Appl Microbiol Biotechnol (2003) 61:8393

DOI 10.1007/s00253-002-1199-x

MINI-REVIEW

D. Bhatnagar K. C. Ehrlich T. E. Cleveland

Molecular genetic analysis and regulation of aflatoxin biosynthesis

Received: 26 August 2002 / Revised: 4 November 2002 / Accepted: 8 November 2002 / Published online: 28 January 2003

Springer-Verlag 2003

Abstract Aflatoxins, produced by some Aspergillus

species, are toxic and extremely carcinogenic furanocoumarins. Recent investigations of the molecular
mechanism of AFB biosynthesis showed that the genes
required for biosynthesis are in a 70 kb gene cluster. They
encode a DNA-binding protein functioning in aflatoxin
pathway gene regulation, and other enzymes such as
cytochrome P450-type monooxygenases, dehydrogenases, methyltransferases, and polyketide and fatty acid
synthases. Information gained from these studies has led
to a better understanding of aflatoxin biosynthesis by
these fungi. The characterization of genes involved in
aflatoxin formation affords the opportunity to examine the
mechanism of molecular regulation of the aflatoxin
biosynthetic pathway, particularly during the interaction
between aflatoxin-producing fungi and plants.

Introduction
Aflatoxins are a group of at least 16 structurally related
polyketide-derived furanocoumarins (Fig. 1). These
metabolites are primarily produced by some isolates of
Aspergillus flavus and A. parasiticus on agricultural
commodities, and less frequently by other Aspergillus
species including A. pseudotamarii, A.bombycis, A.
nomius, and an unnamed taxon from West Africa
(Kurtzman et al. 1987; Cotty and Cardwell 1999; Ito et
al. 2001; Peterson et al. 2001), and an isolate of A.
ochraceoroseus (Frisvad 1985; Klich et al. 2000). There
are four major aflatoxins: B1, B2, G1 and G2. Toxinproducing isolates of A. flavus, A. ochraceoroseus and A.
pseudotamarii produce only the B-type, whereas the other
species produce both B and G-type aflatoxins.

D. Bhatnagar ()) K. C. Ehrlich T. E. Cleveland

Southern Regional Research Center, ARS,
USDA, New Orleans, LA 70124, USA
e-mail: dbhatnag@srrc.ars.usda.gov
Fax: +1-504-2864419

Aflatoxins are potent carcinogens when ingested by

animals and humans (Eaton and Groopman 1994). Since
their discovery over 40 years ago, aflatoxins have been
shown to be immunosuppressive, mutagenic, teratogenic
and hepatocarcinogenic in both experimental animals and
humans (Eaton and Groopman 1994). Contamination of
foods and feeds remains a serious worldwide problem but
is not a threat in most developed countries because of
careful commodity screening (Bhatnagar et al. 2000;
McAlpin et al. 2002). Aflatoxin contamination can arise
from improper storage of commodities as well as before
harvest in corn, peanuts, cottonseed and tree nuts. In this
article we have reviewed current research on the molecular regulation of aflatoxin biosynthesis, as well as the
biological and evolutionary significance of aflatoxin
production. Previous reviews on this topic have covered
the literature up to 1998 (Minto and Townsend 1997;
Payne and Brown 1998; Woloshuk and Prieto 1998;
Bhatnagar et al. 2000; Cary et al. 2000a).

Biochemistry and molecular genetics

of aflatoxin biosynthesis
There are 21 enzymatic steps required for aflatoxin
biosynthesis and the genes for these enzymes have been
cloned. Genes (aflR and aflJ) coding for proteins shown
to be involved in transcriptional activation of most of the
structural genes are also part of the cluster (Table 1) (Cary
et al. 2000a). Restriction mapping of cosmid and lambda
phage libraries of A. flavus and A. parasiticus DNAs
showed that all the genes are clustered within a 70-kb
region of the fungal genome (Fig. 2) (Trail et al. 1995b;
Yu et al. 1995). Several other Aspergilli, e.g., A. nidulans,
make aflatoxin precursors such as sterigmatocystin (ST).
The ST biosynthetic pathway is homologous to that in A.
flavus and A. parasiticus, but the order of genes on the
chromosomes is different (Fig. 2) (Brown et al. 1996).

84

assigned to a specific gene in the cluster. Three genes

are possible candidates for individual steps: cypX, moxY,
and avfA, as indicated in Table 1. Two genes ver1
(encoding a ketoreductase; Skory et al. 1992) and verA
(encoding a cytochrome P-450 monooxygenase) are
required for the conversion of versicolorin A (VERA) to
demethylsterigmatocystin (DMST). The final step in the
formation of aflatoxins is the conversion of O-methylsterigmatocystin (OMST) or dihydro-O-methylsterigmatocystin (DHOMST) to aflatoxins B1, B2, G1 and G2,
requiring the presence of a NADPH-dependent monooxygenase, ordA (Prieto and Woloshuk 1997; Yu et al.
1998). The formation of the G toxins involves an
additional step, possibly involving the enzyme encoded
by ordB (Yu et al. 1998; Yabe et al. 1999). Another gene,
aflT, encodes an ABC transporter protein that may be
necessary for aflatoxin efflux from the cells.

Transcriptional regulation of aflatoxin biosynthesis

Fig. 1AD Chemical structures of the aflatoxins. A The Btype

aflatoxins are characterized by a cyclopentane E-ring. These
compounds fluoresce blue under long-wavelength UV light. B
The G-type aflatoxins have a xanthone ring in place of the
cyclopentane and fluoresce green. C Aflatoxins of the B2 and G2type have a saturated bisfuranyl ring. D Aflatoxins B2a and G2a
have a hydrated bisfuranyl structure

The production of aflatoxin by toxigenic Aspergilli is

affected by environmental and nutritional factors such as
temperature, pH, carbon and nitrogen source, stress
factors, lipids, and certain metal salts (Cary et al.
2000a). Some of these factors may affect expression of
genes in the aflatoxin pathway. Of the 23 genes so far
isolated that code for proteins involved in aflatoxin
biosynthesis, only one, aflR, appears to encode a
transcription factor (Ehrlich et al. 1999b). Globally acting
transcription factors that respond to nutritional and
environmental signals (Tag et al. 2000) may regulate
expression of some of these genes (Ehrlich and Cotty
2002).
Isolation of the pathway-specific regulator gene, aflR

Aflatoxin formation
The synthesis of aflatoxin occurs through a series of
highly organized oxidation-reduction reactions (Dutton
1988; Bhatnagar et al. 1992; Minto and Townsend 1997).
The currently accepted scheme is shown in Fig. 2.
Aflatoxin biosynthesis begins with conversion of malonylCoA to a condensed polyketide noranthrone by the
products of two fatty acid synthase genes (fas-1 and fas-2)
and a polyketide synthase gene (pksA) (Cary et al. 2000a).
No specific enzyme has yet been linked to the conversion
of noranthrone to norsolorinic acid (NOR), the first stable
metabolite that can be isolated, but the conversion may
involve a monooxygenase (possibly cypA) and a dehydrogenase (possibly norB) (Bhatnagar et al. 1992). The
conversion from NOR to averantin (AVN) involves a
dehydrogenase, encoded by the gene nor-1 (Chang et al.
1992; Trail et al. 1994), but can also be catalyzed by the
dehydrogenase encoded by norA (Cary and Bhatnagar
1995). Some of the catalytic steps in the conversion of
AVR to versicolorin B (VERB) have not yet been

aflR was first cloned from an A. flavus cosmid library by

showing that it could restore aflatoxin-producing ability
to a mutant blocked in all steps of aflatoxin biosynthesis
(Payne et al. 1993). A homolog was subsequently isolated
from A. parasiticus transformed with a DNA fragment
containing aflR that caused the transformants to become
orange-pigmented due to overproduction of aflatoxin
biosynthetic intermediates (Chang et al. 1993, 1995c).
Overexpression of aflR in A. flavus up-regulated aflatoxin
pathway gene transcription and aflatoxin accumulation in
a fashion similar to that found in A. parasiticus, but
colored colonies were not observed, a result suggesting
that subtle differences in biosynthesis occur in the two
fungi (Flaherty and Payne 1997). These results suggested
that an increase in the copy number of aflR somehow
altered normal regulation of aflatoxin biosynthesis. Metabolite feeding studies showed that a functional aflR
allele is required for accumulation of NOR, the first stable
intermediate in the aflatoxin biosynthetic pathway (Payne
et al. 1993). When aflR was disrupted, the fungi were
incapable of aflatoxin metabolite production or transcrip-

85
Table 1 Genes in the Aspergillus parasiticus aflatoxin gene cluster
and their inferred protein sequence homology. AFB1 Aflatoxin B1,
AFG1 aflatoxin G1, AVN averantin, AVR averufin, DMST demethylsterigmatocystin, HAVN 5'-hydroxyaverantin, HVN hydroxyversi-

conal, NOR norsolorinic acid, OMST O-methylsterigmatocystin, ST

sterigmatocystin, VERA versicolorin A, VERB versicolorin B, VHA
versiconal hemiacetal acetate, VHOH versiconal hemiacetal hydroxide

Gene

Protein homology

Possible Function

Stable
intermediate

Reference

nor1
norA
norB
cypA
aflT
pksA

Dehydrogenasea
Dehydrogenase
Dehydrogenase
P450 monooxygenase
ABC transporter
Polyketide synthase

Reduce NOR to AVNb

Convert HAVN to AVR
Convert NOR anthrone to NOR
Hydroxylate NOR-anthrone
Export aflatoxin from cell
Convert hexanoylCoA to dodecaketide

AVN
AVR
NOR
None
None
None

hexA
hexB
afIR
aflJ
adhA
estA
ver1
verA
verB
avnA
avfA
omtA
omtB
ordA

Fatty acid synthase

Fatty acid synthase
Cys6Zn2 DNAbindinga
Unknown
Alcohol dehydrogenase
Esterasea
Dehydrogenase
Monooxygenase
Desaturase
P450 monooxygenase
Dehydrogenase
O-Methyl transferasea
O-Methyl transferase
Oxidoreductase

Convert malonylCoA to hexaketide

Reduce hexaketide to hexanoylCoA
Regulate expression of genes in clusterb
AflR co-regulator
Convert HAVN to AVRb
Convert VHA to VHOHb
Convert VERA to DMSTb
Convert VHA to ST
Convert VERB to VERAb
Convert AVN to HAVNb
Convert AVR to VHAb
Convert ST to OMSTb
Convert DMST to STb
Convert OMST to AFB1

None
None
None
None
AVR
VHOH
DMST
Unknown
VERA
HAVN
VHA
OMST
ST
AFB1

vbs
cypX
moxY
ordB

Dehydratasea
P450 monooxygenase
Monooxygenase
Oxidoreductase

Convert
Convert
Convert
Convert

Zhou and Linz 1999

Cary et al. 1996
J. Yu, unpublished
J. Yu, unpublished
P.-K. Chang, unpublished
Chang et al. 1995b;
Feng and Leonard 1995
Watanabe et al. 1996
Watanabe et al. 1996
Cary et al. 2000a
Meyers et al. 1998
Chang et al. 2000b
Kusumoto and Hsieh 1996
Liang et al. 1996
Matsushima et al. 1994
Yabe et al. 1991
Yu et al. 1997
Yu et al. 2000a
Bhatnagar et al. 1988
Yu et al. 2000a
Prieto and Woloshuk 1997;
Yu et al. 1998
Silva et al. 1996
Keller et al. 2000
Keller et al. 2000
D.B., K.C.E. and T.E.C.
unpublished

VHOH to VERBb
AVR to HVNb
VERA to DMST
OMST to AFG1

VERB
HVN
HVN
AFG1

a
Protein
b

function confirmed
Function confirmed by gene knockout in either Aspergillus parasiticus, Aspergillus flavus, or Aspergillus nidulans

tion of nor-1, but otherwise grew normally. An aflR

homolog with only 31% identity to that of A. parasiticus
or A. flavus was isolated from A nidulans, a fungus that
produces ST rather than aflatoxin (Yu et al. 1996).
Induced expression of A. flavus aflR in A. nidulans, under
conditions in which ST biosynthesis is normally suppressed, resulted in activation of genes in the ST
biosynthetic pathway. These studies demonstrated that
aflR function is conserved in widely different Aspergillus
spp. Homologs of aflR have been identified from all
aflatoxin and ST-producing Aspergilli so far examined
(Chang et al. 1995a; Watson et al. 1999; Ehrlich et al.
2002b)
Characterization of AflR and its role
in aflatoxin pathway gene expression
The deduced amino acid sequence of the 444 aa protein,
AflR, contains a cysteine-rich motif, CTSCASSKVRCTKEKPACARCIERGLAC, near its N-terminus (Woloshuk et al. 1994; Chang et al. 1995c). This domain is
homologous to Cys6-Zn2 domains of other fungal and
yeast GAL4-type transcription factors involved in the
regulation of many catabolic pathways (Burger et al.
1991; Suarez et al. 1991; Kulmberg et al. 1992; Lamb et

al. 1996; Todd et al. 1998). Mutation of Cys6 to Trp,

destroyed AflR function (Ehrlich et al. 1998). Preceding
the Cys6-Zn2 domain is an arginine-rich domain, RRARK,
that is homologous to nuclear localization regions in yeast
and fungal GAL4-type proteins. As with mutation of the
zinc cluster region, mutation of the amino acids in the
nuclear localization region resulted in non-functional
AflR, presumably because the protein can no longer be
translocated to the nucleus. In some A. parasiticus
isolates, a partial duplication of the gene cluster has been
found (Chang and Yu 2002). The copy of aflR in this
cluster is predicted to encode a protein defective in its
nuclear localization domain (Cary et al. 2002). Comparison of A. flavus and A. nidulans AflRs showed that, while
overall amino acid identity is only 31%, the nuclear
localization signal domain and the Cys6-Zn2 domain are
71% identical. Much higher identities were found
between the amino acid sequences of AflRs of other
aflatoxin-producing species of Aspergillus (>96%). The
zinc cluster region and the neighboring amino acids
immediately downstream (linker region) have been shown
to be necessary for sequence-specific DNA-binding in
proteins of this type (Reece and Ptashne 1993). Nonconservative substitution of amino acids in the linker
region also resulted in defective AflR (Ehrlich et al.
1998).

86
Fig. 2AC Generally accepted
pathway for sterigmatocystin
(ST) and aflatoxin biosynthesis.
The genes and their corresponding enzymes are shown. A
Aflatoxin biosynthetic pathway
gene cluster in Aspergillus parasiticus and A. flavus. B ST
biosynthetic pathway gene
cluster in A. nidulans. The gene
names are labeled on the side of
the cluster. Arrows indicate the
direction of gene transcription.
Homologous genes in both
clusters have the same number.
A putative hexose utilization
gene cluster is shown 3' to the
aflatoxin pathway gene cluster.
C Synthesis of aflatoxin. AFB1
Aflatoxin B1, AFB2 aflatoxin
B2, AFG1 aflatoxin G1, AFG2
aflatoxin G2, AVN averantin,
AVNN averufanin, AVR averufin, DHDMST dihydrodemethylstengmatocystin,
DHOMST dihydro-O-methylsterigmatocystin, DHST dihydrosterigmatocystin, DMST
demethylsterigmatocystin,
HAVN 5'-hydroxyaverantin, Me
transferase methyltransferase,
NOR norsolorinic acid, OMST
O-methylsterigmatocystin, VAL
versiconal, VERA versicolorin
A, VERB versicolorin B, VHA
versiconal hemiacetal acetate

Several other domains appear to be highly conserved

in AflRs from aflatoxin and ST-producing Aspergilli and
may constitute a portion of the transcription activation
domain. One of the conserved domains ranged from
aa 303 to aa 333 and contained the 66% identical
aa sequence, ILXCXCAXDXYXXXLVXLIVXKVLXWYXAAA (where X = non-identical aa). This region, with
predominantly neutral amino acids, is predicted to have a
coiled rather than a helical structure and may be involved
in interaction with basal transcription activating factors,
such as the TATA-box-binding factor. An acidic amino

acid-rich domain (aa 349368) with the sequence

EERVLHHPSMVGEDCVDEED is homologous to acidic
C-terminal regions in most GAL4-type transcription
factors and is believed to be characteristic of the
activation domain (Xiao et al. 1995). The C-terminal
38 aa region (residues 408444) has runs of His,Arg, and
acidic amino acids (HHPASPFSLLGFSGLEANLRHRLRAVSSDIIDYLHRE) that again are probably important
for transcription regulation (Xie et al. 2000). Pre-termination of aflR in Aspergillus sojae at aa 384, in which the
predicted protein would be missing the C-terminal 37 aa,

87

resulted in a protein with only 15% of the ability of wildtype AflR to activate expression (Matsushima et al. 2001;
Takahashi et al. 2002). When the acidic amino acids
Glu423 and Asp439, were substituted with the basic
amino acids Lys and His, the resulting AflR-carboxyterminal GAL4-DNA binding domain fusion protein had
a 10- to 15-fold lower ability to activate expression of
GAL1::lacZ in Saccharomyces cerevisiae than did the
intact AflR C-terminal fusion protein. Deletion of the
acidic amino acids Asp365, Glu366, and Glu367 reduced
the activation potential by 33% and substitution of
Arg427, Arg429, Arg431 with Leu caused a 50% drop
in activation, whereas deletion of Glu444 had no effect. A
mutation in which Asp436 (highlighted above) was
changed to His abolished activation entirely. These
results demonstrate that the carboxy terminal region is
critical to the transcription activation ability of AflR
(Chang et al. 1999a). When the aflR region encoding
aa 221444 (AFLRC) was fused to the A. parasiticus niiA
(nitrite reductase) promoter, transformants overexpressed
aflatoxin pathway precursors in a manner similar to that
found when extra copies of intact AflR were introduced
into the fungus. Since the AFLRC lacks the AflR DNAbinding domain, the increased metabolite production
could result from titration of a putative AflR repressor
(Chang et al. 1999b), thereby relieving inhibition. A
second possibility is that the C-terminal domain of AflR
can stimulate expression with reduced efficiency even
when the protein is missing a DNA-binding domain
(Chang et al. 1999a). A third possibility relates to the
earlier observation that, in A. flavus, a 1.0 kb antisense
AflR (aflRas) transcript that overlaps the aflR promoter
region was detected. Although the role, if any, of aflRas is
still unknown, it would be expected that increased levels
of AflR transcript would titrate out the antisense transcript
and thereby relieve possible inhibition (Woloshuk et al.
1994).
Two other domains may be involved in aspects of the
fine-tuning of regulation of AflR activity under different
environmental conditions. One unique site in AflR, which
is not present in other GAL4-type transcription factors so
far isolated from yeast and fungi, is a His-rich region
(HAHRQAHTHAHAHSH, aa 103117). It is found in A.
parasiticus and A. flavus AflRs, but is reduced in size in
the AflRs of other aflatoxin- or ST-producing Aspergilli
(Ehrlich et al. 2002a). This region is predicted to be a
metal-binding domain, but its function in AflR, so close
to the zinc binuclear motif (aa 2956), is still unknown.
Another motif close to the carboxy terminus is not present
in A. parasiticus or A. flavus AflRs, but is found in AflRs
from other aflatoxin-producing Aspergilli. This sequence
is Ser-rich and has a PEST value of +11.9, and should be
functionally comparable to PEST sites in other transcription factors such as mammalian Sp1. PEST sequences
help to target a protein for degradation by proteolysis
(Rechsteiner 1988). Many regulatory proteins have PEST
sequences that allow their fast turnover to reflect the
requirements for a rapid response to external metabolic
signals. The turnover is probably mediated by a cascade

of phosphorylation that promotes ubiquitination and

ultimately proteolytic degradation. Such events may
reflect differences in the stability of aflatoxin production
by different types of aflatoxin- and ST-producing fungi.
Phosphorylation may play a more direct role in AflR
activity, just as has been found for other Gal4-type
transcription factors (Parthun and Jaehning 1992). Phosphorylation of key sites might prevent binding to either a
repressor protein or to the putative co-activator, AflJ (see
below). AflR expressed in yeast has been shown to have
at least five phosphorylated sites based on the observation
that five differently migrating species detected on SDSpolyacrylamide gels collapsed to a single band after
phosphatase treatment (K.C. Ehrlich and J.W. Cary,
unpublished results).
DNA-binding by AflR
AflR from both A. nidulans and A. parasiticus binds to the
palindromic sequence 5'-TCGN5CGA-3' (Fernandes et al.
1998; Ehrlich et al. 1999b). When possible sites containing this motif at 168, and 81 were mutated in the A.
nidulans stcU promoter, reporter expression was reduced
6-fold, but when only one of these sites was mutated,
expression was essentially unchanged. A putative farupstream site at 762 appears to play little or no role in
expression. Based on mobility shift assays (EMSA), A.
parasiticus AflR bound to the 5'-TCGN5CGA-3' motif in
the promoter regions of 11 of the aflatoxin biosynthesis
pathway genes known in A. parasiticus at the time of the
study. Some of these genes, like stcU of A. nidulans, have
more than one AflR binding site. Unlike stcU, when either
of the two AflR sites in the pksA promoter were
individually mutated, expression of the reporter construct
was reduced more than 100-fold indicating that, in this
case, both AflR binding sites are critical to gene
expression. In the promoter of the gene encoding the
P450 monooxygenase that oxidizes averantin to hydroxyaverantin, avnA, of the two possible AflR-binding sites,
only mutation of the site closest to the transcription start
site affected expression (Cary et al. 2000b). In this case
the binding site is 5'-TCGN5CGG-3'. Footprinting analysis showed that AflR protects a region 45 bp upstream
of the recognition motif, and that the preferred binding is
to sequences with 5'-TCGG/CNNNC/GCGR-3'. The
amino acids most likely to make contact with the DNA
in the zinc-binding domain of A. nidulans AflR, (aa 32
37, SRSKVK) are functionally identical to those in the
zinc finger domain of A. parasiticus AflR (aa 3338,
ASSKVR). In addition, the linker domains of the two
types of AflR possess the same number and distribution of
Lys and Arg residues, the residues critical for sequencespecific interaction. Therefore, it is not surprising that the
two proteins recognize the same sequence even though
their homology in this region is only 70%. By analogy to
most Gal4-type proteins that bind to a partially palindromic site, AflR probably binds to its recognition site as
a dimer. However, no easily recognizable leucine repeat

88

region is apparent in the sequence beyond the linker

region, although the His-rich domain, which would be
expected to be a-helical, might be involved in dimerization as well as some other, poorly understood, function
within this protein.

AflR coactivator but further work is necessary to prove

this assumption.
Environmental and nutritional effects on aflatoxin
production and the role of signaling pathways on aflatoxin
pathway gene expression

Characterization of the aflR promoter

An intergenic region of 758 bp is located between the
bidirectionally transcribed aflR and aflJ genes. To analyze
promoter function of the aflR gene, the entire 758-bp
intergenic region as well as truncated forms of this region
were used to drive expression of the Escherichia coli bglucuronidase (GUS)-encoding gene uidA (Ehrlich et al.
1999a). Removal of sequences in the promoter from 758
to 280 had no apparent effect on promoter activity, but
further truncation to 118 enhanced gene expression
nearly 5-fold. Therefore, there appears to be a negative
regulatory element in the region from 280 to 118.
Further removal of bases 118 to 100 almost entirely
eliminated GUS gene expression. When the region from
118 to 107 was deleted, there two-thirds of this activity
was lost. Therefore, sequences in the 18-bp region from
100 to 118 appear to be critical for aflR promoter
activity. This region overlaps a 10-bp palindrome (120
to 111) and a purine-rich region. EMSA using nuclear
extracts from A. parasiticus and oligonucleotide ligands
covering the region from 81/173 revealed the presence
of a putative PACC-binding site (5'-GCCARG-3') (Espeso
and Arst 2000). Binding to the aflR PacC site is consistent
with the function of this protein in repressing transcription
of acid-expressed genes under alkaline conditions (Tilburn et al. 1995). Aflatoxin biosynthesis in A. flavus
occurs in acidic media, but is inhibited in alkaline media
(Cotty 1988). It is possible that PacC binding to the 148/
173 site has a negative effect on aflR expression.
The gene divergently transcribed from the aflR
promoter, aflJ, and the protein it encodes, AflJ, may
affect AflR activity (Meyers et al. 1998; Chang et al.
2000a; Ehrlich and Cotty 2002). AflJ has no known
sequence homologies with proteins identified in the yeast
or fungal databases. Meyers et al. (1998) found that
disruption of aflJ in A. flavus resulted in failure to
produce any aflatoxin pathway metabolites even though
transcripts for many of the aflatoxin pathway genes were
still made. This result suggests that AflJ does not affect
AflR activity. Previously, Chang et al. (1995c) had found
that aflR expression was enhanced in A. parasiticus
transformants with aflR in which the aflJ region was
present, compared to transformants in which this region
was missing. In a different study Chang et al. (2000a)
found that the nitrogen regulatory protein, AreA, bound to
GATA sites in the aflR-aflJ intergenic region, suggesting
that nitrogen regulation of aflatoxin production could be
linked to AreA control of aflR and aflJ expression. Using
a yeast two-hybrid system, Chang et al. (1999a) were able
to show that AflJ binds to the carboxy-terminal region of
AflR. From the above results it is possible that AflJ is an

Since sites in the aflR-aflJ intergenic region are recognized by transcription factors that are themselves regulated by environmental signals [pH regulates the activity
of PacC (Tilburn et al. 1995) and nitrate regulates AreA],
it is probable that, at least for nitrate, the effects on
aflatoxin pathway gene transcription may, in part, be
directly caused by changes in the expression of aflR or
aflJ resulting from activation by these factors. To support
this observation we found that certain strains of aflatoxinproducing Aspergilli respond differently to nitrate than do
other strains, and that the differences could be correlated
with differences in the number of possible GATA sites
(ranging from five to nine) near the aflJ start site (Ehrlich
et al. 2002b). Other genes in the aflatoxin biosynthetic
cluster have AreA and PacC binding sites at key positions
in their promoters that may affect their expression. For
example, the1.7 kb intergenic region separating the nor-1
and pksA genes has two adjacent PacC sites nearly in the
middle that, from site-directed mutagenesis studies, affect
expression of pksA, which encodes the pathway-specific
polyketide synthase necessary for the first steps in
formation of the polyketide backbone (Ehrlich et al.
2002a). In A. nidulans, the promoter region of the gene
stcU, which is necessary for conversion of VERA to
DMST, contains a PacC-binding site immediately upstream of its AflR-binding site and is probably involved in
expression of this gene.
The mechanism of nitrate suppression is not clear. The
effects of nitrate on aflatoxin formation indicate that
regulation of aflatoxin biosynthesis may be part of the
nitrogen control circuit. Nitrate assimilation in fungi is a
tightly regulated process. The expression of nitrate
reductase and nitrite reductase genes requires both the
lifting of nitrogen metabolite repression and specific
induction by nitrate (Marzluf 1997). Expression of genes
involved in nitrate utilization is transcriptionally activated
by the global positive-acting regulatory factor, AreA.
Marzluf (1997) suggested that nitrate increases the
cytoplasmic NADPH/NADP ratio, which favors biosynthetic reductive reactions, and thus promotes utilization of
malonyl coenzyme A and NADPH for fatty acid synthesis
rather than for polyketide synthesis. Kachholz and
Demain (1983) suggested that nitrate represses formation
of active enzymes involved in the synthesis of alternariol
monomethyl ether in A. alternara. The transcription of
aflatoxin pathway genes is higher in nitrate medium in
transformants containing an additional copy of aflR than
in the untransformed fungus (Chang et al. 1995c; Flaherty
and Payne 1997). This might be due to increased aflR
copy number, which elevates the basal levels of AFLR in
the transformants. More AFLR would then be available to

89

bind to promoter sites, thereby increasing expression of

other aflatoxin pathway genes. Activation of transcription
could be modulated by the highly acidic domain of
AFLR. Preliminary data have shown that AFLR1 (a
recombinant version of AFLR, but containing an intact
zinc finger) also binds to sites in the promoter regions of
several aflatoxin biosynthetic genes and may thereby
activate their transcription (Ehrlich et al. 1999b).
The role of carbon utilization in the regulation of
expression of genes involved in aflatoxin biosynthesis is
not, as yet, well understood. Unlike the biosynthesis of
many other secondary metabolites, aflatoxin gene expression is induced by the presence of simple carbohydrates,
for example glucose, sucrose, or maltose, but not by
peptone, sorbose, or lactose (Payne and Brown 1998). It
should be noted that all of the aflatoxin pathway genes so
far studied lack CreA sites in their promoters and,
therefore, would not be expected to be subject to carbon
catabolite repression mediated by the transcription factor
CreA. However, an interesting possible role for CreA in
aflR expression could be control of expression of the
antisense aflR mRNA transcript, since two tandem CreAbinding sites, GCGGGGaGTGGGG, are present at the
start of this reported transcript. If carbon catabolite
repression prevents the expression of this transcript, no
decrease in the amount of AflR protein could occur.
Another transcription factor that responds to simple
sugars is Rgt1, a positively acting factor shown to be
necessary for regulation of glucose transporter molecule
expression (Ozcan et al. 1996). In S. cerevisiae, Rgt1
functions as a transcriptional repressor in the absence of
glucose, but in the presence of high concentrations of
glucose it functions as a transcriptional activator. A
possible Rgt1 site is present in the promoter region of A.
parasiticus aflJ (which, as discussed above, may encode
an AflR cofactor) and may be involved in regulation of its
expression.
Another indirect effect of glucose utilization on
aflatoxin pathway gene expression could be the activation
of a four gene sugar cluster downstream of the aflatoxin
gene cluster (Fig. 2) (Yu et al. 2000b). Activation of
genes in this sugar cluster by an external hexose signal
may create a region of active chromatin that includes the
neighboring aflatoxin gene cluster (Muro-Pasteur et al.
1999). To support this observation, we and others found
that when individual aflatoxin biosynthetic genes insert at
sites other than the aflatoxin gene cluster following fungal
transformation, the expression of these genes is much
lower (500-fold) than it is when the genes insert into the
aflatoxin cluster (Liang et al. 1997). Also, expression of
aflR in a partial duplicate copy of the aflatoxin pathway
cluster in some A. parasiticus isolates was not detected
even though its promoter region was essentially intact
(Chang and Yu 2002). Coordinated expression of these
two clusters could explain why the clustering of aflatoxin
genes is necessary for aflatoxin production and why the
cluster has been conserved in widely different taxa
ranging from A. nidulans to A. flavus (Walton 2000).

Another way that carbon source utilization could affect

aflatoxin gene expression may be by inducing Ga proteindependent signaling in Aspergillus cells (Daniel et al.
1998). High glucose levels should increase the level of
cAMP, which in turn activates cAMP-dependent protein
kinases. The level of these kinases is elevated in
aflatoxin-producing cultures (Jayashree et al. 2000). A
correlation between increased pool size of cAMP and
aflatoxin production had been observed previously (Khan
and Venkitasubramanian 1986, 1987), and treatment of
cultures of A. parasiticus with dibutyryl cAMP increased
aflatoxin biosynthesis (Bhatnagar et al. 1999). ST
production by A. nidulans appears to require inhibition
of FadA-dependent signaling (Hicks et al. 1997). FadA is
the alpha subunit of the A. nidulans heterotrimeric G
protein. When FadA is bound to GTP, i.e., in its active
form, ST production (and sporulation) was repressed.
However, in the presence of FlbA, a protein similar to
RGS (regulators of G protein signaling)-type proteins, the
intrinsic GTPase activity of FadA is stimulated, thereby
leading to GTP hydrolysis, inactivation of FadA-dependent signaling, and stimulation of ST production. The
activity of RGS-type proteins is mediated by extracellular
signals that begin the signaling process by binding to a
cell-surface receptor. For the ST signaling cascade in A.
nidulans, one possible product is synthesized by a GSItype glutamine synthatase, FluG. G protein signaling
mediates the levels of cAMP, which probably acts as the
second messenger, affecting the activity of protein
kinases, which, in turn, may directly modulate the activity
of AflR. Since AflR is probably active in its phosphorylated state, regulation of G protein signaling, cAMP
levels, and the activity of key protein kinases all should
ultimately affect aflatoxin and ST production by toxinproducing Aspergilli.

Biological/evolutionary significance
of aflatoxin production
Aflatoxin does not appear to be essential to the growth
and/or life-cycle of the fungus, and much speculation
about its role has been published (Bennett and Christiansen 1983; Ciegler 1983; Lillehoj 1991; Jarvis and
Miller 1996; Demain and Fang 2000). BuLock (1965)
postulated that aflatoxin production was a mechanism for
the organism to release excess carbon when the fungus is
growing in a carbon-rich environment, but little evidence
exists to support or defend this hypothesis and atoxigenic
fungi can compete with toxigenic fungi on the same
medium (Cotty 1989).
Role of the cluster organization
Aflatoxin production may be a vestigal trait that has
survived due to the clustered gene organization. One
hypothesis suggested that such an organization of genes
may allow coordinated regulation of the pathway (Walton

90

2000). Another hypothesis is that the cluster organization

ensures survival of the cluster by providing a mechanism
for horizontal gene transfer (Geiser et al. 1998, 2000).
Evidence for such a transfer is still speculative. Many
species of Aspergilli contain some of the genes for
synthesis of aflatoxin and its precursors. Besides A. flavus
and A. parasiticus, A. nomius, A. pseudotamarii, A.
bombycis, and A. ochraceoroseus (Klich et al. 2000;
Ehrlich et al. 2002b) make aflatoxins, while A. nidulans,
A. ustus, A. variecolor, A. versicolor and possibly certain
Bipolaris spp. make ST (Cole and Cox 1987). The
arrangement of genes in the A. nidulans ST gene cluster is
different from that in A. flavus or A. parasiticus (Fig. 2)
and horizontal transfer of the different types of cluster
between these species has not been found. The availability of a sexual stage, which facilitates recombination, in
A. nidulans may account for the differences in gene
organization of the cluster in these species. Horizontal
gene transfer may explain how genes similar to the
aflatoxin pathway genes are present in species of fungi
quite distant from Aspergilli, such as Dothistroma pini, a
pine forest fungus from New Zealand which makes
VERA (Bradshaw et al. 2002). An argument against
horizontal gene transfer of aflatoxin pathway genes is that
the genes in the cluster quite closely follow the expected
evolutionary lineage of other non-clustered, essential
genes in the same species isolated from widely divergent
geographical locations (Ehrlich et al. 2002b). A. flavus
isolates separated by a vegetative compatibility system
that precludes genetic mixing, also show no evidence for
recombination events that would be expected if these
events were frequent rather than rare.
Aflatoxins as chemical signals
Aflatoxins could be chemical signals between species in
an ecological niche (Lillehoj 1991) or serve to signal
fungal development (Cotty 1988; Beppu 1992; Trail et al.
1995a; Kale et al. 1996). Some of the genes involved in
aflatoxin production appear to developmentally regulated
(Mayorga and Timberlake 1992; Chang et al. 1995b) and
an association between sclerotial formation and aflatoxin
formation has been advanced (Bennett et al. 1978, 1979;
Cotty 1988; Wang et al. 1996).
Role in defense
Aflatoxins toxicity may protect the fungus from competitors in the soil or during crop invasion (Demain and
Fang 2000). Aflatoxins are not particularly phytotoxic
(McLean et al. 1995; Hasan 1999) and there is no
evidence that they serve as virulence factors, since
atoxigenic isolates are equally able to invade susceptible
crop species (Cotty 1989). However, the atoxigenic
isolates that are successful competitors retain the ability
to produce some of the aflatoxin biosynthesis enzymes
(Cotty and Bhatnagar 1994). This result implies that some

of the biosynthetic proteins may be involved in other

metabolic processes that do contribute to fungal virulence.
Aflatoxins are toxic to insects and some aflatoxinproducing species have been associated with insect debris
(Matsumura and Knight 1967; Reiss 1975; Wright et al.
1982; Jarvis et al. 1984; Kurtzman et al. 1987; Llewellyn
et al. 1988; Drummond and Pinnock 1990). The lethal
concentration of aflatoxin for insects is quite high [ppm
levels; (Dowd 1992)] and such concentrations are rarely
found in plants. These concentrations are found in
individual fungal colonies and could be toxic to feeding
insects. In spite of this, insects appear to be excellent
vectors for mycotoxigenic fungi during plant invasion
(Dowd 1992), suggesting that insects and aflatoxigenic
fungi have adapted to one another. Fungi in the A. flavus
group are thought to overwinter in the soil, perhaps as
sclerotia on crop debris, and overwintering structures
(e.g., sclerotia) that contain aflatoxin may have a survival
advantage compared to those from atoxigenic isolates.

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