same amount of AFG1 was pro-
duced when used NADH instead of NADPH (data not shown),
whereas a smaller amount of AFG1 was detected in the absence
of either cofactors. These results indicated that NADA is the precur-
sor of AFG1 and that the cytosol fraction of the wild-type strain
contained an enzyme that catalyzed the reaction from NADA to
AFG1. In the same experiment, a small amount of AFG2 was also
produced in the presence of NADPH, likely due to contamination
of a dihydro-NADA in the NADA sample prepared through TLC.
When the cytosol fraction of the nadA-deletion mutant instead
of the wild-type strain was incubated with NADA, AFG1 formation
was not detected, irrespective of the presence of NADPH (Table 3).
These results indicated that the NadA protein encoded by the nadA
gene is the enzyme involved in the reaction from NADA to AFG1,
which is the last step in G-aflatoxin biosynthesis.
4. Discussion
4.1. The nadA gene in aflatoxin biosynthesis
This work demonstrated that the nadA gene is involved in afla-
toxin biosynthesis, and that the cytosol enzyme involved in G-afla-
toxin formation from OMST corresponds to the NadA enzyme. The
nadA gene was expressed in aflatoxin inductive medium, but not in
the non-inductive medium in RT-PCR analysis. Expression of the
nadA gene depended on the presence of aflR gene, which has been
also observed in transcription profiling using DNA microarray anal-
ysis (Price et al., 2006). Deletion of the nadA gene of A. parasiticus
caused decrease of G-group aflatoxins, but not changed B-group
aflatoxins in the culture (Fig. 4) as well as in cell-free experiment
(Table 2). The nadA-deletion mutant accumulated a novel interme-
1090 J. Cai et al. / Fungal Genetics and Biology 45 (2008) 1081–1093

Table 3
Formation of AFG1 from the NADA pigment by the NadA enzyme

Cytosol source NADPH (mM) Aflatoxins (ng/50 ll reaction mixture)a,b

AFG1c AFG2c
1 — 4 — —
2 SYS-4 4 37.6 ± 4.1 0.24 ± 0.03
3 nadA 4 — —
4 SYS-4 0 11.6 ± 1.7 0.06 ± 0.01
5 nadA 0 — —
a
The pigment-removed cytosol from SYS-4 strain or nadA-deletion mutant was
incubated with NADA intermediate in the presence or absence of NADPH for
40 min. The aflatoxins produced were measured by HPLC. Values are
means ± standard deviations.
b
B-group aflatoxins (AFB1 and AFB2) were not significantly formed.
c
The amounts of AFG1 (16.5 ± 1.2 ng 50 ll 1) or AFG2 (0.19 ± 0.02 ng 50 ll 1),
which was non-enzymatically produced in the absence of the cytosol, was sub-
tracted from each value.

derivative of OMST, is the precursor of AFG2, which is the dihydro-

Fig. 6. Physicochemical properties of NADA pigment. (A) HPLC spectrum of the
NADA pigment in LC–MS analysis. Peaks of NADA (retention time, 18.0 min) and
AFG1 that was non-enzymatically produced from NADA (16.7 min) were detected.
Monitored at 360 nm. (B) Mass signals of the NADA pigment at 200 °C (upper,
positive; lower, negative). (C) Mass signals of the NADA pigment at 250 °C (upper,
positive; lower, negative). The ratio of molecular mass of NADA (360) to AFG1 (328)
decreased by increasing the nebulizer temperature. (D) Absorption spectra of either
peak of AFG1 or NADA in LC–MS analysis.
diate, NADA, which was here confirmed to be an AFG1 precursor.
Although we previously demonstrated that DHOMST, the dihydro-
derivative of AFG1 (Yabe et al., 1988a), it was supposed that a
dihydroderivative of NADA (named DHNADA) serves as a precursor
of AFG2. The reaction pathway proposed is shown in Fig. 7.
As for the pathway for B-group aflatoxins, Udwary et al. re-
ported that HOMST is produced from OMST by the OrdA enzyme,
and HOMST is further converted to AFB1 by only the OrdA enzyme
through formation of the 4-substitute 4-methoxy-3-butenoic acid
intermediate (the intermediate (1)) (Udwary et al., 2002). Interme-
diate 1 was suggested to be successively converted to intermediate
2 by decarboxylation, to intermediate 3 by dehydration, and to
AFB1 by demethylation. They also suggested an alternative scheme
in which tautomerization of the intermediate 1 would give a stabi-
lized intermediate, and the resulting intermediate is changed to
AFB1 by successive reactions of demethylation following by decar-
boxylation (Udwary et al., 2002).
In contrast, the detailed pathway for G-group aflatoxins has not
been clarified. G-group aflatoxins are produced from OMST by a
certain branching step between B-group and G-group aflatoxins
(Yabe et al., 1999; Ehrlich et al., 2004). We recently found that
G-group aflatoxins are also produced from HOMST (data not
shown), suggesting that the branching step between B- and G-
group aflatoxins is present after HOMST. A certain intermediate
at the branching point would change to NADA by the OrdA and
CypA enzymes. Ehrlich et al. showed that disruption of the cypA
gene caused a complete absence of G-group aflatoxin formation,
and that the lack of G-aflatoxin productivity in A. flavus was likely
due to the deletion of the promoter region of the cypA gene in the
genome of the same species (Ehrlich et al., 2004). They predicted
that CypA enzyme oxidizes the double bond of the intermediate
1 (Fig. 7) generated from HOMST to give an epoxide, which may
be subsequently rearranged to AFG1.

Table 2
Aflatoxin formation from OMST depending on the soluble NadA enzyme in cytosol

Cytosol Microsome Time (min) NADPH (mM) Aflatoxinsa (ng/50 ll reaction mixture)
AFG1 AFB1
1 — + 0 4 — —
2 — + 40 4 0.92 ± 0.13 21.5 ± 2.7
3 SYS-4 + 40 4 1.99 ± 0.16b 15.8 ± 1.0
4 nadA + 40 4 0.95 ± 0.16 15.3 ± 2.4
5 — + 40 0 0.01 ± 0.01 0 ± 0.0
6 SYS-4 + 40 0 0.07 ± 0.00 0.6 ± 0.0
7 nadA + 40 0 0.05 ± 0.01 0.2 ± 0.0
a
The pigment-removed cytosol from SYS-4 strain or nadA-deletion mutant was incubated with OMST in the presence of the microsome fraction of NIAH-26 and/or NADPH.
The aflatoxins produced were measured by HPLC. Values are means ± standard deviations.
b
Production of AFG1 was significantly enhanced.
 J. Cai et al. / Fungal Genetics and Biology 45 (2008) 1081–1093 1091

Fig. 7. Postulated pathway from OMST to aflatoxin G1 in aflatoxin biosynthesis. The intermediates 1, 2, or 3 can work as a possible branching point for G-aflatoxin formation.
Order of the decarboxylation, dehydration, and epoxydation can be changed depending on the branching intermediate. Epoxydation of the intermediate causes a novel
intermediate containing an epoxide (intermediate 4), and then further hydroxylation of the resulting substance makes a novel intermediate (NADA) with a molecular mass is
360. NADA shows equilibrium between open and closed forms. Demethylation of the NADA intermediate seemed to cause the formation of AFG1. NADA was proposed to be
the last intermediate in the conversion of OMST to AFG1. For biosynthesis of AFG2 from dihydro-O-methylsterigmatocystin (DHOMST), dihydroderivatives of the same
intermediates including dihydro-NADA (DHNADA) are likely involved in the same enzyme pathway. The NadA enzyme may catalyze both the reaction from NADA to AFG1
and that from DHNADA to AFG2.

We herein hypothesized a reaction scheme in which some
intermediates (structures 1–6) were involved in G-group aflatoxin
formation (Fig. 7). We hypothesized that each or all of the interme-
diates 1, 2, and 3 might work as the intermediate or intermediates
for epoxidation by the CypA enzyme to form intermediates 4, 5,
and 6, respectively. The resulting intermediate 6 might be con-
verted to intermediate 7 through oxidation, probably by the OrdA
enzyme, because our preliminary study indicated that the OrdA en-
zyme was also required for G-group formation from the branching
step for G-aflatoxin formation (data not shown). The intermediates
7 and 8 would connect to each other through tautomerism in those
structures. The NadA enzyme may catalyze the subsequent
demethylation reaction from NADA to AFG1. The LC–MS analysis
showed that the molecular mass of the NADA was 360 Da, which
was larger than AFG1 by 32 Da, suggesting deletion of a methanol.
NADA pigment isolated is proposed to be the intermediate 7, which
is 3,4,7aa,10 aa-tetrahydro-1-hydroxy-1,5-dimethoxy-1H,12H-
furo[30 ,20 :4,5]furo[2,3-h]pyrano[3,4-c][1]benzopyran-12-one. The
spontaneous conversion of NADA to AFG1 strongly suggested that
NADA is the last precursor just before AFG1. In fact, although the
wild SYS-4 strain accumulated small amount of NADA in YES med-
ium (Figs. 4 and 5), the cypA-deleted mutant, which was kindly
gifted from Dr. Kenneth C. Ehrlich (Ehrlich et al., 2004), did not
accumulate any NADA (data not shown), suggesting that the nadA
gene is involved in a step downstream of the cypA-gene step. How-
ever, the detailed mechanism of G-aflatoxin formation remains to
be studied.
The instability of NADA as well as of the hypothesized DHNADA
may be why disruption of the nadA-deletion did not completely in-
hibit G-aflatoxin production in the tip culture (Fig. 4). Since NADA
as well as probably the hypothesized DHNADA were non-enzymat-
ically changed to AFG1 and AFG2 in the culture, respectively
(Fig. 5D), the large parts of AFG1 and AFG2 detected in the mutants
likely corresponded to artificial products from these substances.
1092 J. Cai et al. / Fungal Genetics and Biology 45 (2008) 1081–1093
However, the cell-free experiments showed that the disruption of
the nadA gene almost completely inhibited the enzyme reaction
from OMST to AFG1 (Table 2) as well as that from NADA to AFG1 (Ta-
ble 3). These results suggested that the NadA enzyme plays a major
part in the reaction from NADA to AFG1 or DHNADA to AFG2 in cells.
Furthermore, The instability found in the cell-free systems does not
directly indicate that this substance is in fact unstable in cells, be-
cause we cannot reproduce the same environment in the cell-free
system as that in cells. Also, NADA transiently formed in cell may
rapidly and efficiently be converted to AFG1, indicating that accu-
mulation of relative amount of NADA would seldom occur. There-
fore, the non-enzymatic conversion of NADA to AFG1 would not
significantly contribute to the same pathway in cells.
4.2. The NadA enzyme
We previously reported that at least three enzymes are involved
in the formation of AFG1 from OMST or AFG2 from DHOMST (Yabe
et al., 1999). Two of them were membrane proteins, probably OrdA
and CypA. The nadA gene was suggested to encode a soluble en-
zyme based on the amino acid sequence deduced from its gene se-
quence using the SOSUI system (http://bp.nuap.nagoya-u.ac.jp/
sosui/) (Hirokawa et al., 1998). This work demonstrated that the
nadA gene in all probability encodes the third soluble enzyme in
the cytosol, which is involved in the last reaction in the formation
of G-aflatoxins in aflatoxin biosynthesis. We previously reported
that the native molecular mass of the soluble enzyme was about
220 kDa based on gel-filtration analysis (Yabe et al., 1999); molec-
ular mass of the NadA was calculated to be 43,720 Da according to
the deduced amino acid sequence. Therefore, this enzyme was sup-
posed to be a homo-tetramer or larger complex composed of the
NadA proteins.
Homologous research into the nadA gene revealed that the nadA
homologous genes are present in A. flavus (AY510452, AY510453,
AY510453, and AY510451) and A. oryzae (AP007159, AB196490,
AB072433, and AP007175), each of which has the homologous re-
gion of the aflatoxin gene cluster. Interestingly, A. fumigatus
(BX649605) and FvN Gibberella moniliformis EST1039750
(DR633771, DR608592, and DR655104) also have the nadA gene
homologs, indicating that the nadA gene was relatively conserved
in many microorganisms. Although the prediction of the conserved
domains showed that the nadA gene may encode a NADH-oxidase
as previously suggested (Yu et al., 2000), NADH oxidase generally
requires either NADH or NADPH. In this work, we found that NADH
and NADPH equally enhanced AFG1 formation. A detailed investi-
gation into this enzyme is now in progress in our laboratory.
Acknowledgments
We thank Dr. Kenneth C. Ehrlich for giving an A. parasiticus
cypA-deletion mutant, and Yohei Ono and Akemi Koma for techni-
cal assistance. DNA search of the GenBank database was performed
with the assistance of Computer Center of Agriculture, Forestry and
Fisheries Research, MAFF, Japan. This work was also supported by
JSPS Postdoctoral Fellowship program of Japan Society for the Pro-
motion of Science, by grant-in-aid BDP (Bio-Design Program) from
the Ministry of Agriculture, Forestry and Fisheries, Japan, and a
grant from National Agriculture and Food Research Organization
(NARO), Japan.
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