Crawford et al., 2008b). Mahanti et al. (Mahanti et al., 1996) cloned, by genetic complementation, a 7.5-kb large transcript which is required for NOR formation in a blocked *A. parasiticus* mutant. Its protein has high degree of similarity (67%) and identity (48%) to the beta-subunit of FASs (FAS1) of *Saccharomyces cerevisiae* and *Yarrowia lipolytica*. Metabolite feeding and gene disruption experiments further confirmed that *uvm8* encodes a subunit of a novel fatty acid synthase (FAS) directly involved in the backbone formation of the polyketide precursor of NOR during aflatoxin biosynthesis, therefore, on the basis of its function, the *uvm8* gene was renamed *fas-1A*. In the revised naming scheme, the *fas-1A* gene was renamed as *fas-1*, it encodes fatty acid synthase-1 in the aflatoxin biosynthetic pathway gene cluster (Figure 1). Another large transcript (*fas-2A*) which encodes an alpha-subunit of fatty acid synthase in the aflatoxin gene cluster was reported (Mahanti et al., 1996). The gene *fas-1A* and *fas-2A* were renamed *fas-1* and *fas-2*. They encode two fatty acid synthases (FASα and FASβ) (Payne, 1998). In *A. nidulans* the involvement of FASs in sterigmatocystin (ST) biosynthesis was also confirmed and were named *stcJ* and *stcK* in the ST cluster (Brown et al., 1996a; Brown et al., 1996b). The biochemical evidence for the role of a fatty acid synthase and a polyketide synthase (PKS) in the biosynthesis of aflatoxin was demonstrated (Watanabe & Townsend, 1996). Further details on the early stage of aflatoxin biosynthesis involving fatty acid synthases and polyketide synthases were reported (Watanabe et al., 1996; Hitchman et al., 2001; Watanabe & Townsend, 2002; Crawford et al., 2006). The Nacetylcysteamine thioester of hexanoic acid was incorporated into NOR in a *fas-1* disrupted transformant. A polyketide synthase gene (*pksA*) in *A. parasiticus* was demonstrated by gene disruption to be required for aflatoxin biosynthesis (Chang et al., 1995a). The predicted amino acid sequences of these PKSs contain the typical four conserved domains commonly found in other known PKS proteins: -ketoacyl synthase (KS), acyltransferase (AT), acyl carrier protein (ACP), and thioesterase (TE) (Chang et al., 1995a). Townsend's group has dissected the functional domains of the PKS for aflatoxin biosynthesis (Crawford et al., 2008a; Crawford et al., 2008b; Crawford et al., 2008c). These include domains for the starter unit acyl transferase (SAT) which recognizes hexanoyl CoA and the N-acetylcysteamine thioester of hexanoic acid, the acyl carrier protein (ACP) , ketosynthase (KS), malonyl-CoA:ACP transacylase (MAT), product template (PT) allowing the iterative steps in forming the polyketide, and a thioesterase/Claisen-like cyclase (TE/CLC) (Crawford et al., 2008a). The predicted product converted by PksA is noranthrone. The conversion of noranthrone to NOR, the first stable intermediate in the pathway (Bennett et al., 1971; Lee et al., 1971; Papa, 1979; Bennett et al., 1981; Papa, 1982; Bennett et al., 1994; Bennett et al., 1997), is poorly defined, but it has been proposed to be catalyzed by a noranthrone oxidase, a monooxygenase, or to occur spontaneously (Dutton, 1988). Sequence analysis and enzymatic studies supports the contention that the *hypC* (a gene in the intergenic region of *pksA* and *nor-1*) gene product is the required noranthrone oxidase involved in the catalysis of the orxidation of norsolorinic acid anthrone to NOR (Ehrlich, 2009) The *fas-1*, *fas-2*, and *pksA* genes were renamed as *aflA*, *aflB* and *aflC* respectively (Wilson, 1989; Yu et al., 2004a; Yu et al., 2004c) (Figure 1). The *aflA*, *aflB* and *aflC* gene homologues in *A. nidulans* are *stcJ*, *stcK*, and *stcA*, respectively (Brown et al., 1996b).

#### **3.2 Norsolorinic acid (NOR) to averantin (AVN)**

The first stable AF intermediate was identified as NOR produced in *A. parasiticus* uvgenerated disruption mutants (Bennett et al., 1971; Lee et al., 1971; Detroy et al., 1973;

Aflatoxin Biosynthetic Pathway and Pathway Genes 47

al., 2010). Woloshuk and Payne (Woloshuk & Payne, 1994) identified an alcohol dehydrogenase gene, *adh1*, in *A. flavus*, expressed concurrently with aflatoxin pathway genes. No further report is made on the role of *A. flavus adh1* gene in aflatoxin synthesis. The *aflH* (*adhA*) gene in *A. flavus* and the *adhA* gene in *A. parasiticus* share no significant

VHA was identified as an aflatoxin precursor formed by oxidation of AVF (Fitzell et al., 1977). The conversion of AVF to VHA involves the cytochrome P450 monooxidase, CypX, and another gene, *aflI* (*avfA*). Although *aflI* is required for the conversion, its oxidative role is unclear (Yu et al., 2000b). *A. nidulans* also has an *aflI* gene homolog (*stcO*) (Brown et al., 1996b; Yu et al., 2000b). Complementation of an averufin-accumulating mutant, *A. parasiticus* SRRC 165, with the *aflI* gene of *A. flavus* restored the strain's ability to convert AVF to VHA and to produce aflatoxins (Yu et al., 2000b). It is likely that the *aflI* (*avfA*) encoded protein along with CypX gene product is involved in the ring-closure step in the formation of hydroxyversicolorone. It is possible that the *avfA* gene product is assocated with the P450 monooxygenase to carry out the conversion as no additional intermediates

**3.6 Versiconal hemiacetal acetate (VHA) to versiconal (VHOH, also abbreviated**

Several research groups have demonstrated that an esterase is involvement in the conversion of VHA to VHOH (VAL) (Schroeder et al., 1974; Yao & Hsieh, 1974; Bennett et al., 1976b; Fitzell et al., 1977; Hsieh et al., 1989; Yabe et al., 1991a; Yabe et al., 1991b; Kusumoto & Hsieh, 1996). The esterase was purified in *A. parasiticus* (Hsieh et al., 1989; Kusumoto & Hsieh, 1996). An esterase gene, *aflJ* (*estA*), in the aflatoxin gene cluster was identified (Yu et al., 2002). The homologous gene in the *A. nidulans* ST biosynthetic gene cluster is *stcI*. In the *A. parasiticus aflJ* (*estA*) deletion mutants, the accumulated metabolites were mainly VHA and versicolorin A (VERA) (Chang et al., 2004). A small amount of versiconol acetate (VOAc) and other downstream aflatoxin intermediates, including VHOH and versicolorin B also accumulated. A metabolic grid containing VHA, VOAc, VHOH, and versiconol (VOH) was previously described and it was suggested that the reactions from VHA to VHOH and from VOAc to VOH are catalyzed by the same esterase (Yabe et al., 1991a). Later, another metabolic grid containing versicolorone (VONE), VOAc, and VHA was identified (Yabe et al., 2003). Indeed, it has now been proven that the *estA*-encoded esterase catalyzes the conversion of both VHA to VHOH and VOAc to VOH during

The enzymatic evidence that VHOH is converted toVERB by a cyclase was first provided by Lin and Anderson (Lin & Anderson, 1992). This enzyme was identified as versicolorin B synthase and was studied intensively by Townsend's laboratory (Zuckerman et al., 1967; Hsieh, 1973; McGuire et al., 1996; Silva et al., 1996; Silva & Townsend, 1997). The gene was cloned and named *vbs* (Zuckerman et al., 1967; Hsieh, 1973; Silva et al., 1996). The expected cyclase activity was demonstrated by the expressed recombinant protein of the *vbs* gene (Silva et al., 1996; Silva & Townsend, 1997). The VHOH cyclase (Lin & Anderson, 1992) and

homology at either the DNA or the amino acid level.

**3.5 Averufin (AVF) to versiconal hemiacetal acetate (VHA)**

other that AVF result from the disruption of either gene.

aflatoxin biosynthesis (Chang et al., 2004).

**3.7 Versiconal (VHOH) to versicolorin B (VER B)**

**as VAL)**

Bennett et al., 1981) and in *A. flavus* (Papa, 1979; Papa, 1982) The NOR-accumulating mutants are leaky mutants whose aflatoxin biosynthesis is not completely blocked. By genetic complementation, the gene, *aflD* (*nor-1*), encoding a reductase was cloned (Chang et al., 1992). A recombinant Nor-1 protein expressed in *E. coli* catalyzed the reduction of NOR. Therefore, *aflD* (*nor-1*) encodes the ketoreductase needed for the conversion of the 1'-keto group in NOR to the 1'-hydroxyl group of AVN (Zhou & Linz, 1999). Disruption of the *aflD*  (*nor-1*) gene also confirmed its involvement in conversion of NOR to AVN in aflatoxin biosynthesis (Trail et al., 1994). The *aflD* (*nor-1*) homologous gene in *A. nidulans* is *stcE* (Brown et al., 1996b). Genes homologous to *aflD* (*nor-1*), in the AF cluster, such as *aflE* (*norA*) and *aflF* (*norB*) are predicted to encode short chain aryl alcohol dehydrogenases. These proteins may also be able to catalyze the reduction of NOR to AVN depending on the reductive environment of the cell and may explain the leakiness of the *nor-1* mutation if they are able to complement Nor-1's function (Cary et al., 1996),

#### **3.3 Averantin (AVN) to 5'-hydroxyaverantin (HAVN)**

Radioisotope incorporation experiments established the earliest evidence for the conversion of AVN to HAVN (Bennett et al., 1980; McCormick et al., 1987). In these studies, three enzymatic steps can account for the conversion of NOR to averufin (AVF) (Yabe et al., 1991a). They are (i) NOR to AVN catalyzed by a reductase, (ii) NOR to HAVN catalyzed by a monooxygenase, and (iii) HAVN to AVF catalyzed by a second dehydrogenase. It was also proposed that the oxidation reactions are reversible and that NADPH was the preferred cofactor (Yabe et al., 1991b). The gene previously named *ord-1* encoding a P-450 monooxygenase was cloned and disrupted (Yu et al., 1997). Substrate feeding studies of the *ord*-1 mutant confirmed that HAVN is the intermediate in the conversion of AVN to AVF. The *ord-1* gene, which has a high degree of sequence similarity to *A. nidulans stcF* (Brown et al., 1996b), was renamed *aflG* (*avnA*).

#### **3.4 5'-Hydroxyaverantin (HAVN) to oxoaverantin (OAVN), and averufin (AVF)**

Numerous studies have established averufin as one of the key intermediates in aflatoxin formation (Lee et al., 1971; Hsieh, 1973; Lin & Hsieh, 1973; Lin et al., 1973; Fitzell et al., 1975; Singh & Hsieh, 1977; Keller et al., 2000). Several intermediates were reported to be involved in the conversion from AVN to AVF (Lin & Hsieh, 1973; Bhatnagar et al., 1992). One of these averufanin (AVNN), based on later studies was considered a shunt metabolite and not a genuine aflatoxin intermediate (Sakuno et al., 2003; Yabe & Nakajima, 2004). Chang et al. (Lee et al., 1971; Lin & Hsieh, 1973; Chang et al., 2000) characterized the cluster gene *aflH*  (*adhA*) in *A. parasiticus* which encodes an alcohol dehydrogenase. It was showed that *adhA* deletion mutants accumulated predominantly HAVN and after prolonged growth the mutants were able to produce small amounts of AVNN consistant with AVNN being a shunt metabolite. Thus, HAVN might be converted directly to AVF or indirectly to AVF by an additional cytosolic enzyme. Sakuno et al. (Sakuno et al., 2003) characterized two cytosolic enzymes and a new aflatoxin intermediate named 5'-oxoaverantin (OAVN) as an intermediate between HAVN and AVF. The enzyme for the conversion from HAVN to OAVN is encoded by the *aflH* (*adhA*) gene. The *adhA* gene deletion mutant is leaky indicating that additional enzyme(s) or gene(s) may be involved in the conversion from OAVN to AVF. The enzymatic steps for aflatoxin biosynthesis and the possible involvement of additional enzymes have also been described (Townsend, 1997; Ehrlich, 2009; Ehrlich et

Bennett et al., 1981) and in *A. flavus* (Papa, 1979; Papa, 1982) The NOR-accumulating mutants are leaky mutants whose aflatoxin biosynthesis is not completely blocked. By genetic complementation, the gene, *aflD* (*nor-1*), encoding a reductase was cloned (Chang et al., 1992). A recombinant Nor-1 protein expressed in *E. coli* catalyzed the reduction of NOR. Therefore, *aflD* (*nor-1*) encodes the ketoreductase needed for the conversion of the 1'-keto group in NOR to the 1'-hydroxyl group of AVN (Zhou & Linz, 1999). Disruption of the *aflD*  (*nor-1*) gene also confirmed its involvement in conversion of NOR to AVN in aflatoxin biosynthesis (Trail et al., 1994). The *aflD* (*nor-1*) homologous gene in *A. nidulans* is *stcE* (Brown et al., 1996b). Genes homologous to *aflD* (*nor-1*), in the AF cluster, such as *aflE* (*norA*) and *aflF* (*norB*) are predicted to encode short chain aryl alcohol dehydrogenases. These proteins may also be able to catalyze the reduction of NOR to AVN depending on the reductive environment of the cell and may explain the leakiness of the *nor-1* mutation if they

Radioisotope incorporation experiments established the earliest evidence for the conversion of AVN to HAVN (Bennett et al., 1980; McCormick et al., 1987). In these studies, three enzymatic steps can account for the conversion of NOR to averufin (AVF) (Yabe et al., 1991a). They are (i) NOR to AVN catalyzed by a reductase, (ii) NOR to HAVN catalyzed by a monooxygenase, and (iii) HAVN to AVF catalyzed by a second dehydrogenase. It was also proposed that the oxidation reactions are reversible and that NADPH was the preferred cofactor (Yabe et al., 1991b). The gene previously named *ord-1* encoding a P-450 monooxygenase was cloned and disrupted (Yu et al., 1997). Substrate feeding studies of the *ord*-1 mutant confirmed that HAVN is the intermediate in the conversion of AVN to AVF. The *ord-1* gene, which has a high degree of sequence similarity to *A. nidulans stcF* (Brown et

**3.4 5'-Hydroxyaverantin (HAVN) to oxoaverantin (OAVN), and averufin (AVF)**

Numerous studies have established averufin as one of the key intermediates in aflatoxin formation (Lee et al., 1971; Hsieh, 1973; Lin & Hsieh, 1973; Lin et al., 1973; Fitzell et al., 1975; Singh & Hsieh, 1977; Keller et al., 2000). Several intermediates were reported to be involved in the conversion from AVN to AVF (Lin & Hsieh, 1973; Bhatnagar et al., 1992). One of these averufanin (AVNN), based on later studies was considered a shunt metabolite and not a genuine aflatoxin intermediate (Sakuno et al., 2003; Yabe & Nakajima, 2004). Chang et al. (Lee et al., 1971; Lin & Hsieh, 1973; Chang et al., 2000) characterized the cluster gene *aflH*  (*adhA*) in *A. parasiticus* which encodes an alcohol dehydrogenase. It was showed that *adhA* deletion mutants accumulated predominantly HAVN and after prolonged growth the mutants were able to produce small amounts of AVNN consistant with AVNN being a shunt metabolite. Thus, HAVN might be converted directly to AVF or indirectly to AVF by an additional cytosolic enzyme. Sakuno et al. (Sakuno et al., 2003) characterized two cytosolic enzymes and a new aflatoxin intermediate named 5'-oxoaverantin (OAVN) as an intermediate between HAVN and AVF. The enzyme for the conversion from HAVN to OAVN is encoded by the *aflH* (*adhA*) gene. The *adhA* gene deletion mutant is leaky indicating that additional enzyme(s) or gene(s) may be involved in the conversion from OAVN to AVF. The enzymatic steps for aflatoxin biosynthesis and the possible involvement of additional enzymes have also been described (Townsend, 1997; Ehrlich, 2009; Ehrlich et

are able to complement Nor-1's function (Cary et al., 1996),

**3.3 Averantin (AVN) to 5'-hydroxyaverantin (HAVN)**

al., 1996b), was renamed *aflG* (*avnA*).

al., 2010). Woloshuk and Payne (Woloshuk & Payne, 1994) identified an alcohol dehydrogenase gene, *adh1*, in *A. flavus*, expressed concurrently with aflatoxin pathway genes. No further report is made on the role of *A. flavus adh1* gene in aflatoxin synthesis. The *aflH* (*adhA*) gene in *A. flavus* and the *adhA* gene in *A. parasiticus* share no significant homology at either the DNA or the amino acid level.

#### **3.5 Averufin (AVF) to versiconal hemiacetal acetate (VHA)**

VHA was identified as an aflatoxin precursor formed by oxidation of AVF (Fitzell et al., 1977). The conversion of AVF to VHA involves the cytochrome P450 monooxidase, CypX, and another gene, *aflI* (*avfA*). Although *aflI* is required for the conversion, its oxidative role is unclear (Yu et al., 2000b). *A. nidulans* also has an *aflI* gene homolog (*stcO*) (Brown et al., 1996b; Yu et al., 2000b). Complementation of an averufin-accumulating mutant, *A. parasiticus* SRRC 165, with the *aflI* gene of *A. flavus* restored the strain's ability to convert AVF to VHA and to produce aflatoxins (Yu et al., 2000b). It is likely that the *aflI* (*avfA*) encoded protein along with CypX gene product is involved in the ring-closure step in the formation of hydroxyversicolorone. It is possible that the *avfA* gene product is assocated with the P450 monooxygenase to carry out the conversion as no additional intermediates other that AVF result from the disruption of either gene.

#### **3.6 Versiconal hemiacetal acetate (VHA) to versiconal (VHOH, also abbreviated as VAL)**

Several research groups have demonstrated that an esterase is involvement in the conversion of VHA to VHOH (VAL) (Schroeder et al., 1974; Yao & Hsieh, 1974; Bennett et al., 1976b; Fitzell et al., 1977; Hsieh et al., 1989; Yabe et al., 1991a; Yabe et al., 1991b; Kusumoto & Hsieh, 1996). The esterase was purified in *A. parasiticus* (Hsieh et al., 1989; Kusumoto & Hsieh, 1996). An esterase gene, *aflJ* (*estA*), in the aflatoxin gene cluster was identified (Yu et al., 2002). The homologous gene in the *A. nidulans* ST biosynthetic gene cluster is *stcI*. In the *A. parasiticus aflJ* (*estA*) deletion mutants, the accumulated metabolites were mainly VHA and versicolorin A (VERA) (Chang et al., 2004). A small amount of versiconol acetate (VOAc) and other downstream aflatoxin intermediates, including VHOH and versicolorin B also accumulated. A metabolic grid containing VHA, VOAc, VHOH, and versiconol (VOH) was previously described and it was suggested that the reactions from VHA to VHOH and from VOAc to VOH are catalyzed by the same esterase (Yabe et al., 1991a). Later, another metabolic grid containing versicolorone (VONE), VOAc, and VHA was identified (Yabe et al., 2003). Indeed, it has now been proven that the *estA*-encoded esterase catalyzes the conversion of both VHA to VHOH and VOAc to VOH during aflatoxin biosynthesis (Chang et al., 2004).

#### **3.7 Versiconal (VHOH) to versicolorin B (VER B)**

The enzymatic evidence that VHOH is converted toVERB by a cyclase was first provided by Lin and Anderson (Lin & Anderson, 1992). This enzyme was identified as versicolorin B synthase and was studied intensively by Townsend's laboratory (Zuckerman et al., 1967; Hsieh, 1973; McGuire et al., 1996; Silva et al., 1996; Silva & Townsend, 1997). The gene was cloned and named *vbs* (Zuckerman et al., 1967; Hsieh, 1973; Silva et al., 1996). The expected cyclase activity was demonstrated by the expressed recombinant protein of the *vbs* gene (Silva et al., 1996; Silva & Townsend, 1997). The VHOH cyclase (Lin & Anderson, 1992) and

Aflatoxin Biosynthetic Pathway and Pathway Genes 49

the methyl from S-adenosylmethionine (SAM) to the hydroxyls of DMST and DHDMST to produce ST and DHST, respectively. This 43-kDa enzyme was purified from *A. parasiticus* and characterized (Yabe et al., 1998; Yabe et al., 1999). The corresponding gene, *dmtA*, was isolated from *A. parasiticus* based on a partial amino acid sequence of the purified enzyme (Motomura et al., 1999). Yu et al. (Yu et al., 2000b) concurrently isolated the same gene but named it *aflO* (*omtB*) (for *O*-methyltransferase B) from *A. parasiticus*, *A. flavus* and *A. sojae*. The predicted *dmtA*-encoded protein contains a consensus SAM-binding motif (Motomura et al., 1999). The *aflO* (*omtB*) homolog in *A. nidulans* was identified as *stcP.* This gene is required for the conversion of DMST to ST in *A. nidulans* as shown by gene disruption

**3.11 Sterigmatocystin (ST) to** *O***-methylsterigmatocystin (OMST) and**

**demethylsterigmatocystin (DMST) to dihydro-***O***-methylsterigmatocystin (DHOMST)**  The gene for *O*-methyltransferase required for the conversion of ST to OMST and DHST to DHOMST was first cloned (Yu et al., 1993) from *A. parasiticus* by reverse genetics using antibodies raised against the purified *A. parasiticus O*-methyltransferase A (Keller et al., 1993). This gene was initially named *omt-1*, then *omtA* and finally renamed *aflP* (*omtA*) (Yu et al., 1993). The recombinant enzyme was expressed in *E. coli* and its activity to convert ST to OMST was demonstrated by substrate feeding studies (Yu et al., 1993). *O*-methyltransferase A has strict substrate-specificity and cannot methylate DMST or DHDMST. Thus, the *O*methyltransferases A encoded by *aflP* (*omtA*) is the enzyme responsible for the conversion of ST to OMST and DMST to DHOMST. The genomic DNA sequence of this gene (*omtA*) was cloned from *A. parasiticus* and *A. flavus* (Yu et al., 1995b). This *aflP* (*omtA*) gene homologue was also detected in other aflatoxigenic and non-aflatoxigenic *Aspergillus* species (Klich et al., 1995). The absence of the *aflP* orthologue in *A. nidulans* is the reason that *A. nidulans* produces ST as

**3.12** *O***-methylsterigmatocystin (OMST) to aflatoxin B1 (AFB1) and aflatoxin G1 (AFG1) and demethyldihydrosterigmatocystin (DMDHST) to aflatoxin B2 (AFB2) and aflatoxin**

The relationship between B-group and G-group aflatoxin formation was proposed based on feeding experiments (Yabe et al., 1988). A P-450 monooxygenase gene in *A. flavus* named *ord-1* was shown to be necessory for this reaction (Prieto et al., 1996; Prieto & Woloshuk, 1997). This P-450 monooxygenase gene, *aflQ* (*ordA*), was cloned in *A. parasiticus* and demonstrated in a yeast system that it is involved in the conversion of OMST to AFB1/AFG1, and DHOMST to AFB2/AFG2 (Yu et al., 1998). Whether *aflQ* (*ordA*) gene product, OrdA, catalyzes two successive monooxygenase reactions in the later steps of aflatoxin biosynthesis is not clear. Studies (Yu et al., 1998) suggested that additional enzyme(s) is required for the synthesis of G-group aflatoxins. After the cloning and characterization of the *cypA* gene, it is clear that *cypA* encoded a cytochrome P450 monooxygenase for the formation of G-group aflatoxins (Ehrlich et al., 2004). Most recently, the *nadA* gene, which was shown, by gene profiling studies using microarray, to be a member of the aflatoxin gene cluster (Price et al., 2006; Yu et al., 2011) rather than belonging to the adjoining sugar utilization cluster as originally proposed (Yu et al., 2000a), was found to play a role in AFG1/AFG2 formation. Yabe's group recently disrupted the *nadA* gene and reported that NadA is a cytosolic enzyme for the conversion from a new aflatoxin intermediate named NADA, which is between OMST and AFG1, to

(Kelkar et al., 1996).

the end product instead of aflatoxins.

**G2 (AFG2)**

VER B synthase (McGuire et al., 1996) were independently isolated from *A. parasiticus*. The enzyme catalyzes the side chain cyclodehydration of racemic VHA to VER B. This is another key step in aflatoxin formation since it closes the bisfuran ring of aflatoxin, the moiety ultimately responsible for aflatoxin's toxicity andcarcinogenicity. . The *vbs* gene was renamed *aflK* (*vbs*) a (Yu et al., 2004c). The homologous gene in the *A. nidulans* ST biosynthetic gene cluster is *stcN*.

#### **3.8 Versicolorin B (VER B) to versicolorin A (VER A)**

VER B is a critical branch point leading to the formation of either AFB1/AFG1 or AFB2/AFG2. Similar to AFB2/AFG2, VER B contains a tetrahydrobisfuran ring and, like AFB1/AFG1, VERA contains a dihydrobisfuran ring. The conversion of VER B to VER A requires desaturation of the bisfuran ring of VER B by an unstable microsomal enzyme that requires NADPH (Yabe et al., 1993). Disruption of *stcL* in *A. nidulans* (Kelkar et al., 1997) abolished ST synthesis and resulted in the accumulation of VER B. The *stcL* gene encodes a cytochrome P-450 monooxygenase. The homologue, *aflL* (*verB*), is present in the aflatoxin gene cluster of *A. parasiticus* and *A. flavus* strains. Cultural conditions appear to markedly affect the activity of VER B desaturase and thereby, the final ratio of AFB1 to AFB2 and AFG1 to AFG2 (Yabe & Nakajima, 2004).

#### **3.9 Versicolorin A (VER A) to demethylsterigmatocystin (DMST) and versicolorin B (VER B) to demethyldihydrosterigmatocystin (DMDHST)**

The formation of DMST and the biochemical conversion steps from VERA to DMST (and VerB to DHDMST) have been described in great detail (Henry & Townsend, 2005). The *aflM* (*ver-1*) gene (Skory et al., 1992), cloned by genetic complementation of VER A-accumulating *A. parasiticus* CS10, was shown to be responsible for the conversion of VER A to an intermediate that has not been isolated. The *aflM* (*ver-1*) gene was predicted to encode a ketoreductase, similar Nor-1. The *ver-1* homologue, *stcU*, (previously named *verA*) was identified in *A. nidulans* (Keller et al., 1994)*.* Double mutation of *stcU* and *stcL* resulted in accumulation of only VER A (Keller et al., 1994). The *stcS* gene (previously named *verB*), another cytochrome P-450 monooxygenase gene, was also identified and studies showed that it is also involved in the conversion of VER A to an intermediate in the formation of DMST (possibly the first intermediate, which is then acted upon by Ver-1). Disruption of *stcS* resulted in the accumulation of VER A as did disruption of Ver-1 (Keller et al., 1995). Thus, both *stcU* and *stcS* are required for the conversion of VER A to DMST. The *stcS* homologue in *A. parasiticus*, named *aflN* (*verA*), has also been identified (Yu et al., 2004a; Yu et al., 2004c). A third enzyme is required for the conversion: hypA (aflY). This gene is predicted to encode a Baeyer-Villiger monooxygenase. Disruption of this gene also led to accumulation of VERA suggesting that, like VER-1, it acts as part of an enzyme complex without allowing the formation of an intermediate. A fourth enzyme, OrdB has also been implicated in the conversion, and like AvfA, its homolog, may be a helper protein for the monooxygenase, CypX.

#### **3.10 Demethylsterigmatocystin (DMST) to sterigmatocystin (ST) and**

#### **demethyldihydrosterigmatocystin (DMDHST) to dihydrosterigmatocystin (DHST)**

Enzyme purification studies revealed that two *O*-methyltransferases, I and II, are involved in aflatoxin biosynthesis (Yabe et al., 1989). *O*-methyltransferase I catalyzes the transfer of

VER B synthase (McGuire et al., 1996) were independently isolated from *A. parasiticus*. The enzyme catalyzes the side chain cyclodehydration of racemic VHA to VER B. This is another key step in aflatoxin formation since it closes the bisfuran ring of aflatoxin, the moiety ultimately responsible for aflatoxin's toxicity andcarcinogenicity. . The *vbs* gene was renamed *aflK* (*vbs*) a (Yu et al., 2004c). The homologous gene in the *A. nidulans* ST biosynthetic

VER B is a critical branch point leading to the formation of either AFB1/AFG1 or AFB2/AFG2. Similar to AFB2/AFG2, VER B contains a tetrahydrobisfuran ring and, like AFB1/AFG1, VERA contains a dihydrobisfuran ring. The conversion of VER B to VER A requires desaturation of the bisfuran ring of VER B by an unstable microsomal enzyme that requires NADPH (Yabe et al., 1993). Disruption of *stcL* in *A. nidulans* (Kelkar et al., 1997) abolished ST synthesis and resulted in the accumulation of VER B. The *stcL* gene encodes a cytochrome P-450 monooxygenase. The homologue, *aflL* (*verB*), is present in the aflatoxin gene cluster of *A. parasiticus* and *A. flavus* strains. Cultural conditions appear to markedly affect the activity of VER B desaturase and thereby, the final ratio of AFB1 to AFB2 and AFG1

**3.9 Versicolorin A (VER A) to demethylsterigmatocystin (DMST) and versicolorin B**

The formation of DMST and the biochemical conversion steps from VERA to DMST (and VerB to DHDMST) have been described in great detail (Henry & Townsend, 2005). The *aflM* (*ver-1*) gene (Skory et al., 1992), cloned by genetic complementation of VER A-accumulating *A. parasiticus* CS10, was shown to be responsible for the conversion of VER A to an intermediate that has not been isolated. The *aflM* (*ver-1*) gene was predicted to encode a ketoreductase, similar Nor-1. The *ver-1* homologue, *stcU*, (previously named *verA*) was identified in *A. nidulans* (Keller et al., 1994)*.* Double mutation of *stcU* and *stcL* resulted in accumulation of only VER A (Keller et al., 1994). The *stcS* gene (previously named *verB*), another cytochrome P-450 monooxygenase gene, was also identified and studies showed that it is also involved in the conversion of VER A to an intermediate in the formation of DMST (possibly the first intermediate, which is then acted upon by Ver-1). Disruption of *stcS* resulted in the accumulation of VER A as did disruption of Ver-1 (Keller et al., 1995). Thus, both *stcU* and *stcS* are required for the conversion of VER A to DMST. The *stcS* homologue in *A. parasiticus*, named *aflN* (*verA*), has also been identified (Yu et al., 2004a; Yu et al., 2004c). A third enzyme is required for the conversion: hypA (aflY). This gene is predicted to encode a Baeyer-Villiger monooxygenase. Disruption of this gene also led to accumulation of VERA suggesting that, like VER-1, it acts as part of an enzyme complex without allowing the formation of an intermediate. A fourth enzyme, OrdB has also been implicated in the conversion, and like AvfA, its homolog, may be a helper protein for the

gene cluster is *stcN*.

to AFG2 (Yabe & Nakajima, 2004).

monooxygenase, CypX.

**3.8 Versicolorin B (VER B) to versicolorin A (VER A)**

**(VER B) to demethyldihydrosterigmatocystin (DMDHST)**

**3.10 Demethylsterigmatocystin (DMST) to sterigmatocystin (ST) and**

**demethyldihydrosterigmatocystin (DMDHST) to dihydrosterigmatocystin (DHST)**  Enzyme purification studies revealed that two *O*-methyltransferases, I and II, are involved in aflatoxin biosynthesis (Yabe et al., 1989). *O*-methyltransferase I catalyzes the transfer of the methyl from S-adenosylmethionine (SAM) to the hydroxyls of DMST and DHDMST to produce ST and DHST, respectively. This 43-kDa enzyme was purified from *A. parasiticus* and characterized (Yabe et al., 1998; Yabe et al., 1999). The corresponding gene, *dmtA*, was isolated from *A. parasiticus* based on a partial amino acid sequence of the purified enzyme (Motomura et al., 1999). Yu et al. (Yu et al., 2000b) concurrently isolated the same gene but named it *aflO* (*omtB*) (for *O*-methyltransferase B) from *A. parasiticus*, *A. flavus* and *A. sojae*. The predicted *dmtA*-encoded protein contains a consensus SAM-binding motif (Motomura et al., 1999). The *aflO* (*omtB*) homolog in *A. nidulans* was identified as *stcP.* This gene is required for the conversion of DMST to ST in *A. nidulans* as shown by gene disruption (Kelkar et al., 1996).

#### **3.11 Sterigmatocystin (ST) to** *O***-methylsterigmatocystin (OMST) and demethylsterigmatocystin (DMST) to dihydro-***O***-methylsterigmatocystin (DHOMST)**

The gene for *O*-methyltransferase required for the conversion of ST to OMST and DHST to DHOMST was first cloned (Yu et al., 1993) from *A. parasiticus* by reverse genetics using antibodies raised against the purified *A. parasiticus O*-methyltransferase A (Keller et al., 1993). This gene was initially named *omt-1*, then *omtA* and finally renamed *aflP* (*omtA*) (Yu et al., 1993). The recombinant enzyme was expressed in *E. coli* and its activity to convert ST to OMST was demonstrated by substrate feeding studies (Yu et al., 1993). *O*-methyltransferase A has strict substrate-specificity and cannot methylate DMST or DHDMST. Thus, the *O*methyltransferases A encoded by *aflP* (*omtA*) is the enzyme responsible for the conversion of ST to OMST and DMST to DHOMST. The genomic DNA sequence of this gene (*omtA*) was cloned from *A. parasiticus* and *A. flavus* (Yu et al., 1995b). This *aflP* (*omtA*) gene homologue was also detected in other aflatoxigenic and non-aflatoxigenic *Aspergillus* species (Klich et al., 1995). The absence of the *aflP* orthologue in *A. nidulans* is the reason that *A. nidulans* produces ST as the end product instead of aflatoxins.

#### **3.12** *O***-methylsterigmatocystin (OMST) to aflatoxin B1 (AFB1) and aflatoxin G1 (AFG1) and demethyldihydrosterigmatocystin (DMDHST) to aflatoxin B2 (AFB2) and aflatoxin G2 (AFG2)**

The relationship between B-group and G-group aflatoxin formation was proposed based on feeding experiments (Yabe et al., 1988). A P-450 monooxygenase gene in *A. flavus* named *ord-1* was shown to be necessory for this reaction (Prieto et al., 1996; Prieto & Woloshuk, 1997). This P-450 monooxygenase gene, *aflQ* (*ordA*), was cloned in *A. parasiticus* and demonstrated in a yeast system that it is involved in the conversion of OMST to AFB1/AFG1, and DHOMST to AFB2/AFG2 (Yu et al., 1998). Whether *aflQ* (*ordA*) gene product, OrdA, catalyzes two successive monooxygenase reactions in the later steps of aflatoxin biosynthesis is not clear. Studies (Yu et al., 1998) suggested that additional enzyme(s) is required for the synthesis of G-group aflatoxins. After the cloning and characterization of the *cypA* gene, it is clear that *cypA* encoded a cytochrome P450 monooxygenase for the formation of G-group aflatoxins (Ehrlich et al., 2004). Most recently, the *nadA* gene, which was shown, by gene profiling studies using microarray, to be a member of the aflatoxin gene cluster (Price et al., 2006; Yu et al., 2011) rather than belonging to the adjoining sugar utilization cluster as originally proposed (Yu et al., 2000a), was found to play a role in AFG1/AFG2 formation. Yabe's group recently disrupted the *nadA* gene and reported that NadA is a cytosolic enzyme for the conversion from a new aflatoxin intermediate named NADA, which is between OMST and AFG1, to

Aflatoxin Biosynthetic Pathway and Pathway Genes 51

The *aflS* (*aflJ*) gene, although not demonstrating significant homology with any other encoded proteins found in databases, is necessary for aflatoxin formation. In the *A. parasiticus aflR* transformants, the production of aflatoxin pathway intermediates was significantly enhanced in transformants that contained an additional *aflR* plus *aflS* (Chang et al., 1995b). Quantitative PCR showed that in the *aflS* knockout mutants, the lack of *aflS* transcript is associated with 5- to 20-fold reduction of expression of some aflatoxin pathway genes such as *aflC* (*pksA*), *aflD* (*nor-1*), *aflM* (*ver-1*), and *aflP* (*omtA*). The mutants lost the ability to synthesize aflatoxin intermediates and no aflatoxins were produced (Meyers et al., 1998). However, deletion of *aflS* (*aflJ*) did not have a discernible effect on *aflR* transcription, and vice versa. Du et al. (Du et al., 2007) showed that overexpression of *A. flavus aflS* (*aflJ*) did not result in elevated transcription of *aflM* (*ver-1*), *aflP* (*omtA*), or *aflR*, but it appears to have some effect on *aflC* (*pksA*), *aflD* (*nor-1*), *aflA* (*fas*-*1*), and *aflB* (*fas-2*) (Du et al., 2007), which are required for the biosynthesis of the early aflatoxin pathway intermediate, averantin. The mechanism(s) by which *aflS* modulates transcription of these pathway genes in concert with *aflR* is under investigation by gene profiling analysis using microarray

The novel global regulatory gene, *laeA* (for **l**ack of *aflR* **e**xpression), was first identified from *A. nidulans* (Bok & Keller, 2004). This gene is well conserved in fungi as shown by its presence in the genomes of all fungi so far sequenced. LaeA is a nuclear protein which contains an S-adenosylmethionine (SAM) binding motif and activates transcription of several other secondary metabolism gene clusters in addition to the AF cluster*.* Examples include the sterigmatocystin and penicillin clusters in *A. nidulans*, the gliotoxin cluster in *A. fumigatus*, and aflatoxin cluster in *A. flavus* (Bok & Keller, 2004; Bouhired et al., 2007). It also regulates genes required for virulence of *A. fumigatus* (Sugui et al., 2007). Perrin et al. (Perrin et al., 2007) carried out a whole-genome comparison of the transcriptional profiles of wildtype and *laeA*-deleted *A. fumigatus* strains and found that LaeA positively controls the expression of 20% to 40% of major classes of secondary metabolite biosynthesis genes. It also regulates some genes not associated with secondary metabolite clusters. Similar results were confirmed in gene expression profiling in *A. flavus* using microarrays to study the genetic mechanism of sclerotia formation (Yu, personal communication). The exact mechanism of how LaeA regulates secondary metabolism gene clusters is not yet known. Interestingly, when an unrelated gene such as *argB* was placed within the boundary of the ST gene cluster, it was co-regulated with other genes in the cluster. But, when a gene in the cluster, such as *aflR* was placed elsewhere in the genome, its regulation was not affected by LaeA (Bok et al., 2006). One proposed regulatory mechanism is that LaeA differentially methylates histone protein and it alters the chromatin structure for gene expression. Unlike the mentioned signaling factors, the primary role of LaeA is to regulate metabolic gene clusters, not sporulation, because *laeA*-deleted strains produced wild-type levels of conidia (Bok & Keller, 2004). Most recent analyses of nonaflatoxigenic *A. parasiticus sec-* (for secondary metabolism negative) variants generated through serial transfer of mycelia of the *sec+* parents show that *laeA* was expressed in both *sec+* and *sec-* strains (Kale et al., 2007). This result suggests that LaeA only exerts its effect on aflatoxin biosynthesis at a certain level and is independent of other regulatory pathways that are involved in fungal development.

**4.2 Regulation by** *aflS* **(***aflJ***)**

technology.

**4.3 Regulation by** *laeA*

AFG1 (Cai et al., 2008). The *aflE* (*norA*) gene was initially believed to be involved in the conversion of NOR due to certain degree of sequence similarity to the *aflD* (*nor-1*) gene (Cary et al., 1996). However, recent studies support the hypothesis that the *aflE* (*norA*) is involved in the final two steps in AFB1 formation (Ehrlich, 2009). In the same report, the transcript, *hypB*, a homolog of *hypC*, may be involved in one of the oxidation steps in the conversion of OMST to aflatoxins. *A. flavus* produces only AFB1 and AFB2, whereas *A. parasiticus* produces all four major aflatoxins, AFB1, AFB2, AFG1, and AFG2. Coincidentally, only the G-group aflatoxin producer, *A. parasiticus*, has intact *nadA* and *norB* genes. Preliminary data suggests that *norB* encodes another enzyme predominantly involved in AFG1/AFG2 formation (Ehrlich et al., 2008).

#### **4. Regulation of aflatoxin biosynthesis**

The aflatoxin pathway genes are found to be clustered in the genome of *A. flavus* and *A. parasiticus* (Yu et al., 1995a; Woloshuk & Prieto, 1998; Yu et al., 2004a; Yu et al., 2004c). These genes are expressed concurrently except for the regulatory gene *aflR*. In this gene cluster, a positive-acting regulatory gene, *aflR*, is located in the middle of the gene cluster. Adjacent to *aflR* a divergently transcribed gene, *aflS* (*aflJ*), was also found to be involved in the regulation of transcription (Meyers et al., 1998; Chang, 2004). Other physically unrelated genes, such as *laeA* and *veA*, also have been shown to exhibit a "global" regulatory role on aflatoxin biosynthesis (Kato et al., 2003; Bok & Keller, 2004; Calvo et al., 2004; Perrin et al., 2007).

#### **4.1 Regulation by** *aflR*

The *aflR* gene, encoding a 47 kDa sequence-specific zinc-finger DNA-binding protein is required for transcriptional activation of most, if not all, the structural genes of the aflatoxin gene cluster (Chang et al., 1993; Payne et al., 1993; Woloshuk et al., 1994; Chang et al., 1995b; Yu et al., 1996a; Yu et al., 1996b; Flaherty & Payne, 1997; Ehrlich et al., 1998; Chang et al., 1999a; Chang et al., 1999b). Like other Gal4-type regulatory proteins that bind to palindromic sequences, functional AflR probably binds as a dimer. It binds to the palindromic sequence 5'-TCGN5CGR-3' in the promoter regions of the structural genes (Ehrlich et al., 1999a; Ehrlich et al., 1999b). The AflR-binding motifs are found to be located from –80 to –600 bp, with the majority at the –100 to –200 bp, relative to the translation start site. AflR binds, in some cases, to a deviated sequence rather than the typical motif such as in the case of *aflG* (*avnA*). When there is more than one binding motif, only one of them is the preferred binding site such as in the case of *aflC* (*pksA*) (Ehrlich et al., 1999a; Ehrlich et al., 1999b). The more upstream motif is found to belong to another gene for turning on the expression of *hypC* (Ehrlich, unpublished observation). Deletion of *aflR* in *A. parasiticus* abolishes the expression of other aflatoxin pathway genes (Cary et al., 2000). Overexpression of *aflR* in *A. flavus* up-regulates aflatoxin pathway gene transcription and aflatoxin accumulation (Flaherty & Payne, 1997) in a fashion similar to that reported for *A. parasiticus* (Chang et al., 1995b). These results demonstrate that AflR is specifically involved in the regulation of aflatoxin biosynthesis. Indeed, all 23 upregulated genes, identified by transcription profiling using DNA microarray assays comparing wild-type and *aflR-*deleted *A. parasiticus* strains, have the consensus AflR binding motif in their promoter regions (Meyers et al., 1998; Price et al., 2006; Wilkinson et al., 2007a; Wilkinson et al., 2007b).

#### **4.2 Regulation by** *aflS* **(***aflJ***)**

50 Aflatoxins – Biochemistry and Molecular Biology

AFG1 (Cai et al., 2008). The *aflE* (*norA*) gene was initially believed to be involved in the conversion of NOR due to certain degree of sequence similarity to the *aflD* (*nor-1*) gene (Cary et al., 1996). However, recent studies support the hypothesis that the *aflE* (*norA*) is involved in the final two steps in AFB1 formation (Ehrlich, 2009). In the same report, the transcript, *hypB*, a homolog of *hypC*, may be involved in one of the oxidation steps in the conversion of OMST to aflatoxins. *A. flavus* produces only AFB1 and AFB2, whereas *A. parasiticus* produces all four major aflatoxins, AFB1, AFB2, AFG1, and AFG2. Coincidentally, only the G-group aflatoxin producer, *A. parasiticus*, has intact *nadA* and *norB* genes. Preliminary data suggests that *norB* encodes another enzyme predominantly

The aflatoxin pathway genes are found to be clustered in the genome of *A. flavus* and *A. parasiticus* (Yu et al., 1995a; Woloshuk & Prieto, 1998; Yu et al., 2004a; Yu et al., 2004c). These genes are expressed concurrently except for the regulatory gene *aflR*. In this gene cluster, a positive-acting regulatory gene, *aflR*, is located in the middle of the gene cluster. Adjacent to *aflR* a divergently transcribed gene, *aflS* (*aflJ*), was also found to be involved in the regulation of transcription (Meyers et al., 1998; Chang, 2004). Other physically unrelated genes, such as *laeA* and *veA*, also have been shown to exhibit a "global" regulatory role on aflatoxin biosynthesis (Kato et al., 2003; Bok & Keller, 2004; Calvo et al.,

The *aflR* gene, encoding a 47 kDa sequence-specific zinc-finger DNA-binding protein is required for transcriptional activation of most, if not all, the structural genes of the aflatoxin gene cluster (Chang et al., 1993; Payne et al., 1993; Woloshuk et al., 1994; Chang et al., 1995b; Yu et al., 1996a; Yu et al., 1996b; Flaherty & Payne, 1997; Ehrlich et al., 1998; Chang et al., 1999a; Chang et al., 1999b). Like other Gal4-type regulatory proteins that bind to palindromic sequences, functional AflR probably binds as a dimer. It binds to the palindromic sequence 5'-TCGN5CGR-3' in the promoter regions of the structural genes (Ehrlich et al., 1999a; Ehrlich et al., 1999b). The AflR-binding motifs are found to be located from –80 to –600 bp, with the majority at the –100 to –200 bp, relative to the translation start site. AflR binds, in some cases, to a deviated sequence rather than the typical motif such as in the case of *aflG* (*avnA*). When there is more than one binding motif, only one of them is the preferred binding site such as in the case of *aflC* (*pksA*) (Ehrlich et al., 1999a; Ehrlich et al., 1999b). The more upstream motif is found to belong to another gene for turning on the expression of *hypC* (Ehrlich, unpublished observation). Deletion of *aflR* in *A. parasiticus* abolishes the expression of other aflatoxin pathway genes (Cary et al., 2000). Overexpression of *aflR* in *A. flavus* up-regulates aflatoxin pathway gene transcription and aflatoxin accumulation (Flaherty & Payne, 1997) in a fashion similar to that reported for *A. parasiticus* (Chang et al., 1995b). These results demonstrate that AflR is specifically involved in the regulation of aflatoxin biosynthesis. Indeed, all 23 upregulated genes, identified by transcription profiling using DNA microarray assays comparing wild-type and *aflR-*deleted *A. parasiticus* strains, have the consensus AflR binding motif in their promoter regions (Meyers et al., 1998; Price et al., 2006; Wilkinson et al., 2007a;

involved in AFG1/AFG2 formation (Ehrlich et al., 2008).

**4. Regulation of aflatoxin biosynthesis**

2004; Perrin et al., 2007).

**4.1 Regulation by** *aflR*

Wilkinson et al., 2007b).

The *aflS* (*aflJ*) gene, although not demonstrating significant homology with any other encoded proteins found in databases, is necessary for aflatoxin formation. In the *A. parasiticus aflR* transformants, the production of aflatoxin pathway intermediates was significantly enhanced in transformants that contained an additional *aflR* plus *aflS* (Chang et al., 1995b). Quantitative PCR showed that in the *aflS* knockout mutants, the lack of *aflS* transcript is associated with 5- to 20-fold reduction of expression of some aflatoxin pathway genes such as *aflC* (*pksA*), *aflD* (*nor-1*), *aflM* (*ver-1*), and *aflP* (*omtA*). The mutants lost the ability to synthesize aflatoxin intermediates and no aflatoxins were produced (Meyers et al., 1998). However, deletion of *aflS* (*aflJ*) did not have a discernible effect on *aflR* transcription, and vice versa. Du et al. (Du et al., 2007) showed that overexpression of *A. flavus aflS* (*aflJ*) did not result in elevated transcription of *aflM* (*ver-1*), *aflP* (*omtA*), or *aflR*, but it appears to have some effect on *aflC* (*pksA*), *aflD* (*nor-1*), *aflA* (*fas*-*1*), and *aflB* (*fas-2*) (Du et al., 2007), which are required for the biosynthesis of the early aflatoxin pathway intermediate, averantin. The mechanism(s) by which *aflS* modulates transcription of these pathway genes in concert with *aflR* is under investigation by gene profiling analysis using microarray technology.

#### **4.3 Regulation by** *laeA*

The novel global regulatory gene, *laeA* (for **l**ack of *aflR* **e**xpression), was first identified from *A. nidulans* (Bok & Keller, 2004). This gene is well conserved in fungi as shown by its presence in the genomes of all fungi so far sequenced. LaeA is a nuclear protein which contains an S-adenosylmethionine (SAM) binding motif and activates transcription of several other secondary metabolism gene clusters in addition to the AF cluster*.* Examples include the sterigmatocystin and penicillin clusters in *A. nidulans*, the gliotoxin cluster in *A. fumigatus*, and aflatoxin cluster in *A. flavus* (Bok & Keller, 2004; Bouhired et al., 2007). It also regulates genes required for virulence of *A. fumigatus* (Sugui et al., 2007). Perrin et al. (Perrin et al., 2007) carried out a whole-genome comparison of the transcriptional profiles of wildtype and *laeA*-deleted *A. fumigatus* strains and found that LaeA positively controls the expression of 20% to 40% of major classes of secondary metabolite biosynthesis genes. It also regulates some genes not associated with secondary metabolite clusters. Similar results were confirmed in gene expression profiling in *A. flavus* using microarrays to study the genetic mechanism of sclerotia formation (Yu, personal communication). The exact mechanism of how LaeA regulates secondary metabolism gene clusters is not yet known. Interestingly, when an unrelated gene such as *argB* was placed within the boundary of the ST gene cluster, it was co-regulated with other genes in the cluster. But, when a gene in the cluster, such as *aflR* was placed elsewhere in the genome, its regulation was not affected by LaeA (Bok et al., 2006). One proposed regulatory mechanism is that LaeA differentially methylates histone protein and it alters the chromatin structure for gene expression. Unlike the mentioned signaling factors, the primary role of LaeA is to regulate metabolic gene clusters, not sporulation, because *laeA*-deleted strains produced wild-type levels of conidia (Bok & Keller, 2004). Most recent analyses of nonaflatoxigenic *A. parasiticus sec-* (for secondary metabolism negative) variants generated through serial transfer of mycelia of the *sec+* parents show that *laeA* was expressed in both *sec+* and *sec-* strains (Kale et al., 2007). This result suggests that LaeA only exerts its effect on aflatoxin biosynthesis at a certain level and is independent of other regulatory pathways that are involved in fungal development.

Aflatoxin Biosynthetic Pathway and Pathway Genes 53

and the regulatory mechanisms of aflatoxin formation. Better understanding of the mechanisms of aflatoxin biosynthesis helps to identify natural inhibitors of fungal growth aflatoxin formation, and eventually will allow design of effective strategies to can reduce or

Allcroft, R., Carnaghan, R.B.A., Sargeant, K. & O'Kelly, J. (1961). A toxic factor in Brazilian

Arsura, M. & Cavin, L.G. (2005). Nuclear factor-kappaB and liver carcinogenesis, *Cancer Lett.*

Azziz-Baumgartner, E., Lindblade, K., Gieseker, K., Rogers, H.S., Kieszak, S., Njapau, H.,

Baertschi, S.W., Raney, K.D., Shimada, T., Harris, T.M. & Guengerich, F.P. (1989).

Bennett, J.W. (1970).  Aflatoxins and anthraquinones from diploids of *Aspergillus parasiticus*,

Bennett, J.W. (1981). Loss of norsolorinic acid and aflatoxin production by a mutant of

Bennett, J.W. (1987). Mycotoxins, mycotoxicoses, mycotoxicology and mycopathologia,

Bennett, J.W., Bhatnagar, D. & Chang, P.K. (1994). The molecular genetics of aflatoxin

Bennett, J.W., Chang, P.K. & Bhatnagar, D. (1997). One gene to whole pathway: the role of norsolorinic acid in aflatoxin research, *Adv. Appl. Microbiol.* 45, 1-15. Bennett, J.W. & Goldblatt, L.A. (1973). The isolation of mutants of *Aspergillus flavus* and *A. parasiticus* with altered aflatoxin producing ability, *Sabouraudia* 11, 235-241.

Bennett, J.W., Kronberg, F. & Gougis, G. (1976a). Pigmented isolates from anthraquinone-

characterization by dry column chromatography, *Mycologia* 75, 202-208. Bennett, J.W. & Lee, L.S. (1979). Mycotoxins - their biosynthesis in fungi: aflatoxins and

Bennett, J.W., Lee, L.S. & Cucullu, A.F. (1976b). Effect of dichlorvos on aflatoxin and versicolorin A production in *Aspergillus parasiticus*, *Bot. Gaz.* 137, 318-324. Bennett, J.W., Lee, L.S., Shoss, S.M. & Boudreaux, G.H. (1980). Identification of averantin as

producing mutants of *Aspergillus parasiticus*, *American Society for Microbiology* 76, 6. Bennett, J.W., Kronberg, F.G., Goodman, L.A. & Seltman, M.A. (1983). Isolation of an

anthraquinoe-accumulating mutant of *Aspergillus parasiticus* and partial

an aflatoxin B1 precursor: placement in the biosynthetic pathway, *Appl. Environ.*

inducing different genetic responses, *Chem. Res. Toxicol.* 2, 114-122.

*Investigative Group. Environ. Health Perspect.* 113, 1779-1783.

*Aspergillus parasiticus*, *J. Gen. Microbiol.* 124, 429- 432.

Bennett, J.W. & Klich, M. (2003). Mycotoxins, *Clin. Microbiol. Rev.* 16, 497-516.

other bisfuranoids, *J Food Protection* 42, 805-809.

Schleicher, R., McCoy, L.F., Misore, A., DeCock, K., Rubin, C. & Slutsker, L. (2005). Case-control study of an acute aflatoxicosis outbreak, Kenya, 2004, *Aflatoxin*

Comparison rates of enzymatic oxidation of aflatoxin B1, aflatoxin G1, and sterigmatocystin and activities of the epoxides in forming guanyl-N adducts and

eliminate aflatoxin contamination of food and feed commodities.

groundnut meal, *Vet. Rec.* 73, 428-429.

*Microscop Offic Proc* 118-131.

*J. Gen. Microbiol.* 113, 127-136.

biosynthesis, *FEMS Symp.* 51-58.

*Mycopathologia* 100, 3-5.

*Microbiol.* 39, 835-839.

**6. References**

229, 157-169.

#### **4.4 Regulation by** *veA*

The *veA* gene in *A. nidulans* (Mooney & Yager, 1990) is a gene initially found to be crucial for light-dependent conidiation. The light dependence is abolished by a mutation (*veA1*) which allows conidiation of *A. nidulans* to occur in the dark. A comparison of the light effect on sterigmatocystin production by *A. nidulans veA+* and *veA1* strains showed that both strains produced sterigmatocystin but the highest amount was produced by the *veA*+ strain grown in darkness. However, *veA*-deleted *A. flavus* and *A. parasiticus* strains completely lost the ability to produce aflatoxin regardless of the illumination conditions (Duran et al., 2007; Stinnett et al., 2007). Under normal growth conditions, some *A. flavus* and all *A. parasiticus* strains produce conidia in both dark and light conditions. Stinnett et al. (Stinnett et al., 2007) showed that VeA contains a bipartite nuclear localization signal (NLS) motif and its migration to the nucleus is light-dependent and requires the importin α carrier protein. In the dark VeA is located mainly in the nucleus; under light it is located both in cytoplasm and nucleus. VeA has no recognizable DNA-binding seuqences and likely exerts its effect on sterigmatosyctin and aflatoxin production through proteinprotein interactions with other regulatory factors. Post-translational modifications such as phosphylation and dephosphorylation may modulate its activity. Lack of VeA production in the *veA*-deleted *A. flavus* and *A. parasiticus* strains consequently abolishes aflatoxin production because a threshold concentration of nuclear VeA might be necessary to initiate aflatoxin biosynthesis.

Fig. 1. Aflatoxin pathway gene cluster in *A. flavus.* This figure shows the order and location of the 30 aflatoxin pathway genes plus an *aflR* antisense gene clustered together in about 80 kb DNA region. The old gene names are labeled on top of the line and the new gene names sysmatically renamed according to gene convention are labeled below the line (Yu et al., 2004c). The transcripts of *hypA*, *hypB*, *hypC*, *hypD*, *hypE* and *aflRas* are identified through *Aspergillus flavus* EST. Arrows indicate the direction of gene transcription.

### **5. Conclusions**

Aflatoxins are toxic and carcinogenic secondary metabolites produced mainly by *Aspergillus flavus* and *Aspergillus parasiticus* that contaminate preharvest crops and post harvest grains. Scientists worldwide have extensively studied biosynthesis of aflatoxins for more than 50 years. Aflatoxin biosynthesis is a complex process involving many intermediates and enzymes. Regulation of aflatoxin gene expression occurs at multiple levels and by multiple regulatory components. There are genetic factors, biotic and abiotic elements that affect aflatoxin formation. Recent studies have shed more light on the functions of the enzymes involved in each of the steps of aflatoxin biosynthesis, the genes encoding those enzymes, and the regulatory mechanisms of aflatoxin formation. Better understanding of the mechanisms of aflatoxin biosynthesis helps to identify natural inhibitors of fungal growth aflatoxin formation, and eventually will allow design of effective strategies to can reduce or eliminate aflatoxin contamination of food and feed commodities.

#### **6. References**

52 Aflatoxins – Biochemistry and Molecular Biology

The *veA* gene in *A. nidulans* (Mooney & Yager, 1990) is a gene initially found to be crucial for light-dependent conidiation. The light dependence is abolished by a mutation (*veA1*) which allows conidiation of *A. nidulans* to occur in the dark. A comparison of the light effect on sterigmatocystin production by *A. nidulans veA+* and *veA1* strains showed that both strains produced sterigmatocystin but the highest amount was produced by the *veA*+ strain grown in darkness. However, *veA*-deleted *A. flavus* and *A. parasiticus* strains completely lost the ability to produce aflatoxin regardless of the illumination conditions (Duran et al., 2007; Stinnett et al., 2007). Under normal growth conditions, some *A. flavus* and all *A. parasiticus* strains produce conidia in both dark and light conditions. Stinnett et al. (Stinnett et al., 2007) showed that VeA contains a bipartite nuclear localization signal (NLS) motif and its migration to the nucleus is light-dependent and requires the importin α carrier protein. In the dark VeA is located mainly in the nucleus; under light it is located both in cytoplasm and nucleus. VeA has no recognizable DNA-binding seuqences and likely exerts its effect on sterigmatosyctin and aflatoxin production through proteinprotein interactions with other regulatory factors. Post-translational modifications such as phosphylation and dephosphorylation may modulate its activity. Lack of VeA production in the *veA*-deleted *A. flavus* and *A. parasiticus* strains consequently abolishes aflatoxin production because a threshold concentration of nuclear VeA might be necessary to

Fig. 1. Aflatoxin pathway gene cluster in *A. flavus.* This figure shows the order and location of the 30 aflatoxin pathway genes plus an *aflR* antisense gene clustered together in about 80 kb DNA region. The old gene names are labeled on top of the line and the new gene names sysmatically renamed according to gene convention are labeled below the line (Yu et al., 2004c). The transcripts of *hypA*, *hypB*, *hypC*, *hypD*, *hypE* and *aflRas* are identified through *Aspergillus flavus* EST. Arrows indicate the direction of gene transcription.

Aflatoxins are toxic and carcinogenic secondary metabolites produced mainly by *Aspergillus flavus* and *Aspergillus parasiticus* that contaminate preharvest crops and post harvest grains. Scientists worldwide have extensively studied biosynthesis of aflatoxins for more than 50 years. Aflatoxin biosynthesis is a complex process involving many intermediates and enzymes. Regulation of aflatoxin gene expression occurs at multiple levels and by multiple regulatory components. There are genetic factors, biotic and abiotic elements that affect aflatoxin formation. Recent studies have shed more light on the functions of the enzymes involved in each of the steps of aflatoxin biosynthesis, the genes encoding those enzymes,

**4.4 Regulation by** *veA*

initiate aflatoxin biosynthesis.

**5. Conclusions**

Aflatoxin Biosynthetic Pathway and Pathway Genes 55

Calvo, A.M., Bok, J.-W., Brooks, W. & Keller, N.P. (2004). VeA is required for toxin and

Cary, J.W., Ehrlich, K.C., Wright, M., Chang, P.K. & Bhatnagar, D. (2000). Generation of aflR

Cary, J.W., Wright, M., Bhatnagar, D., Lee, R. & Chu, F.S. (1996). Molecular characterization

Centers for Disease Control and Prevention (2004). Outbreak of aflatoxin poisoning - eastern and central province, Kenya, *MMWR Morb Mortal Weekly Report* 53, 790-792. Chang, P.K. (2004). Lack of interaction between AFLR and AFLJ contributes to

Chang, P.K., Bennett, J.W. & Cotty, P.J. (2002). Association of aflatoxin biosynthesis and sclerotial development in *Aspergillus parasiticus*, *Mycopathologia* 153, 41-48. Chang, P.K., Cary, J.W., Bhatnagar, D., Cleveland, T.E., Bennett, J.W., Linz, J.E., Woloshuk,

Chang, P.K., Cary, J.W., Yu, J., Bhatnagar, D. & Cleveland, T.E. (1995a). The *Aspergillus*

Chang, P.K., Ehrlich, K.C., Yu, J., Bhatnagar, D. & Cleveland, T.E. (1995b). Increased

Chang, P.K., Horn, B.W. & Dorner, J.W. (2005). Sequence breakpoints in the aflatoxin

Chang, P.K., Skory, C.D. & Linz, J.E. (1992). Cloning of a gene associated with aflatoxin B1

Chang, P.K., Yabe, K. & Yu, J. (2004). The *Aspergillus parasiticus estA*- encoded esterase

versiconol in aflatoxin biosynthesis, *Appl. Environ. Microbiol.* 70, 3593-3599. Chang, P.K., Yu, J., Bhatnagar, D. & Cleveland, T.E. (1999a). The carboxy-terminal portion of

Chang, P.K., Yu, J., Bhatnagar, D. & Cleveland, T.E. (1999b). Repressor- AFLR interaction

Chang, P.K., Yu, J., Ehrlich, K.C., Boue, S.M., Montalbano, B.G., Bhatnagar, D. & Cleveland,

hydroxyaverantin to averufin, *Appl. Environ. Microbiol.* 66, 4715-4719.

biosynthesis in *Aspergillus parasiticus*, *Curr. Genet.* 21, 231-233.

required for aflatoxin B1 biosynthesis, *Mol. Gen. Genet.* 248, 270-277.

biosynthesis gene cluster, *Appl. Environ. Microbiol.* 62, 360-366.

nonaflatoxigenicity of *Aspergillus sojae*, *J. Biotechnol.* 107, 245-253.

4739.

3273-3279.

2508-2512.

112.

*Microbiol.* 61, 2372-2377.

isolates, *Fungal Genet. Biol.* 42, 914-923.

sclerotial production in *Aspergillus parasiticus*, *Appl. Environ. Microbiol.* 70, 4733-

disruption mutants of *Aspergillus parasiticus*, *Appl. Microbiol. Biotechnol.* 53, 680-684.

of an *Aspergillus parasiticus* dehydrogenase gene, *norA*, located on the aflatoxin

C.P. & Payne, G.A. (1993). Cloning of the *Aspergillus parasiticus apa-2* gene associated with the regulation of aflatoxin biosynthesis, *Appl. Environ. Microbiol.* 59,

*parasiticus* polyketide synthase gene *pksA*, a homolog of *Aspergillus nidulans wA*, is

expression of *Aspergillus parasiticus aflR*, encoding a sequence-specific DNAbinding protein, relieves nitrate inhibition of aflatoxin biosynthesis, *Appl. Environ.*

biosynthesis gene cluster and flanking regions in nonaflatoxigenic *Aspergillus flavus*

converts versiconal hemiacetal acetate to versiconal and versiconol acetate to

the aflatoxin pathway regulatory protein AFLR of *Aspergillus parasiticus* activates *GAL1::lacZ* gene expression in *Saccharomyces cerevisiae*, *Appl. Environ. Microbiol.* 65,

modulates aflatoxin biosynthesis in *Aspergillus parasiticus*, *Mycopathologia* 147, 105-

T.E. (2000). *adhA* in *Aspergillus parasiticus* is involved in conversion of 5'-

Bennett, J.W., Lee, L.S. & Vinnett, C.H. (1971). The correlation of aflatoxin and norsolorinic

Bennett, J.W., Leong, P.M., Kruger, S.J. & Keyes, D. (1986). Sclerotial and low aflatoxigenic

Bennett, J.W. & Papa, K.E. (1988). The aflatoxigenic *Aspergillus* spp., *Adv. Plant Pathol.* 6, 265-

Bennett, J.W., Silverstein, R.B. & Kruger, S.J. (1981). Isolation and characterization of two

Bhatnagar, D., Brown, R., Ehrlich, K. & Cleveland, T.E. (2002). Mycotoxins Contaminating

Bhatnagar, D. & Cleveland, T.E. (1991). Aflatoxin biosynthesis: developments in chemistry,

Bhatnagar, D., Cleveland, T.E. & Lillehoj, E.B. (1989). Enzymes in aflatoxin B1 biosynthesis:

Bhatnagar, D., Ehrlich, K.C. & Cleveland, T.E. (1992). Oxidation- reduction reactions in

Bok, J.-W. & Keller, N.P. (2004). LaeA, a regulator of secondary metabolism in *Aspergillus*

Bok, J.W., Noordermeer, D., Kale, S.P. & Keller, N.P. (2006). Secondary metabolic gene cluster silencing in *Aspergillus nidulans*, *Mol. Microbiol.* 61, 1636-1645. Bouhired, S., Weber, M., Kempf-Sontag, A., Keller, N.P. & Hoffmeister, D. (2007). Accurate

Bressac, B., Kew, M., Wands, J. & Ozturk, M. (1991). Selective G to T mutations of p53 gene in hepatocellular carcinoma from southern Africa, *Nature* 350, 429-431. Brown, D.W., Adams, T.H. & Keller, N.P. (1996a). *Aspergillus* has distinct fatty acid

Brown, D.W., Yu, J.H., Kelkar, H.S., Fernandes, M., Nesbitt, T.C., Keller, N.P., Adams, T.H.

Busby, W.F., Jr. & Wogan, G.N. (1981). Aflatoxins. In: Shank, R.C. (Ed.), *Mycotoxins and Nnitrosocompounds: Environmental Risks*. CRC Press, Boca Raton, pp. 3-45. Cai, J., Zeng, H., Shima, Y., Hatabayashi, H., Nakagawa, H., Ito, Y., Adachi, Y., Nakajima, H.

in *Aspergillus parasiticus*, *Fungal Genet Biol* 45, 1081-1093.

strategies for identifying pertinent genes, *Mycopathologia* 107, 75-83.

the transcriptional regulator LaeA, *Fungal Genet. Biol.* 44, 1134-1145.

morphological variants from haploid and diploid *Aspergillus parasiticus Experientia*

nonaflatoxigenic classes of morphological variants of *Aspergillus parasiticus*, *J.*

Cereal Grain Crops: Their Occurrence and Toxicity. In: Khachatourians, G.G. & Arora, D.K. (Eds.), *Applied Mycology and Biotechnology*. Elsevier B.V., New York, pp.

biochemistry, and genetics, *Res. Bull. Iowa State Univ. Agric. Home Econ. Exp. Stn.*

biosynthesis of secondary metabolites. In: Bhatnagar, D., Lillehoj, E.B. & Arora, D.K. (Eds.), *Mycotoxins in Ecological Systems*. Marcel Dekker, New York, pp. 255-285.

prediction of the *Aspergillus nidulans* terrequinone gene cluster boundaries using

synthases for primary and secondary metabolism, *Proc. Natl. Acad. Sci. U. S. A.* 93,

& Leonard, T.J. (1996b). Twenty-five coregulated transcripts define a sterigmatocystin gene cluster in *Aspergillus nidulans*, *Proc. Natl. Acad. Sci. U. S. A.*

& Yabe, K. (2008). Involvement of the *nadA* gene in formation of G-group aflatoxins

acid production, *J. American Oil Chemists Society* 48, 368-370.

42, 848-851.

*American Oil Chemists Society* 952.

spp., *Eukaryot. Cell* 3, 527-535.

279.

171-196.

391-405.

14873-14877.

93, 1418-1422.

Aflatoxin Biosynthetic Pathway and Pathway Genes 57

Denning, D.W., Riniotis, K., Dobrashian, R. & Sambatakou, H. (2003). Chronic cavitary and

Detroy, R.W., Freer, S. & Ciegler, A. (1973). Aflatoxin and anthraquinone biosynthesis by

Du, W., Obrian, G.R. & Payne, G.A. (2007). Function and regulation of *aflJ* in the

Duran, R.M., Cary, J.W. & Calvo, A.M. (2007). Production of cyclopiazonic acid, aflatrem,

Dutton, M.F. (1988). Enzymes and aflatoxin biosynthesis, *Microbiol. Rev.* 52, 274-295. Eaton,

Eaton, D.L. & Groopman, J.D. The toxicology of aflatoxins: human health, veterinary, and

Ehrlich, K.C., Cary, J.W. & Montalbano, B.G. (1999a). Characterization of the promoter for

Ehrlich, K.C., Chang, P.-K., Scharfenstein, J.S.L., Cary, J.W., Crawford, J.M. & Townsend,

Ehrlich, K.C., Montalbano, B.G. & Cary, J.W. (1999b). Binding of the C6- zinc cluster protein,

Ehrlich, K.C., Scharfenstein, J.S.L., Montalbano, B.G. & Chang, P.-K. (2008). Are the genes

Ehrlich, K.C. & Yu, J. (2009). Aflatoxin-like gene clusters and how they evolved. In: Varma,

Fitzell, D.L., Singh, R., Hsieh, D.P. & Motell, E.L. (1977). Nuclear magnetic resonance

FAO (1995), Worldwide regulations for mycotoxins 1995, A Compendium. Italy, Rome. Fitzell, D.L., Hsieh, D.P., Yao, R.C. & La Mar, G.N. (1975). Biosynthesis of averufin from

acetate by A*spergillus parasiticus*, *J. Agric. Food Chem.* 23, 442-444.

sclerotial formation, *Appl. Microbiol. Biotechnol.* 73, 1158-1168.

*parasiticus* transformants, *Fungal Genet. Biol.* 23, 279-287.

Heidelberg, Dordrecht, London, New York, pp. 65-76.

biosynthesis, *J. Agric. Food. Chem.* 25, 1193-1197.

and review, *Clin. Infect. Dis.* 37, S265-280.

*Addit. Contam.* 24, 1043-1050.

*Pharmacol. Toxicol.* 34, 135-172.

*Biochim. Biophys. Acta.* 1444, 412-417.

*parasiticus*, *Gene* 230, 249-257.

*Molecular Sciences* 9, 1717- 1729.

1373-1378.

1, 37-58.

fibrosing pulmonary and pleural aspergillosis: Case series, proposed nomenclature

nitrosoguanidine-derived mutants of *Aspergillus parasiticus*, *Can. J. Microbiol.* 19,

accumulation of aflatoxin early pathway intermediate in *Aspergillus flavus*, *J. Food*

and aflatoxin by *Aspergillus flavus* is regulated by *veA*, a gene necessary for

D. & Gallagher, E. (1994). Mechanisms of aflatoxin carcinogenesis, *Annu. Rev.*

agricultural significance. Academic Press, San Diego, CA, 1994. Ehrlich, K.C. (2009). Predicted roles of uncharacterized clustered genes in aflatoxin biosynthesis, *Toxins*

the gene encoding the aflatoxin biosynthetic pathway regulatory protein AFLR,

C.A. (2010). Absence of the aflatoxin biosynthesis gene, *norA*, allows accumulation of deoxyaflatoxin B1 in *Aspergillus flavus* cultures, *FEMS Microbiol. Lett.* 305, 65-70. Ehrlich, K.C., Chang, P.K., Yu, J. & Cotty, P.J. (2004). Aflatoxin biosynthesis cluster gene *cypA* is required for G aflatoxin formation, *Appl. Environ. Microbiol.* 70, 6518-6524. Ehrlich, K.C., Montalbano, B.G., Bhatnagar, D. & Cleveland, T.E. (1998). Alteration of

different domains in AFLR affects aflatoxin pathway metabolism in *Aspergillus*

AFLR, to the promoters of aflatoxin pathway biosynthesis genes in *Aspergillus*

nadA and norB involved in formation of aflatoxin G1, *International Journal of*

A.K. & Rai, M.K. (Eds.), *Mycotoxins in Food, Feed, and Bioweapons*. Springer Verlag,

identification of versiconal hemiacetal acetate as an intermediate in aflatoxin

Chen, C.J., Wang, L.Y., Lu, S.N., Wu, M.H., You, S.L., Zhang, Y.J., Wang, L.W. & Santella,

Chen, C.J., Yu, M.W., Liaw, Y.F., Wang, L.W., Chiamprasert, S., Matin, F., Hirvonen, A., Bell,

Cleveland, T.E. & Bhatnagar, D. (1990). Evidence for de novo synthesis of an aflatoxin

Cleveland, T.E., Lax, A.R., Lee, L.S. & Bhatnagar, D. (1987). Appearance of enzyme activities

Council for Agricultural Science and Technology (2003), Mycotoxins: Risk in plant, animal,

Coursaget, P., Depril, N., Chabaud, M., Nandi, R., Mayelo, V., LeCann, P. & Yvonnet, B.

Crawford, J.M., Dancy, B.C., Hill, E.A., Udwary, D.W. & Townsend, C.A. (2006).

Crawford, J.M., Vagstad, A.L., Ehrlich, K.C. & Townsend, C.A. (2008b). Starter unit

Crawford, J.M., Vagstad, A.L., Whitworth, K.P., Ehrlich, K.C. & Townsend, C.A. (2008c).

Denning, D.W., Follansbee, S.E., Scolaro, M., Norris, S., Edelstein, H. & Stevens, D.A. (1991).

Aspergillus parasiticus cultures, *Appl. Environ. Microbiol.* 53, 1711-1713. Cotty, P. (1988). Aflatoxin and sclerotial production by *Aspergillus flavus*: influence of pH,

of sterigmatocystin to aflatoxin B1, *Can. J. Microbiol.* 33, 1108-1112.

carcinoma, *Hepatology* 24, 38- 42.

pp. 205- 228.

*Phytopathol.* 78, 1250-1253.

and human systems, Ames, IA.

function, *Science* 320, 243-246.

*Chem.* 36, 16-22.

*Med.* 324, 654-662.

carcinomas from Senegal, *Br. J. Cancer* 67, 1395-1397.

the classical "Starter-Unit Effect", *Chembiochem.*

Denning, D.W. (1998). Invasive aspergillosis, *Clin. Infect. Dis.* 26, 781-805.

R.M. (1996a). Elevated aflatoxin exposure and increased risk of hepatocellular

D.A. & Santella, R.M. (1996b). Chronic hepatitis B carriers with null genotypes of glutathione S transferase MI and TI polymorphisms who are exposed to aflatoxin are at increased risk of hepatocellular carcinoma, *Am. J. Human Genet.* 59, 128-134. Cleveland, T.E. & Bhatnagar, D. (1987). Individual reaction requirements of two enzyme

activities, isolated from Aspergillus parasiticus, which together catalyze conversion

pathway methyltransferase near the cessation of active growth and the onset of aflatoxin biosynthesis in *Aspergillus parasiticus* mycelia, *Can. J. Microbiol.* 36, 1-5. Cleveland, T.E. & Bhatnagar, D. (1991). Molecular regulation of aflatoxin biosynthesis. In: Bray, G.A. & Ryan, D.H. (Eds.), *Proc Symp Mycotoxins Cancer Health*, pp. 270-287. Cleveland, T.E. & Bhatnagar, D. (1992). Molecular strategies for reducing aflatoxin levels in

crops before harvest. In: Bhatnagar, D. & Cleveland, T.E. (Eds.), *Molecular Approaches to Improving Food Quality and Safety*. Van Nostrand Reinhold, New York,

catalyzing conversion of sterigmatocystin to aflatoxin B1 in late-growth-phase

(1993). High prevalence of mutations at codon 249 of the p53 gene in hyptocellular

Identification of a starter unit acyl-carrier protein transacylase domain in an iterative type I polyketide synthase, *Proc. Natl. Acad. Sci. U. S. A.* 103, 16728-16733. Crawford, J.M., Thomas, P.M., Scheerer, J.R., Vagstad, A.L., Kelleher, N.L. & Townsend,

C.A. (2008a). Deconstruction of iterative multidomain polyketide synthase

specificity directs genome mining of polyketide synthase pathways in fungi, *Bioorg.*

Synthetic strategy of nonreducing Iterative polyketide synthases and the origin of

Pulmonary aspergillosis in the acquired immunodeficiency syndrome, *N. Engl. J.*

Aflatoxin Biosynthetic Pathway and Pathway Genes 59

Jelinek, C.F., Pohland, A.E. & Wood, G.E. (1989). Worldwide occurrence of mycotoxins in

Kale, S.P., Cary, J.W., Hollis, N., Wilkinson, J.R., Bhatnagar, D., Yu, J., Cleveland, T.E. &

Kato, N., Brooks, W. & Calvo, A.M. (2003). The expression of sterigmatocystin and penicillin

Kelkar, H.S., Keller, N.P. & Adams, T.H. (1996). *Aspergillus nidulans stcP* encodes an *O*-

Kelkar, H.S., Skloss, T.W., Haw, J.F., Keller, N.P. & Adams, T.H. (1997). *Aspergillus nidulans*

Keller, N.P., Dischinger, H.C., Jr., Bhatnagar, D., Cleveland, T.E. & Ullah, A.H. (1993).

Keller, N.P., Kantz, N.J. & Adams, T.H. (1994). *Aspergillus nidulans verA* is required for

Keller, N.P., Segnar, S., Bhatnagar, D. & Adams, T.H. (1995). *stcS*, a putative P-450

Kelly, J.D., Eaton, D.L., Guengerich, F.P. & Coulombe, R.A., Jr. (1997). Aflatoxin B1

Klich, M.A. (1998). Soil fungi of some low-altitude desert cotton fields and ability of their

Klich, M.A., Yu, J., Chang, P.K., Mullaney, E.J., Bhatnagar, D. & Cleveland, T.E. (1995).

Kusumoto, K.I. & Hsieh, D.P.H. (1996). Purification and characterization of the esterases

Lancaster, M.D., Jenkins, F.P. & Philip, J.M. (1961). Toxicity associated with certain samples

Lee, L.S., Bennett, J.W., Goldblatt, L.A. & Lundin, R.E. (1971). Norsolorinic acid from a mutant strain of *Aspergillus parasiticus*, *J. American Oil Chemists Society* 48, 93-94.

and non-aflatoxigenic aspergilli, *Appl. Microbiol. Biotechnol.* 44, 439-443. Krishnamachari, K.A., Bhat, R.V., Nagarajan, V. & Tilak, T.B. (1975). Hepatitis due to

by *Aspergillus nidulans*, *Appl. Environ. Microbiol.* 66, 359-362.

activation in human lung, *Toxicol. Appl. Pharmacol.* 144, 88-95.

extracts to inhibit *Aspergillus flavus*, *Mycopathologia* 142, 97-100.

Bennett, J.W. (2007). Analysis of aflatoxin regulatory factors in serial transferinduced non-aflatoxigenic *Aspergillus parasiticus*, *J. Food Addit. Contam.* 24, 1061-

genes in *Aspergillus nidulans* is controlled by *veA*, a gene required for sexual

methyltransferase that is required for sterigmatocystin biosynthesis, *Appl. Environ.*

*stcL* encodes a putative cytochrome P- 450 monooxygenase required for bisfuran desaturation during aflatoxin/sterigmatocystin biosynthesis, *J. Biol. Chem.* 272,

Purification of a 40-kilodalton methyltransferase active in the aflatoxin biosynthetic

production of the mycotoxin sterigmatocystin, *Appl. Environ. Microbiol.* 60, 1444-

monooxygenase, is required for the conversion of versicolorin A to sterigmatocystin in *Aspergillus nidulans*, *Appl. Environ. Microbiol.* 61, 3628-3632. Keller, N.P., Watanabe, C.M., Kelkar, H.S., Adams, T.H. & Townsend, C.A. (2000).

Requirement of monooxygenase-mediated steps for sterigmatocystin biosynthesis

Hybridization of genes involved in aflatoxin biosynthesis to DNA of aflatoxigenic

aflatoxicosis: an outbreak of hepatitis in parts of western India, *Lancet.* 1, 1061-1063.

involved in aflatoxin biosynthesis in *Aspergillus parasiticus*, *Can. J. Microbiol.* 42, 804-

food and feeds--an update, *J. Assoc. Off. Anal. Chem.* 72, 223-230.

development, *Eukaryot. Cell* 2, 1178-1186.

pathway, *Appl. Environ. Microbiol.* 59, 479-484.

of ground nuts, *Nature* 192, 1095-1096.

*Microbiol.* 62, 4296- 4298.

1069.

1589- 1594.

1450.

810.

Flaherty, J.E. & Payne, G.A. (1997). Overexpression of *aflR* leads to upregulation of pathway

Fung, F. & Clark, R.F. (2004). Health effects of mycotoxins: a toxicological overview, *J.*

Gong, Y., Hounsa, A., Egal, S., Turner, P.C., Sutcliffe, A.E., Hall, A.J., Cardwell, K. & Wild,

Groopman, J.D., Wogan, G.N., Roebuck, B.D. & Kensler, T.W. (1994). Molecular biomarkers

Hall, A.J. & Wild, C.P. (1994). Epidemiology of aflatoxin-related disease. In: Eaton, D.L. &

Henry, K.M. & Townsend, C.A. (2005). Ordering the reductive and cytochrome P450

Hitchman, T.S., Schmidt, E.W., Trail, F., Rarick, M.D., Linz, J.E. & Townsend, C.A. (2001).

Hsieh, D.P. (1973). Inhibition of aflatoxin biosynthesis of dichlorvos, *J. Agric. Food Chem.* 21,

Hsieh, D.P., Cullen, J.M., Hsieh, L.S., Shao, Y. & Ruebner, B.H. (1985). Cancer risks posed by

Hsieh, D.P., Lin, M.T. & Yao, R.C. (1973). Conversion of sterigmatocystin to aflatoxin B1 by

Hsieh, D.P., Lin, M.T., Yao, R.C. & Singh, R. (1976). Biosynthesis of aflatoxin. Conversion of

Hsieh, D.P. & Mateles, R.I. (1970). The relative contribution of acetate and glucose to

Hsieh, D.P.H. (1989). Potential human health hazards of mycotoxins. In: Natori, S.,

Hsu, I.C., Metcalf, R.A., Sun, T., Welsh, J.A., Wang, N.J. & Harris, C.C. (1991). Mutational hotspot in the p53 gene in human hepatocellular carcinomas, *Nature* 350, 427-428. Huang, J.H. & Hsieh, D.P. (1988). Comparative study of aflatoxins M1 and B1 production in

Hsieh, D.P., Wan, C.C. & Billington, J.A. (1989). A versiconal hemiacetal acetate converting

norsolorinic acid and other hypothetical intermediates into aflatoxin B1, *J. Agric.*

Hashimoto, H. & Ueno, Y. (Eds.), *Mycotoxins and Phycotoxins*. Elsevier, Amsterdam,

solid-state and shaking liquid cultures, *Proc. Natl. Sci. Counc. Repub. China* 12, 34-42.

*Aspergillus parasiticus*, *Biochem. Biophys. Res. Commun.* 52, 992-997.

aflatoxin biosynthesis, *Biochem. Biophys. Acta.* 208, 482- 486.

enzyme in aflatoxin biosynthesis, *Mycopathologia* 107, 121-126.

*Agricultural Significance*. Academic Press, San Diego, pp. 233-258.

background, control and implications. Acaderitic Press, New York.

*Environ. Microbiol.* 63, 3995-4000.

4038.

191.

3724-3733.

468-470.

pp. 69-80.

biosynthesis, *Bioorg. Chem.* 29, 293-307.

*Food Chem.* 24, 1170-1174.

aflatoxin M1, *Princess Takamatsu Symp.* 16, 57-65.

gene expression and increased aflatoxin production in *Aspergillus flavus*, *Appl.*

*Toxicol. Clin. Toxicol.* 42, 217-234. Goldblatt, L.A. (1969). Aflatoxin-scientific

C.P. (2004). Postweaning exposure to aflatoxin results in impaired child growth: a longitudinal study in Benin, West Africa, *Environ Health Perspect* 112, 1134-1138. Goto, T., Wicklow, D.T. & Ito, Y. (1996). Aflatoxin and cyclopiazonic acid production by a

sclerotium-producing *Aspergillus tamarii* strain, *Appl. Environ. Microbiol.* 62, 4036-

for aflatoxins and their application to human cancer prevention, *Cancer Res.* 54, 190-

Groopman, J.D. (Eds.), *The Toxicology of Aflatoxins: Human Health, Veterinary, and*

oxidative steps in demethylsterigmatocystin formation yields general insights into the biosynthesis of aflatoxin and related fungal metabolites, *J. Am. Chem. Soc.* 127,

Hexanoate synthase, a specialized type I fatty acid synthase in aflatoxin B1

Aflatoxin Biosynthetic Pathway and Pathway Genes 61

Ozturk, M. (1991). p53 mutation in hepatocellular carcinoma after aflatoxin exposure, *Lancet.*

Papa, G. (1982). Norsolorinic acid mutant of *Aspergillus flavus*, *J. Gen. Microbiol.* 128, 1345-

Papa, K.E. (1979). Genetics of *Aspergillus flavus*: complementation and mapping of aflatoxin

Papa, K.E. (1984). Genetics of *Aspergillus flavus*: linkage of aflatoxin mutants, *Can. J.*

Payne, G.A. (1998). Process of contamination by aflatoxin-producing fungi and their impacts

Payne, G.A., Nystrom, G.J., Bhatnagar, D., Cleveland, T.E. & Woloshuk, C.P. (1993). Cloning

Peers, F., Bosch, X., Kaldor, J., Linsell, A. & Pluijmen, M. (1987). Aflatoxin exposure, hepatitis B virus infection and liver cancer in Swaziland, *Int. J .Cancer* 39, 545-553. Perrin, R.M., Fedorova, N.D., Bok, J.W., Cramer, R.A., Wortman, J.R., Kim, H.S., Nierman,

Polychronaki, N., West, R.M., Turner, P.C., Amra, H., Abdel-Wahhab, M., Mykkänen, H. &

Price, M.S., Yu, J., Nierman, W.C., Kim, H.S., Pritchard, B., Jacobus, C.A., Bhatnagar, D.,

milk of selected Egyptian mothers, *Food Chem Toxicol* 45, 1210-1215.

on crops. In: Sinha, K.K. & Bhatnagar, D. (Eds.), *Mycotoxins in Agriculture and Food*

of the *afl-2* gene involved in aflatoxin biosynthesis from *Aspergillus flavus*, *Appl.*

W.C. & Keller, N.P. (2007). Transcriptional regulation of chemical diversity in

El-Nezami, H. (2007). A longitudinal assessment of aflatoxin M1 excretion in breast

Cleveland, T.E. & Payne, G.A. (2006). The aflatoxin pathway regulator AflR induces gene transcription inside and outside of the aflatoxin biosynthetic cluster, *FEMS*

Papa, K.E. (1976). Linkage groups in *Aspergillus flavus*, *Mycologia* 68, 159-165.

*Safety*. Marcel Dekker, New York, pp. 279-306.

*Aspergillus fumigatus* by LaeA, *PLoS Pathog.* 3, e50.

*Nature* 438, 1151-1156.

mutants, *Genet. Res.* 34, 1-9.

*Environ. Microbiol.* 59, 156-162.

*Microbiol. Lett.* 255, 275-279.

*Microbiol.* 30, 68-73.

338, 1356-1359.

1348.

Coulsen, R., Davies, R., Dyer, P.S., Farman, M., Fedorova, N., Fedorova, N., Feldblyum, T.V., Fischer, R., Fosker, N., Fraser, A., Garcia, J.L., Garcia, M.J., Goble, A., Goldman, G.H., Gomi, K., Griffith-Jones, S., Gwilliam, R., Haas, B., Haas, H., Harris, D., Horiuchi, H., Huang, J., Humphray, S., Jimenez, J., Keller, N., Khouri, H., Kitamoto, K., Kobayashi, T., Konzack, S., Kulkarni, R., Kumagai, T., Lafon, A., Latge, J.P., Li, W., Lord, A., Lu, C., Majoros, W.H., May, G.S., Miller, B.L., Mohamoud, Y., Molina, M., Monod, M., Mouyna, I., Mulligan, S., Murphy, L., O'Neil, S., Paulsen, I., Penalva, M.A., Pertea, M., Price, C., Pritchard, B.L., Quail, M.A., Rabbinowitsch, E., Rawlins, N., Rajandream, M.A., Reichard, U., Renauld, H., Robson, G.D., Rodriguez de Cordoba, S., Rodriguez-Pena, J.M., Ronning, C.M., Rutter, S., Salzberg, S.L., Sanchez, M., Sanchez-Ferrero, J.C., Saunders, D., Seeger, K., Squares, R., Squares, S., Takeuchi, M., Tekaia, F., Turner, G., Vazquez de Aldana, C.R., Weidman, J., White, O., Woodward, J., Yu, J.H., Fraser, C., Galagan, J.E., Asai, K., Machida, M., Hall, N., Barrell, B. & Denning, D.W. (2005). Genomic sequence of the pathogenic and allergenic filamentous fungus *Aspergillus fumigatus*,

Lewis, L., Onsongo, M., Njapau, H., Schurz-Rogers, H., Luber, G., Kieszak, S., Nyamongo, J.,

Lin, M.T. & Hsieh, D.P. (1973). Averufin in the biosynthesis of aflatoxin B, *J. Am Chem. Soc.*

Lin, M.T., Hsieh, D.P., Yao, R.C. & Donkersloot, J.A. (1973). Conversion of averufin into

Mahanti, N., Bhatnagar, D., Cary, J.W., Joubran, J. & Linz, J.E. (1996). Structure and function

McCormick, S.P., Bhatnagar, D. & Lee, L.S. (1987). Averufanin is an aflatoxin B1 precursor

McGlynn, K.A., Hunter, K., LeVoyer, T., Roush, J., Wise, P., Michielli, R.A., Shen, F.M.,

McGuire, S.M., Silva, J.C., Casillas, E.G. & Townsend, C.A. (1996). Purification and

Meyers, D.M., Obrian, G., Du, W.L., Bhatnagar, D. & Payne, G.A. (1998). Characterization of

Minto, R.E. & Townsend, C.A. (1997). Enzymology and molecular biology of aflatoxin

Mooney, J.L. & Yager, L.N. (1990). Light is required for conidiation in *Aspergillus nidulans*,

Mori, T., Matsumura, M., Yamada, K., Irie, S., Oshimi, K., Suda, K., Oguri, T. & Ichinoe, M.

Motomura, M., Chihaya, N., Shinozawa, T., Hamasaki, T. & Yabe, K. (1999). Cloning and

Ngindu, A., Johnson, B.K., Kenya, P.R., Ngira, J.A., Ocheng, D.M., Nandwa, H., Omondi,

Nierman, W.C., Pain, A., Anderson, M.J., Wortman, J.R., Kim, H.S., Arroyo, J., Berriman, M.,

in aflatoxin biosynthesis, *Appl. Environ. Microbiol.* 65, 4987-4994.

of *fas-1A*, a gene encoding a putative fatty acid synthetase directly involved in aflatoxin biosynthesis in *Aspergillus parasiticus*, *Appl. Environ. Microbiol.* 62, 191-195.

between averantin and averufin in the biosynthetic pathway, *Appl. Environ.*

Evans, A.A., London, W.T. & Buetow, K.H. (2003). Susceptibility to aflatoxin B1 related primary hepatocellular carcinoma in mice and humans, *Cancer Res.* 63, 4594-

characterization of versicolorin B synthase from *Aspergillus parasiticus*. Catalysis of the stereodifferentiating cyclization in aflatoxin biosynthesis essential to DNA

*aflJ*, a gene required for conversion of pathway intermediates to aflatoxin, *Appl.*

(1998). Systemic aspergillosis caused by an aflatoxin-producing strain of *Aspergillus*

characterization of the *O-*methyltransferase I gene (*dmtA*) from *Aspergillus parasiticus* associated with the conversions of demethylsterigmatocystin to sterigmatocystin and dihydrodemethylsterigmatocystin to dihydrosterigmatocystin

T.N., Jansen, A.J., Ngare, W., Kaviti, J.N., Gatei, D. & Siongok, T.A. (1982). Outbreak of acute hepatitis caused by aflatoxin poisoning in Kenya., *Lancet.* 1, 1346-

Abe, K., Archer, D.B., Bermejo, C., Bennett, J., Bowyer, P., Chen, D., Collins, M.,

*Aspergillus parasiticus*, *Arch. Biochem. Biophys.* 293, 67-70.

aflatoxins by *Aspergillus parasiticus*, *Biochemistry* 12, 5167-5171.

95, 1668-1669.

*Microbiol.* 53, 14-16.

interaction, *Biochemistry* 35, 11470-11486.

biosynthesis, *Chem. Rev.* 97, 2537-2556.

*Environ. Microbiol.* 64, 3713-3717.

*Genes Dev.* 4, 1473-1482.

*flavus*, *Med. Mycol.* 36, 107-112.

4601.

1348.

Backer, L., Dahiye, A.M., Misore, A., DeCock, K. & Rubin, C. (2005). Aflatoxin contamination of commercial maize products during an outbreak of acute aflatoxicosis in eastern and central Kenya, *Environ Health Perspect* 113, 1763- 1767. Lin, B.K. & Anderson, J.A. (1992). Purification and properties of versiconal cyclase from Coulsen, R., Davies, R., Dyer, P.S., Farman, M., Fedorova, N., Fedorova, N., Feldblyum, T.V., Fischer, R., Fosker, N., Fraser, A., Garcia, J.L., Garcia, M.J., Goble, A., Goldman, G.H., Gomi, K., Griffith-Jones, S., Gwilliam, R., Haas, B., Haas, H., Harris, D., Horiuchi, H., Huang, J., Humphray, S., Jimenez, J., Keller, N., Khouri, H., Kitamoto, K., Kobayashi, T., Konzack, S., Kulkarni, R., Kumagai, T., Lafon, A., Latge, J.P., Li, W., Lord, A., Lu, C., Majoros, W.H., May, G.S., Miller, B.L., Mohamoud, Y., Molina, M., Monod, M., Mouyna, I., Mulligan, S., Murphy, L., O'Neil, S., Paulsen, I., Penalva, M.A., Pertea, M., Price, C., Pritchard, B.L., Quail, M.A., Rabbinowitsch, E., Rawlins, N., Rajandream, M.A., Reichard, U., Renauld, H., Robson, G.D., Rodriguez de Cordoba, S., Rodriguez-Pena, J.M., Ronning, C.M., Rutter, S., Salzberg, S.L., Sanchez, M., Sanchez-Ferrero, J.C., Saunders, D., Seeger, K., Squares, R., Squares, S., Takeuchi, M., Tekaia, F., Turner, G., Vazquez de Aldana, C.R., Weidman, J., White, O., Woodward, J., Yu, J.H., Fraser, C., Galagan, J.E., Asai, K., Machida, M., Hall, N., Barrell, B. & Denning, D.W. (2005). Genomic sequence of the pathogenic and allergenic filamentous fungus *Aspergillus fumigatus*, *Nature* 438, 1151-1156.

Aflatoxin Biosynthetic Pathway and Pathway Genes 63

Stinnett, S.M., Espeso, E.A., Cobeno, L., Araujo-Bazan, L. & Calvo, A.M. (2007). *Aspergillus*

Sugui, J.A., Pardo, J., Chang, Y.C., Zarember, K.A., Nardone, G., Galvez, E.M., Mullbacher,

Trail, F., Chang, P.-K., Cary, J. & Linz, J.E. (1994). Structural and functional analysis of the

Trail, F., Mahanti, N. & Linz, J. (1995a). Molecular biology of aflatoxin biosynthesis,

Trail, F., Mahanti, N., Rarick, M., Mehigh, R., Liang, S.H., Zhou, R. & Linz, J.E. (1995b).

Turner, P.C., Collinson, A.C., Cheung, Y.B., Gong, Y., Hall, A.J., Prentice, A.M. & Wild, C.P.

van Egmond, H.P. (1989). Current situation on regulations for mycotoxins. Overview of

van Egmond, H.P. & Jonker, M.A. (2005). Worldwide regulations on aflatoxins. In: Abbas, H.K. (Ed.). CRC Press, Taylor and Francis Group, Boca Raton, FL, pp. 77-93. van Egmond, H.P., Schothorst, R.C. & Jonker, M.A. (2007). Regulations relating to

Watanabe, C.M. & Townsend, C.A. (1996). Incorporation of molecular oxygen in aflatoxin B1

Watanabe, C.M. & Townsend, C.A. (2002). Initial characterization of a type I fatty acid

Watanabe, C.M., Wilson, D., Linz, J.E. & Townsend, C.A. (1996). Demonstration of the

Wild, C.P., Shrestha, S.M., Anwar, W.A. & Montesano, R. (1992). Field studies of aflatoxin

immunosuppressed with hydrocortisone, *Eukaryot. Cell* 6, 1562- 1569. Tandon, H.D., Tandon, B.N. & Ramalingaswami, V. (1978). Epidemic of toxic hepatitis in India of possible mycotoxic origin, *Arch. Pathol. Lab. Med.* 102, 372-376. Townsend, C.A. (1997). Progress towards a biosynthetic rationale of the aflatoxin pathway,

and on light, *Mol. Microbiol.* 63, 242-255.

*Pure Appl. Chem.* 58, 227-238.

*Microbiol.* 141, 755-765.

*Epidemiol* 36, 1119-1125.

*Contam.* 6, 139-188.

*Chem* 389, 147-157.

3, 463-469.

64-65, 455-461.

*Environ. Microbiol.* 60, 4078-4085.

pathway, *Appl. Environ. Microbiol.* 61, 2665-2673.

biosynthesis, *J. Org. Chem.* 61, 1990-1993.

of aflatoxin B1, *Chem. Biol.* 9, 981-988.

*nidulans* VeA subcellular localization is dependent on the importin alpha carrier

A., Gallin, J.I., Simon, M.M. & Kwon- Chung, K.J. (2007). Gliotoxin is a virulence factor of *Aspergillus fumigatus*: *gliP* deletion attenuates virulence in mice

*nor-1* gene involved in the biosynthesis of aflatoxins by *Aspergillus parasiticus*, *Appl.*

Physical and transcriptional map of an aflatoxin gene cluster in *Aspergillus parasiticus* and functional disruption of a gene involved early in the aflatoxin

(2007). Aflatoxin exposure in utero causes growth faltering in Gambian infants, *Int J*

tolerances and status of standard methods of sampling and analysis, *J. Food Addit.*

mycotoxins in food: Perspectives in a global and European context, *Anal Bioanal*

synthase and polyketide synthase multienzyme complex NorS in the biosynthesis

catalytic roles and evidence for the physical association of type I fatty acid synthases and a polyketide synthase in the biosynthesis of aflatoxin B1, *Chem. Biol.*

exposure, metabolism and induction of genetic alterations in relation to HBV infection and hepatocellular carcinoma in The Gambia and Thailand, *Toxicol. Lett.*

 *http://www.apsnet.org/online/feature/mycotoxin/top.html* 2-8.

Prieto, R. & Woloshuk, C.P. (1997). *ord1*, an oxidoreductase gene responsible for conversion

Prieto, R., Yousibova, G.L. & Woloshuk, C.P. (1996). Identification of aflatoxin biosynthesis

Raisuddin, S., Singh, K.P., Zaidi, S.I., Paul, B.N. & Ray, P.K. (1993). Immunosuppressive

Richard, J.L. & Payne, G.A. (2003), Mycotoxins: Risks in Plant, Animal and Human Systems,

Robens, J.F. & Cardwell, K. (2005). The cost of mycotoxin management in the United States.

Ronning, C.M., Fedorova, N.D., Bowyer, P., Coulson, R., Goldman, G., Kim, H.S., Turner, G.,

Genomics of *Aspergillus fumigatus*, *Rev. Iberoam Micol. (RIAM)* 22, 223-228. Sakuno, E., Yabe, K. & Nakajima, H. (2003). Involvement of two cytosolic enzymes and a

averufin in aflatoxin biosynthesis, *Appl. Environ. Microbiol.* 69, 6418-6426. Scholl, P. & Groopman, J.D. (1995). Epidermiology of human exposures and its relationship

Schroeder, H.W., Cole, R.J., Grigsby, R.D. & Hein, H., Jr. (1974). Inhibition of aflatoxin

Silva, J.C. & Townsend, C.A. (1997). Heterologous expression, isolation, and characterization

Singh, R. & Hsieh, D.P. (1977). Aflatoxin biosynthetic pathway: elucidation by using blocked

Skory, C.D., Chang, P.K., Cary, J. & Linz, J.E. (1992). Isolation and characterization of a gene

acetate" from treatment with dichlorvos, *Appl. Microbiol.* 27, 394-399. Silva, J.C., Minto, R.E., Barry, C.E., 3rd, Holland, K.A. & Townsend, C.A. (1996). Isolation

aflatoxin B1 biosynthetic pathway, *J. Biol. Chem.* 272, 804-813.

*Aspergillus flavus*, *Appl. Environ. Microbiol.* 66, 320-324.

carcinogens, *Carcinogenesis* 11, 507-518.

aflatoxin gene cluster, *Appl. Environ. Microbiol.* 62, 3567-3571.

effects of aflatoxin in growing rats, *Mycopathologia* 124, 189-194.

*Microbiol.* 63, 1661-1666.

pp. 169-182.

13608.

in the United States, *APSnet Feature*

 *http://www.apsnet.org/online/feature/mycotoxin/top.html* 2-8.

of *O*- methylsterigmatocystin to aflatoxin in *Aspergillus flavus*, *Appl. Environ.*

genes by genetic complementation in an *Aspergillus flavus* mutant lacking the

Council for Agricultural Science and Technology (CAST), Ames, IA. Robens, J.F. (2001). The costs of mycotoxin management to the USA: Management of aflatoxins

In: Abbas, H.K. (Ed.), *Aflatoxin and Food Safety*. CRC Press, Boca Raton, FL, pp. 1-12.

Wortman, J.R., Yu, J., Anderson, M.J., Denning, D.W. & Nierman, W.C. (2005).

novel intermediate, 5'-oxoaverantin, in the pathway from 5'-hydroxyaverantin to

to liver cancer. In: Eklund, M., Richard, J.L. & Mise, K. (Eds.), *Molecular Approaches to Food Safety: Issues Involving Toxic Microorganisms*. Alaken, Inc., Fort Collins, Co.,

production and tentative identification of an aflatoxin intermediate "versiconal

and characterization of the versicolorin B synthase gene from *Aspergillus parasiticus*. Expansion of the aflatoxin B1 biosynthetic gene cluster, *J. Biol. Chem.* 271, 13600-

of versicolorin B synthase from *Aspergillus parasiticus*. A key enzyme in the

mutants of *Aspergillus parasiticus*, *Arch. Biochem. Biophys.* 178, 285-292. Skipper, P.L. & Tannenbaum, S.R. (1990). Protein adducts in the molecular dosimetry of chemical

from *Aspergillus parasiticus* associated with the conversion of versicolorin A to sterigmatocystin in aflatoxin biosynthesis, *Appl. Environ. Microbiol.* 58, 3527-3537. St Leger, R.J., Screen, S.E. & Shams-Pirzadeh, B. (2000). Lack of host specialization in

Aflatoxin Biosynthetic Pathway and Pathway Genes 65

Yabe, K., Nakamura, Y., Nakajima, H., Ando, Y. & Hamasaki, T. (1991b). Enzymatic

Yao, R.C. & Hsieh, D.P. (1974). Step of dichlorvos inhibition in the pathway of aflatoxin

Yu, J., Bhatnagar, D. & Cleveland, T.E. (2004a). Completed sequence of aflatoxin pathway

Yu, J., Bhatnagar, D. & Cleveland, T.E. (2004b). Genetics and biochemistry of aflatoxin

Yu, J., Cary, J.W., Bhatnagar, D., Cleveland, T.E., Keller, N.P. & Chu, F.S. (1993). Cloning and

Yu, J., Chang, P.K., Bhatnagar, D. & Cleveland, T.E. (2000a). Cloning of a sugar utilization gene cluster in *Aspergillus parasiticus*, *Biochim. Biophys. Acta.* 1493, 211-214. Yu, J., Chang, P.K., Bhatnagar, D. & Cleveland, T.E. (2002). Cloning and functional

Yu, J., Chang, P.K., Cary, J.W., Bhatnagar, D. & Cleveland, T.E. (1997). *avnA*, a gene

Yu, J., Chang, P.K., Cary, J.W., Wright, M., Bhatnagar, D., Cleveland, T.E., Payne, G.A. &

Yu, J., Chang, P.K., Ehrlich, K.C., Cary, J.W., Montalbano, B., Dyer, J.M., Bhatnagar, D. &

Yu, J., Chang, P.K., Payne, G.A., Cary, J.W., Bhatnagar, D. & Cleveland, T.E. (1995b).

aflatoxin biosynthesis, *Appl. Environ. Microbiol.* 70, 1253-1262.

gene cluster in *Aspergillus parasiticus*, *FEBS Lett.* 564, 126-130.

*Microbiol.* 57, 1340-1345.

242.

3564-3571.

4841.

biosynthesis, *Appl. Microbiol.* 28, 52-57.

*Environ. Microbiol.* 63, 1349-1356.

*Aspergillus* species, *Gene* 248, 157- 167.

conversion of norsolorinic acid to averufin in aflatoxin biosynthesis, *Appl. Environ.*

formation and genomics approach for eliminating aflatoxin contamination. In: Romeo, J.T. (Ed.), *Recent Advances in Phytochemistry* Elsevier, New York, pp. 224-

characterization of a cDNA from *Aspergillus parasiticus* encoding an *O*methyltransferase involved in aflatoxin biosynthesis, *Appl. Environ. Microbiol.* 59,

expression of an esterase gene in *Aspergillus parasitcus*, *Mycopathologia* 156, 227-234.

encoding a cytochrome P-450 monooxygenase, is involved in the conversion of averantin to averufin in aflatoxin biosynthesis in *Aspergillus parasiticus*, *Appl.*

Linz, J.E. (1995a). Comparative mapping of aflatoxin pathway gene clusters in *Aspergillus parasiticus* and *Aspergillus flavus*, *Appl. Environ. Microbiol.* 61, 2365-2371. Yu, J., Chang, P.K., Ehrlich, K.C., Cary, J.W., Bhatnagar, D., Cleveland, T.E., Payne, G.A.,

Linz, J.E., Woloshuk, C.P. & Bennett, J.W. (2004c). Clustered pathway genes in

Cleveland, T.E. (1998). Characterization of the critical amino acids of an *Aspergillus parasiticus* cytochrome P- 450 monooxygenase encoded by *ordA* that is involved in the biosynthesis of aflatoxins B1, G1, B2, and G2, *Appl. Environ. Microbiol.* 64, 4834-

Comparison of the *omtA* genes encoding *O*-methyltransferases involved in aflatoxin biosynthesis from *Aspergillus parasiticus* and *A. flavus*, *Gene* 163, 121-125. Yu, J., Fedorova, F., Montalbano, B.G., Bhatnagar, D., Cleveland, T.E., Bennett, J.W. &

Nierman, W.C. (2011). Mycotoxin gene expression in *Aspergillus flavus* in response to temperature as detected by RNA- Sequencing *FEMS Microbiol. Lett.* submitted. Yu, J., Woloshuk, C.P., Bhatnagar, D. & Cleveland, T.E. (2000b). Cloning and

characterization of *avfA* and *omtB* genes involved in aflatoxin biosynthesis in three

Wilkinson, J.R., Yu, J., Abbas, H.K., Scheffler, B.E., Kim, H.S., Nierman, W.C., Bhatnagar, D.

Wilkinson, J.R., Yu, J., Bland, J.M., Nierman, W.C., Bhatnagar, D. & Cleveland, T.E. (2007b).

Wilson, D.M. (1989). Analytical methods for aflatoxins in corn and peanuts, *Arch. Environ.*

Woloshuk, C.P., Foutz, K.R., Brewer, J.F., Bhatnagar, D., Cleveland, T.E. & Payne, G.A.

Woloshuk, C.P. & Payne, G.A. (1994). The alcohol dehydrogenase gene *adh1* is induced in

Woloshuk, C.P. & Prieto, R. (1998). Genetic organization and function of the aflatoxin B1

Wong, J.J., Singh, R. & Hsieh, D.P. (1977). Mutagenicity of fungal metabolites related to

Yabe, K., Ando, Y. & Hamasaki, T. (1988). Biosynthetic relationship among aflatoxins B1, B2,

Yabe, K., Ando, Y. & Hamasaki, T. (1991a). A metabolic grid among versiconal hemiacetal

Yabe, K., Ando, Y., Hashimoto, J. & Hamasaki, T. (1989). Two distinct *O*-methyltransferases

Yabe, K., Chihaya, N., Hamamatsu, S., Sakuno, E., Hamasaki, T., Nakajima, H. & Bennett,

Yabe, K., Matsushima, K., Koyama, T. & Hamasaki, T. (1998). Purification and

Yabe, K., Matsuyama, Y., Ando, Y., Nakajima, H. & Hamasaki, T. (1993). Stereochemistry

Yabe, K. & Nakajima, H. (2004). Enzyme reactions and genes in aflatoxin biosynthesis, *Appl.*

Yabe, K., Nakamura, M. & Hamasaki, T. (1999). Enzymatic formation of G-group aflatoxins

in aflatoxin biosynthesis, *Appl. Environ. Microbiol.* 55, 2172-2177.

acetate, versiconol acetate, versiconol and versiconal during aflatoxin biosynthesis,

J.W. (2003). Enzymatic conversion of averufin to hydroxyversicolorone and elucidation of a novel metabolic grid involved in aflatoxin biosynthesis, *Appl.*

characterization of O-methyltransferase I involved in conversion of demethylsterigmatocystin to sterigmatocystin and of dihydrodemethylsterigmatocystin to dihydrosterigmatocystin during aflatoxin

during aflatoxin biosynthesis: conversion of norsolorinic acid to averufin, *Appl.*

and biosynthetic relationship between G- and B- group aflatoxins, *Appl. Environ.*

hepatocellular carcinoma in humans, *Cancer Res.* 52, 2114-2118.

biosynthesis, *Appl. Environ. Microbiol.* 60, 2408-2414.

biosynthetic genes, *FEMS Microbiol. Lett.* 160, 169- 176.

aflatoxin biosynthesis, *Mutat. Res.* 44, 447- 450.

G1, and G2, *Appl. Environ. Microbiol.* 54, 2101-2106.

biosynthesis, *Appl. Environ. Microbiol.* 64, 166-171.

*Appl. Microbiol. Biotechnol.* 74, 1308-1319.

*Environ. Microbiol.* 60, 670-676.

*J. Gen. Microbiol.* 137, 2469-2475.

*Environ. Microbiol.* 69, 66-73.

*Environ. Microbiol.* 59, 2486- 2492.

*Microbiol. Biotechnol.* 64, 745-755.

*Microbiol.* 65, 3867-3872.

1060.

& Cleveland, T.E. (2007a). Aflatoxin formation and gene expression in response to carbon source media shift in *Aspergillus parasiticus*, *J. Food Addit. Contam.* 24, 1051-

Amino acid supplementation reveals differential regulation of aflatoxin biosynthesis in *Aspergillus flavus* NRRL 3357 and *Aspergillus parasiticus* SRRC 143,

*Contam. Toxicol.* 18, 308-314. Wogan, G.N. (1992). Aflatoxins as risk factors for

(1994). Molecular characterization of *aflR*, a regulatory locus for aflatoxin

*Aspergillus flavus* grown on medium conducive to aflatoxin biosynthesis, *Appl.*

**4**

*USA*

**Conserved Regulatory**

Ana M. Calvo and Sourabh Dhingra

*Northern Illinois University,*

**Mechanisms Controlling Aflatoxin**

**and Sterigmatocystin Biosynthesis**

Filamentous fungi produce a wide array of compounds known as secondary metabolites or natural products (i.e. Adrio and Demain, 2003; Reverberi et al 2010; Brakhage and Schroeckh 2011). The exact function of these compounds is unknown but it is postulated that they provide fungi with an advantage in their ecological niche (Keller et al., 2005; Georgianna and Payne 2009). The broad group of natural products includes compounds such as antibiotics, pigments and also mycotoxins (Fig.1). Among mycotoxins, aflatoxins (AF), specifically AF B1, are the most potent carcinogenic natural compounds known, and they are mainly produced by the opportunistic plant pathogens *Aspergillus flavus* and *Aspergillus parasiticus* (Squire 1989; Sweeny and Dobson 1999; Payne and Brown, 1998). Other AFproducers among *Aspergilli* include *Aspergillus nomius*, *Aspergillus ochraceoroseus*, *Aspergillus bombycis* and *Aspergillus pseudotamari* (Cary and Ehrlich, 2006). *Aspergillus flavus* and *Aspergillus parasiticus* have the ability to colonize oil seed crops of agricultural importance, such as corn, cotton, peanuts, sorghum and tree nuts. Ingestion of aflatoxin contaminated food can cause hepatocellular carcinoma, immunotoxicity, and teratogenic effects (Dvorackova and Kusak, 1990; Trail et al., 1995; Wogan et al., 1992). Various developed countries have strict regulation on the amount of AF allowed in food commodities. Contaminated crops above the permitted limit have to be destroyed, resulting in economic losses. The annual estimated loss due to AF contamination is attributed at approximately

It is known that AF and a related mycotoxin called sterigmatocystin (ST) (Fig.2) are synthesized through the same conserved biosynthetic pathway, in which ST is the penultimate precursor. ST is produced by several *Aspergillus* species, including *Aspergillus nidulans*, one of the most characterized eukaryotic systems that has been used as a model organism for more than 60 years (Pontecorvo et al., 1953)*.* The elevated number of characterized *A. nidulans* genes and mutant strains makes this model fungus ideal for genetic and molecular studies. There is a physical and genetic map of the eight chromosomes in *A. nidulans.* The whole *A. nidulans* genome has been sequenced and annotated, and has been compared with other *Aspergillus* genomes (David et al., 2008; Galagan et al., 2005). Structural and signaling pathway genes controlling ST production in *A. nidulans* are also found in AF-producer *Aspergillus* spp. (Hicks et al., 1997; reviewed by

\$270 million in the USA alone (Richard and Payne, 2003).

**1. Introduction**

### **Conserved Regulatory Mechanisms Controlling Aflatoxin and Sterigmatocystin Biosynthesis**

Ana M. Calvo and Sourabh Dhingra *Northern Illinois University, USA*

#### **1. Introduction**

66 Aflatoxins – Biochemistry and Molecular Biology

Yu, J.H., Butchko, R.A., Fernandes, M., Keller, N.P., Leonard, T.J. & Adams, T.H. (1996a).

Yu, J.H., Wieser, J. & Adams, T.H. (1996b). The *Aspergillus* FlbA RGS domain protein

Zhou, R. & Linz, J.E. (1999). Enzymatic function of the nor-1 protein in aflatoxin biosynthesis

Zhu, J.Q., Zhang, L.S., Hu, X., Xiao, Y., Chen, J.S., Xu, Y.C., Fremy, J. & Chu, F.S. (1987).

Zuckerman, A.J., Rees, K.R., Inman, D. & Petts, V. (1967). Site of action of aflatoxin on

in Aspergillus parasiticus, *Appl. Environ. Microbiol.* 65, 5639-5641.

*Aspergillus nidulans* and *A. flavus*, *Curr. Genet.* 29, 549-555.

*EMBO J.* 15, 5184-5190.

urine, *Cancer Res.* 47, 1848-1852.

human liver cells in culture, *Nature* 214, 814-815.

Conservation of structure and function of the aflatoxin regulatory gene *aflR* from

antagonizes G protein signaling to block proliferation and allow development,

Correlation of dietary aflatoxin B1 levels with excretion of aflatoxin M1 in human

Filamentous fungi produce a wide array of compounds known as secondary metabolites or natural products (i.e. Adrio and Demain, 2003; Reverberi et al 2010; Brakhage and Schroeckh 2011). The exact function of these compounds is unknown but it is postulated that they provide fungi with an advantage in their ecological niche (Keller et al., 2005; Georgianna and Payne 2009). The broad group of natural products includes compounds such as antibiotics, pigments and also mycotoxins (Fig.1). Among mycotoxins, aflatoxins (AF), specifically AF B1, are the most potent carcinogenic natural compounds known, and they are mainly produced by the opportunistic plant pathogens *Aspergillus flavus* and *Aspergillus parasiticus* (Squire 1989; Sweeny and Dobson 1999; Payne and Brown, 1998). Other AFproducers among *Aspergilli* include *Aspergillus nomius*, *Aspergillus ochraceoroseus*, *Aspergillus bombycis* and *Aspergillus pseudotamari* (Cary and Ehrlich, 2006). *Aspergillus flavus* and *Aspergillus parasiticus* have the ability to colonize oil seed crops of agricultural importance, such as corn, cotton, peanuts, sorghum and tree nuts. Ingestion of aflatoxin contaminated food can cause hepatocellular carcinoma, immunotoxicity, and teratogenic effects (Dvorackova and Kusak, 1990; Trail et al., 1995; Wogan et al., 1992). Various developed countries have strict regulation on the amount of AF allowed in food commodities. Contaminated crops above the permitted limit have to be destroyed, resulting in economic losses. The annual estimated loss due to AF contamination is attributed at approximately \$270 million in the USA alone (Richard and Payne, 2003).

It is known that AF and a related mycotoxin called sterigmatocystin (ST) (Fig.2) are synthesized through the same conserved biosynthetic pathway, in which ST is the penultimate precursor. ST is produced by several *Aspergillus* species, including *Aspergillus nidulans*, one of the most characterized eukaryotic systems that has been used as a model organism for more than 60 years (Pontecorvo et al., 1953)*.* The elevated number of characterized *A. nidulans* genes and mutant strains makes this model fungus ideal for genetic and molecular studies. There is a physical and genetic map of the eight chromosomes in *A. nidulans.* The whole *A. nidulans* genome has been sequenced and annotated, and has been compared with other *Aspergillus* genomes (David et al., 2008; Galagan et al., 2005). Structural and signaling pathway genes controlling ST production in *A. nidulans* are also found in AF-producer *Aspergillus* spp. (Hicks et al., 1997; reviewed by

Conserved Regulatory Mechanisms Controlling Aflatoxin and Sterigmatocystin Biosynthesis 69

In addition to the structural genes, two regulatory genes are also included in these clusters, *aflR* and *aflS* (formerly known as *aflJ)*. The gene products AflR and AflS, are specific regulators for AF/ST gene cluster activation and for concomitant AF/ST production. AflR, encodes a transcription factor with a cysteine rich Gal4 type bi-nuclear zinc finger cluster (Chang et al., 1995; Yu et al., 1996). AflR has been shown to control most of the steps of the ST/AF biosynthetic pathway. AflR binds DNA at the consensus motif 5'TCGN5CGA3' in the promoter of biosynthetic genes (Fernandes et al., 1998). More than one AflR binding site is common in promoters of AF genes (Cary et al., 2009; Cary et al., 2000). Despite the differences between *A. nidulans* and *A. flavus* AflR, the function is conserved (Yu et al., 1996). Deletion *aflR* (∆*aflR*) strain in *A. flavus* and *A. nidulans* fails to produce AF and ST respectively. Additionally, heterologously expressed *A. flavus aflR* in the deletion *A. nidulans* ∆*aflR* strain is able to activate the ST gene cluster, supporting functional conservation (Yu et al., 1996). In *A. parasiticus*, a microarray comparison of ∆*aflR* and wild-type strains revealed eighteen genes involved in AF pathway to be differentially expressed (Price et al., 2006). Constitutive expression of *aflR* was shown to increase the transcript of AF cluster genes along with 50-fold greater aflatoxin production

In addition to AflR, AflS also plays a role in the regulation of AF biosynthesis (Meyers et al., 1998; Chang et al., 1993; 2002; Chang, 2003). *aflS* lies next to *aflR* in the AF cluster. The gene products AflR and AflS were found to interact in *A. parasiticus* (Chang, 2003). Deletion of *aflS* has been shown to decrease expression of several biosynthetic genes in the cluster (Meyers et al., 1998), but does not alter *aflR* expression (Chang, 2003). The ∆*aflS* mutant still produces reduced but detectable levels of AF in *A. parasiticus* (Meyers et al., 1998)*.* Interestingly, overexpression of *aflS*, particularly in combination with overexpression of *aflR* synergically increased expression of biosynthetic genes and AF production (Chang, 2003). *Aspergillus sojae*, is unable to produce AF in spite possessing the AF gene cluster in its genome; its *aflR* gene has a mutation that results in a truncated protein, which fails to interact with AflS (Chang,

Our genomic sequence analysis indicates that *aflS* is also present in *A. nidulans*, and that it is located next to *aflR* in the ST gene cluster (Calvo et al., unpublished data). As in the case of *aflS* in the AF cluster, *A. nidulans aflS* transcriptional direction is opposite with respect to

Global regulatory mechanisms that control different cell functions, including morphogenesis and secondary metabolism tend to be conserved across fungal species. The *velvet* gene or *veA* is an example of global regulation. VeA is unique to fungi and is highly conserved in Ascomycetes (Myung et al., 2011).  Interestingly, our laboratory showed for the first time that VeA also controls the production of numerous secondary metabolites, including ST and AF production in *A. nidulans,* and *A. flavus* and *A. parasiticus* (Kato et al., 2003; Calvo et al., 2004; Duran et al., 2007; Carry et al., 2007). In *A. nidulans* and later in *A. flavus* it has been demonstrated that

**3. AflR and AflS**

(Flaherty and Payne 1997).

**4. Global regulation**

that of *aflR*, sharing an intergenic region of 599 bp.

**4.1 VeA and VeA-interacting proteins**

2004).

Calvo et al., 2002; Calvo, 2008). For these reasons this model system is especially productive in the study of the AF/ST gene clusters and regulatory pathways directing mycotoxin production. In this review we will focus on these common regulatory mechanisms governing AF and ST biosynthesis in *A. flavus* and *A. parasiticus,* and in *A. nidulans*.

#### **2. AF/ST gene clusters**

ST/AF gene clusters remain some of the best characterized mycotoxin gene clusters. Both encode enzymes participating in 29 metabolic steps along with two regulatory proteins, AflR and AflS. (Cary et al., 2009). Both gene clusters extend to approximately 70 kb in the genome (Brown et al., 1996; Cary and Ehrlich, 2006; Ehrlich et al., 2005; Cary et al., 2009). The order and direction of genes in AF and ST clusters is conserved for all genes except four in AF pathway and three in ST which are inverted. It has been hypothesized that these differences might have been the result of gene reorganization from an ST-producing ancestor by recombination and duplications of near-telomeric regions where these clusters are found (Cary et al., 2009; Carbone et al., 2007). Based on phylogenetic analysis, the clusters may be 450 million years old (Galagan et al., 2005).

The enzymatic reactions and chemistry involved in the AF/ST biosynthetic pathway have already been extensively covered in previous reviews (Bhatnagar et al., 2003; Bennett and Klich, 2003; Hicks et al., 2002; Huffman et al., 2010; Keller et al., 2005; Minto and Townsend, 1997; Payne and Brown, 1998; Yabe and Nakajima, 2004), and will not be addressed in this review. Beyond the genus *Aspergillus*, other fungi present semi-conserved pathways, for example *Dothistroma septosporum*, which produces a metabolite that resembles versicolorin B, a precursor of both ST and AF. This suggests further cluster conservation across fungal genera (Bradshaw and Zhang, 2006).

Fig. 1. Chemical structures of the mycotoxins aflatoxin and sterigmatocystin.

#### **3. AflR and AflS**

68 Aflatoxins – Biochemistry and Molecular Biology

Calvo et al., 2002; Calvo, 2008). For these reasons this model system is especially productive in the study of the AF/ST gene clusters and regulatory pathways directing mycotoxin production. In this review we will focus on these common regulatory mechanisms

ST/AF gene clusters remain some of the best characterized mycotoxin gene clusters. Both encode enzymes participating in 29 metabolic steps along with two regulatory proteins, AflR and AflS. (Cary et al., 2009). Both gene clusters extend to approximately 70 kb in the genome (Brown et al., 1996; Cary and Ehrlich, 2006; Ehrlich et al., 2005; Cary et al., 2009). The order and direction of genes in AF and ST clusters is conserved for all genes except four in AF pathway and three in ST which are inverted. It has been hypothesized that these differences might have been the result of gene reorganization from an ST-producing ancestor by recombination and duplications of near-telomeric regions where these clusters are found (Cary et al., 2009; Carbone et al., 2007). Based on phylogenetic analysis, the

The enzymatic reactions and chemistry involved in the AF/ST biosynthetic pathway have already been extensively covered in previous reviews (Bhatnagar et al., 2003; Bennett and Klich, 2003; Hicks et al., 2002; Huffman et al., 2010; Keller et al., 2005; Minto and Townsend, 1997; Payne and Brown, 1998; Yabe and Nakajima, 2004), and will not be addressed in this review. Beyond the genus *Aspergillus*, other fungi present semi-conserved pathways, for example *Dothistroma septosporum*, which produces a metabolite that resembles versicolorin B, a precursor of both ST and AF. This suggests further cluster conservation across fungal

Fig. 1. Chemical structures of the mycotoxins aflatoxin and sterigmatocystin.

governing AF and ST biosynthesis in *A. flavus* and *A. parasiticus,* and in *A. nidulans*.

clusters may be 450 million years old (Galagan et al., 2005).

**2. AF/ST gene clusters**

genera (Bradshaw and Zhang, 2006).

In addition to the structural genes, two regulatory genes are also included in these clusters, *aflR* and *aflS* (formerly known as *aflJ)*. The gene products AflR and AflS, are specific regulators for AF/ST gene cluster activation and for concomitant AF/ST production. AflR, encodes a transcription factor with a cysteine rich Gal4 type bi-nuclear zinc finger cluster (Chang et al., 1995; Yu et al., 1996). AflR has been shown to control most of the steps of the ST/AF biosynthetic pathway. AflR binds DNA at the consensus motif 5'TCGN5CGA3' in the promoter of biosynthetic genes (Fernandes et al., 1998). More than one AflR binding site is common in promoters of AF genes (Cary et al., 2009; Cary et al., 2000). Despite the differences between *A. nidulans* and *A. flavus* AflR, the function is conserved (Yu et al., 1996). Deletion *aflR* (∆*aflR*) strain in *A. flavus* and *A. nidulans* fails to produce AF and ST respectively. Additionally, heterologously expressed *A. flavus aflR* in the deletion *A. nidulans* ∆*aflR* strain is able to activate the ST gene cluster, supporting functional conservation (Yu et al., 1996). In *A. parasiticus*, a microarray comparison of ∆*aflR* and wild-type strains revealed eighteen genes involved in AF pathway to be differentially expressed (Price et al., 2006). Constitutive expression of *aflR* was shown to increase the transcript of AF cluster genes along with 50-fold greater aflatoxin production (Flaherty and Payne 1997).

In addition to AflR, AflS also plays a role in the regulation of AF biosynthesis (Meyers et al., 1998; Chang et al., 1993; 2002; Chang, 2003). *aflS* lies next to *aflR* in the AF cluster. The gene products AflR and AflS were found to interact in *A. parasiticus* (Chang, 2003). Deletion of *aflS* has been shown to decrease expression of several biosynthetic genes in the cluster (Meyers et al., 1998), but does not alter *aflR* expression (Chang, 2003). The ∆*aflS* mutant still produces reduced but detectable levels of AF in *A. parasiticus* (Meyers et al., 1998)*.* Interestingly, overexpression of *aflS*, particularly in combination with overexpression of *aflR* synergically increased expression of biosynthetic genes and AF production (Chang, 2003). *Aspergillus sojae*, is unable to produce AF in spite possessing the AF gene cluster in its genome; its *aflR* gene has a mutation that results in a truncated protein, which fails to interact with AflS (Chang, 2004).

Our genomic sequence analysis indicates that *aflS* is also present in *A. nidulans*, and that it is located next to *aflR* in the ST gene cluster (Calvo et al., unpublished data). As in the case of *aflS* in the AF cluster, *A. nidulans aflS* transcriptional direction is opposite with respect to that of *aflR*, sharing an intergenic region of 599 bp.

#### **4. Global regulation**

#### **4.1 VeA and VeA-interacting proteins**

Global regulatory mechanisms that control different cell functions, including morphogenesis and secondary metabolism tend to be conserved across fungal species. The *velvet* gene or *veA* is an example of global regulation. VeA is unique to fungi and is highly conserved in Ascomycetes (Myung et al., 2011).  Interestingly, our laboratory showed for the first time that VeA also controls the production of numerous secondary metabolites, including ST and AF production in *A. nidulans,* and *A. flavus* and *A. parasiticus* (Kato et al., 2003; Calvo et al., 2004; Duran et al., 2007; Carry et al., 2007). In *A. nidulans* and later in *A. flavus* it has been demonstrated that

Conserved Regulatory Mechanisms Controlling Aflatoxin and Sterigmatocystin Biosynthesis 71

adenylyl cyclase and cAMP-dependent kinase PKA. Activation of FadA or overexpression of PKA results in an increase in vegetative growth and reduction of asexual development,as well as reduction in ST production (Hicks et al., 1997; Shimizu and Keller, 2000). It has been shown that overexpression of PKA represses *aflR* expression (Shimizu and Keller, 2000). In addition to transcriptional regulation of ST genes, PKA also regulates ST production at posttranslational level. PKA negatively regulates the localization of AflR protein in the nucleus by phosphorylation (Shimizu et al., 2003). In *A. parasiticus* PKA also negatively regulates AF production (Roze et al., 2004). Additionally, expression of the FadA constitutive active form also resulted in a decrease of AF intermediates and a reduction in conidiation in *A*. *parasiticus* (Hicks et al., 1997). These findings strongly indicate a conservation of the FadA-PKA signaling pathway in regulating ST and AF

Fig. 2. *Aspergillus flavus* (70S) and *Aspergillus nidulans* (FGSC 4) wild type strains. A) Point-inoculated cultures incubated for five days. B) Thin layer chromatography analysis of the mycotoxins aflatoxin and sterigmatocystin. The chemical analysis was carried out as previously described (Kato et al., 2003; Duran et al., 2007). Std. = Standard,

In the wild type, FadA is regulated by FluG, which activates transcription of *flbA*, encoding a GTPase activating protein that negatively regulates FadA (Lee and Adams, 1994; 1996; Seo et al., 2003. Both, *fluG* and *flbA* are necessary for ST production (Calvo et al., 2002).

Further studies showed of role of the FadA-interacting proteins, SfaD and GpgA subunits, and PhnA, a positive regulator of Gβγ, as positive regulators of *aflR* and consequently ST

AF = Aflatoxin, ST = Sterigmatocystin. 1-3 represent replicates.

biosynthesis (Seo and Yu, 2006).

Homologs are also present in the *A. flavus* genome (data not shown).

production.

*veA* is necessary for *aflR* transcription, and therefore controls the expression of the ST/AF clustered genes (Kato et al., 2003; Duran et al., 2007).

The effect of VeA on secondary metabolism is broad. In *A. nidulans* the production of other metabolites, including penicillin, is also affected by the absence of *veA* (Kato et al., 2003). In *A. flavus*, the biosynthesis of other mycotoxins is also *veA*-dependent, such as the case of cyclopiazonic acid and aflatrem, where we showed that expression of aflatrem genes requires a *veA* wild-type allele (Duran et al., 2007).

VeA is also conserved across fungal genera, for example the *veA* homolog FvVE1 in *Fusarium verticilliodies* (Li et al., 2006; Myung et al., 2009), *AcveA* in *Acremonium chrysogenum* (Dreyer et al., 2007) and *ve-1* in *Neurospora crassa* (Bayram et al., 2008). Studies in these organisms also link *veA* to secondary metabolism. The *veA* regulatory system has been extensively reviewed (Calvo, 2008). VeA is transported to the nucleus (Stinnett et al., 2007) where it forms interactions with other regulatory proteins that also influence morphogenesis, and secondary metabolism, including production of mycotoxin (Purschwitz et al., 2008; Calvo, 2008; Bayram et al., 2008; Bayram et al., 2010).

In *A. nidulans* it has been shown that VeA interacts with a protein called LaeA (Bayram et al., 2008). *laeA* is also necessary for ST/AF production. Unlike *veA*, which has been shown to control *A. nidulans* asexual/sexual morphological development, *laeA* has a mild effect on morphogenesis, influencing Hülle cell numbers (Bayram et al., 2010). However, in *A. flavus* both *veA* and *laeA* have an important role in morphogenesis, being necessary for sclerotial production (Duran et al., 2007; Duran et al., 2009; Kale et al., 2008). LaeA is a putative methyl transferase, with an S-adenosylmethionine binding domain (Bok and Keller, 2004). In *A. flavus* it has been shown that *laeA* is a negative regulator of *veA* transcription (Kale et al., 2008). In addition, increased expression of *veA* (in a strain with multiple *veA* copies) results in decreased *laeA* expression (Amaike and Keller, 2009), suggesting a mutual negative transcriptional regulatory feedback control.

In addition to transcriptional regulation between *veA* and *laeA*, recent studies also suggest posttranslational regulation (Bayram et al., 2010); a new form of the VeA protein with higher molecular weight has been detected in *laeA* strains, indicating that in the wild type, *laeA* prevents a modification of the VeA protein (Bayram et al., 2010).

Another protein from the *velvet* family, VelB, has been shown to interact with VeA. The *∆velB* strain produces a reduced and delayed but still detectable amount of ST. VelB protein also interacts with VosA, a positive regulator of sporogenesis (Ni and Yu 2007; Bayram et al., 2010). Homologs of *velB* and *vosA* are present in *A. flavus* genome (data not shown). Other VeA-interacting proteins, such as the light-response mediating proteins FphA, LreA and LreB (Purschwitz et al., 2008), also influence morphological and metabolic changes in response to environmental stimuli affecting mycotoxin production (see section below corresponding to the effect of light).

#### **4.2 G protein signalling**

Several cellular functions, including fungal growth, morphogenesis and secondary metabolism are governed by G-protein signalling pathways. For example, the *A. nidulans fadA* gene encodes an α subunit of an heterotrimeric G protein complex where FadA interacts with Gβ (SfaD) and Gγ (GpgA) subunits (Rosen et al., 1999; Seo et al., 2005). Mutations in FadA blocking the intrinsic GTPase activity results in a permanently active stage of this protein (Hicks et at., 1997). It is likely that activation of FadA upregulates

*veA* is necessary for *aflR* transcription, and therefore controls the expression of the ST/AF

The effect of VeA on secondary metabolism is broad. In *A. nidulans* the production of other metabolites, including penicillin, is also affected by the absence of *veA* (Kato et al., 2003). In *A. flavus*, the biosynthesis of other mycotoxins is also *veA*-dependent, such as the case of cyclopiazonic acid and aflatrem, where we showed that expression of aflatrem genes

VeA is also conserved across fungal genera, for example the *veA* homolog FvVE1 in *Fusarium verticilliodies* (Li et al., 2006; Myung et al., 2009), *AcveA* in *Acremonium chrysogenum* (Dreyer et al., 2007) and *ve-1* in *Neurospora crassa* (Bayram et al., 2008). Studies in these organisms also link *veA* to secondary metabolism. The *veA* regulatory system has been extensively reviewed (Calvo, 2008). VeA is transported to the nucleus (Stinnett et al., 2007) where it forms interactions with other regulatory proteins that also influence morphogenesis, and secondary metabolism, including production of mycotoxin (Purschwitz

In *A. nidulans* it has been shown that VeA interacts with a protein called LaeA (Bayram et al., 2008). *laeA* is also necessary for ST/AF production. Unlike *veA*, which has been shown to control *A. nidulans* asexual/sexual morphological development, *laeA* has a mild effect on morphogenesis, influencing Hülle cell numbers (Bayram et al., 2010). However, in *A. flavus* both *veA* and *laeA* have an important role in morphogenesis, being necessary for sclerotial production (Duran et al., 2007; Duran et al., 2009; Kale et al., 2008). LaeA is a putative methyl transferase, with an S-adenosylmethionine binding domain (Bok and Keller, 2004). In *A. flavus* it has been shown that *laeA* is a negative regulator of *veA* transcription (Kale et al., 2008). In addition, increased expression of *veA* (in a strain with multiple *veA* copies) results in decreased *laeA* expression (Amaike and Keller, 2009), suggesting a mutual

In addition to transcriptional regulation between *veA* and *laeA*, recent studies also suggest posttranslational regulation (Bayram et al., 2010); a new form of the VeA protein with higher molecular weight has been detected in *laeA* strains, indicating that in the wild type, *laeA*

Another protein from the *velvet* family, VelB, has been shown to interact with VeA. The *∆velB* strain produces a reduced and delayed but still detectable amount of ST. VelB protein also interacts with VosA, a positive regulator of sporogenesis (Ni and Yu 2007; Bayram et al., 2010). Homologs of *velB* and *vosA* are present in *A. flavus* genome (data not shown). Other VeA-interacting proteins, such as the light-response mediating proteins FphA, LreA and LreB (Purschwitz et al., 2008), also influence morphological and metabolic changes in response to environmental stimuli affecting mycotoxin production (see section below

Several cellular functions, including fungal growth, morphogenesis and secondary metabolism are governed by G-protein signalling pathways. For example, the *A. nidulans fadA* gene encodes an α subunit of an heterotrimeric G protein complex where FadA interacts with Gβ (SfaD) and Gγ (GpgA) subunits (Rosen et al., 1999; Seo et al., 2005). Mutations in FadA blocking the intrinsic GTPase activity results in a permanently active stage of this protein (Hicks et at., 1997). It is likely that activation of FadA upregulates

clustered genes (Kato et al., 2003; Duran et al., 2007).

requires a *veA* wild-type allele (Duran et al., 2007).

negative transcriptional regulatory feedback control.