The Biosynthetic Pathways of Aflatoxins and Sterigmatocystin

2.3.1 Acetate to Norsolorinic Acid (NOR) Trail et al.


Molecular evidence supports the plausible hypothesis that two fatty acid synthases (FAS) and a polyketide synthase (PKS) are involved in the synthesis of polyketide from acetate (Bhatnagar et al. 1992; Brown et al. 1996a; Townsend et al. 1984; Trail et al. 1995a). Complementation of an aflatoxin blocked UV mutant, UVM8, Mahanti et al. (1996) identified a gene named uvm8, which is required for NOR biosynthesis and aflatoxin production in A. parasiticus. The predicted amino acids of the gene uvm8 shares high degree of similarity (67%) and identity (48%) to the beta-subunit of FASs (FAS1) from Saccharomyces cerevisiae and Yarrowia lipolytica (Trail et al. 1995a,b). Complementation, metabolite feeding and disruption experiments by Mahanti et al. (1996) confirmed that the 7.5-kb large transcript of the gene uvm8 encodes one subunit of a novel FAS directly involved in the backbone formation prior to nor-1 of aflatoxin synthesis. For the proposed function, the gene uvm8 was renamed fas-1A. The fas-1A gene was renamed fas-1 encoding FAS-1 in the aflatoxin biosynthetic pathway gene cluster (Figure 1, panel A). Sequence analyses have shown that there are two large genes, fas-1A and fas-2A, encoding for beta- and alpha-subunit of FAS, respectively (Mahanti et al. 1996, unpublished, personal communication). The gene fas-1A and fas-2A were renamed fas-1 and fas-2 in the aflatoxin pathway gene cluster encoding for FAS-1 (FASa) and FAS-2 (FASp), respectively (Payne and Brown 1998). Brown et al. (1996a) proposed the involvement of FAS in ST biosynthesis in A. nidulans. They identified two genes, stcJ and stcK in the ST cluster, that are homologous to FASs, fas-2 and fas-1 of aflatoxin pathway genes, respectively. Disruption of stcJ and stcK encoding FASa and FASb subunits (FAS-2 and FAS-1), respectively in A. nidulans, stopped ST synthesis. Watanabe et al. (1996) provided the biochemical evidence for the role of a FASs and PKSs in the biosynthesis of aflatoxin. The N-acetylcysteamine thioester of hexanoic acid could be incorporated into NOR in a disrupted fas-1 transformant. Mayorga and Timberlake (1992) identified a gene wA encoding the PKS in A. nidulans. Chang et al. (1995a) confirmed that a PKS is required for aflatoxin biosynthesis by cloning the pksA gene encoding a PKS for the synthesis of polyketide from A. parasiticus. Feng and Leonard (1995) also isolated apksL1 gene for PKS, the equivalent of pksA gene, by PCR from a pool of 19 A. parasiticus clones. Disruption of the pksL1 gene produced no aflatoxin or any aflatoxin intermediates. The pksL1 gene is 99% identical to pksA and they are believed to be the same gene. The 1% sequence discrepancy may possibly be due to strain variation and sequencing errors. Yu (1995) isolated a gene, pksST that encodes a PKS from A. nidulans. The nucleotide sequence of pksST from A. nidulans is identical to stcA (Brown et al. 1996b). However, no significant sequence homology was found between wA and stcA (pks ST). The predicted amino acid sequences of these PKS contains a typical four conserved domains common to other known PKS proteins: p-ketoacyl synthase (KS), acyltransferase (AT), acyl carrier protein (ACP), and thioesterase (TE). The gene for PKS from A. parasiticus (pksA or pksLl) was designated as pksA (Yu et al. 1995a) in the aflatoxin pathway gene cluster and its homolog in A. nidulans was designated as stcA (Brown et al. 1996b). NOR is the first stable intermediate in the pathway (Bennett 1981; Bennett et al. 1997; Papa 1979; 1982). However, the predicted conversion product of PKS in the aflatoxin pathway is believed to be a noranthrone. The conversion for noranthrone to NOR is poorly defined, but it has been proposed to occur by a noranthrone oxidase (Vederas and Nakashima 1980), a monooxygenase (Bhatnagar et al. 1992), or spontaneously (Dutton 1988).

2.3.2 NOR to Averantin (AVN)

Using NOR-accumulating mutants, it was demonstrated by Papa (1979); Papa (1982) in A. flavus and by Bennett (1981), Detroy et al. (1973) in A. parasiticus that NOR was an intermediate in aflatoxin biosynthetic pathway (Bennett et al. 1997). It was found that the NOR-accumulating mutants were always leaky without completely blocking aflatoxin biosynthesis. Bhatnagar et al. (1992) proposed the involvement of dehydrogenase in the conversion of NOR to AVN and Chang et al. (1992) identified and cloned the gene, nor-1, that complemented a NOR-non-producing mutant in A. parasiticus. Trail et al. (1994) further characterized this gene to encode an enzyme that functions as a ketoreductase for the conversion of NOR to AVN. Cary et al. (1996) characterized a gene, norA encoding an aryl-alcohol dehydrogenase in the aflatoxin pathway gene cluster, which were involved in the conversion of NOR to AVN (Bennett et al. 1980; Bennett 1981; Bhatnagar et al. 1992). An additional gene, norB, which was also found to be homologous to the nor-1 and norA genes in the aflatoxin pathway gene cluster in A. parasiticus (Yu et al. unpublished). The enzymatic function, and coordinated genetic regulation of the three NOR-converting genes are to be further investigated. The nor-1 homologous gene in A. nidulans is stcE (Brown et al. 1996b).

2.3.3 AVN to 5'hydroxyaverantin (HAVN)

Earliest evidence for the conversion of AVN to HAVN was elucidated via radiotracing by Bennett et al. (1980), McCormick et al. (1987). Subsequently, Yabe et al. (1991b), based on the enzymatic studies, proposed three enzymatic steps in two possible routes involved in the conversion of aflatoxin intermediates from NOR to AVF: (a) conversion from NOR to AVN catalyzed by a dehydrogenase; (b) conversion from NOR to HAVN by a monooxygenase; (c) HAVN then would be converted to AVF by a second dehydrogenase. Yabe et al. (1991b) also proposed that this reaction was reversible and that the NADPH was the preferred cofactor. Bhatnagar et al. (1992) proposed two alternate routes requiring 3-4 enzymes for the conversion of NOR to AVF. Yu et al. (1997) cloned and disrupted a gene that encodes a P-450 monooxygenase (previously named ord-1, Yu et al. 1995a). Using disruption and substrate feeding it was defined that HAVN and averufanin (AVNN) are the conversion intermediate products from AVN to averufin (AVF). This gene was designated avnA, which has high sequence similarity to the stcF gene in the A. nidulans ST cluster (Brown et al. 1996b).

2.3.4 HAVN to AVNN and AVF

In the proposed scheme by Yabe et al. (1991b; 1993), AVNN was considered a shunt metabolite and not an aflatoxin intermediate. Bhatnagar et al. (1992) proposed both AVN and AVNN were the intermediates from NOR to AVF based on radiolabeling experiment. Chang et al. (2000b) cloned and characterized a gene, adhA, encoding an alcohol dehydro-genase in A. parasiticus. Gene disruption and feeding experiments demonstrated that AVN might be converted directly to AVF or indirectly to AVF through an intermediate substrate AVNN. The exact nature of the enzymatic function and the possible involvement of additional enzymes are still unclear. Woloshuk and Payne (1994) identified an alcohol dehydrogenase gene, adhl, in A. flavus that is expressed concurrently with aflatoxin pathway genes. The adh1 gene in A. flavus and adhA gene in A. parasiticus shares no significant homology at both DNA and amino acid level. The involvement of the adhl from A. flavus in aflatoxin synthesis is to be investigated.

2.3.5 AVF to Versiconal Hemiacetal Acetate (VHA)

The conversion from AVF to VHA is thought to involve an oxidase (Bhatnagar et al. 1992). Yu et al. (2000b) cloned the avfA gene from both A. parasiticus and an AVF-accumulating A. flavus strain as well as A. sojae strain that was shown by gene complementation experiment to encode for an enzyme (homologous to oxidase) responsible for the conversion from AVF to VHA. The avfA gene homolog in A. nidulans was identified to be stcO (Yu et al. 2000b).

2.3.6 VHA to Versiconal (VAL)

The evidence for the involvement of an esterase in the conversion of VHA to VAL in aflatoxin biosynthesis was demonstrated by Bennett et al. (1976), Fitzell et al. (1977), Schroeder et al. (1974), Yabe et al. (1991a,b), and Yao and Hsieh (1974), when the A. parasiticus was treated with the organophosphorus pesticide dichlorvos. The esterase has been purified in A. parasiticus by Hsieh et al. (1989); Kusumoto and Hsieh (1996). Yu et al. (2003) cloned a gene, estA, which could well be the gene for this esterase. The homologous gene in A. nidulans ST biosynthetic gene cluster is stcl. Attempts to disrupt the estA or stcl have not resulted in any conclusive evidence for the involvement of these genes in toxin synthesis.

2.3.7 VAL to Versicolorin B (VER B)

Yabe and Hamasaki (1993) provided enzymatic evidence for the conversion of VAL to VER B. Silva et al. (1996), Silva and Townsend (1996), and McGuire et al. (1996) cloned, characterized and expressed the vbs gene in the aflatoxin pathway gene cluster in A. parasiticus. It was demonstrated that the VER B synthase catalyzes the side chain cyclodehydration of racemic VHA to VER B. This is a key step in the aflatoxin formation since it closed the bisfuran ring of aflatoxin for binding to DNA. The homologous gene in A. nidulans ST biosynthetic gene cluster is stcN.

2.3.8 VER B to Versicolorin A (VER A)

In the aflatoxin biosynthetic pathway, the VER B is a critical branch point (Bhatnagar et al. 1991) leading either to AFB1 and AFG1 or to AFB2 and AFG2. The conversion of VER B to VER A was proposed to require a desaturation of the bisfuran ring of VER B (Bhatnagar et al. 1993; Yabe and Hamasaki 1993). Disruption of stcL in A. nidulans by Kelkar et al.

(1997) stopped ST synthesis but resulted in the accumulation of VER B. The stcL gene, for a P-450 monooxygenase, was shown to be required for the conversion of VER B to VER A. A homologous gene to A. nidulans stcL in the aflatoxin pathway gene cluster, verB, encoding a P-450 mono-oxygenase/desaturase, was cloned from A. parasiticus and A. flavus strains (Bhatnagar et al., unpublished, GenBank accession numbers: AF106958, AF106959, and AF106960 respectively).

2.3.9 VER a to Demethylsterigmatocystin (DMST) and VER B to

Demethyldihydrosterigmatocystin (DMDHST)

Another cloned gene involved in a key step aflatoxin synthesis is the ver-1 gene (Skory et al. 1992) in A. parasiticus, this gene was shown by complementing the ver-1 mutant to be required for the conversion of VER A to DMST in A. parasiticus. Keller et al. (1994) identified a gene stcU (formerly named verA) in A. nidulans, which is a homolog of ver-1 in A. parasiticus for a ketoreductase, required for the conversion of VER A to DMST. Double mutation of stcU and stcL resulted accumulation of only VER B. Keller et al. (1995a) also identified stcS(formerly named verB, Keller et al. 1995b), which is homologous to P-450 monooxygenases, to be involved in the conversion of VER A to DMST. Disruption of this gene resulted in the accumulation of VER A. Thus, both stcU and stcS are required for the conversion of VER B to DMST. The stcS homolog, named verA in A. parasiticus strain, was also identified (Yu, unpublished).

2.3.10 DMST to ST and DMDHST to DHST

Yabe et al. (1989) demonstrated a distinct methyltransferase activity in A. parasiticus for the conversion of DMST to ST. This enzyme may also be responsible for the conversion of DMDHST to DHST. The enzyme was purified and characterized (Yabe et al. 1998; Yabe et al. 1999). Disruption of stcP in A. nidulans by Kelkar et al. (1996) showed the requirement of this gene for the conversion from DMST to ST. The gene responsible for the conversion of DMST to ST and DMDHST to DHST were cloned by Motomura et al. (1999), named dmtA for O-methyltransferase I in A. parasiticus and concurrently by Yu et al. (2000b), named omtB for O-methyltransferase B in A. parasiticus, A. flavus and A. sojae.

2.3.11 ST To O-methylsterigmatocystin (OMST) and DMST to Dihydro-O-methylsterigmatocystin (DHOMST)

Several researchers (Bhatnagar et al. 1988; Keller et al. 1993; Yabe et al. 1989) reported the presence of an O-methyltransferase for the conversion of ST to OMST and DHST to DHOMST. Yu et al. (1993) cloned the cDNA

sequence from A. parasiticus (initially named omt-1, later renamed as omtA) by reverse genetics. The enzyme was expressed in Escherichia coli and its activity to convert ST to OMST was demonstrated by substrate feeding studies. The genomic DNA sequence of this gene (omtA) was cloned from A. parasiticus and A. flavus (Yu et al. 1995b). This omtA gene homologue was also detected in other aflatoxigenic and nonaflatoxigenic Aspergillus species (Klich et al. 1995).

2.3.12 OMST to Aflatoxin B1 (AFB1) and Aflatoxin G1 (AFG1) and DMDHST to Aflatoxin B2 (AFB2) and Aflatoxin G2 (AFG2)

By feeding experiments, Yabe et al. (1988a) proposed the relationship between B-Group (AFB1 and AFB2) and G-Group (AFG1 and AFG2) aflatoxin biosynthesis. It was also predicted by Bhatnagar et al. (1992) that in the late stages of aflatoxin biosynthesis, a NADPH-dependent mono-oxygenase is required for the conversion of OMST to AFB1. Prieto et al. (1996), Prieto and Woloshuk (1997) reported in A. flavus that a P-450 monooxygenase gene, ord-1, is required for this reaction. Yu et al. (1998) cloned the P-450 monooxygenase gene, ordA, from A. parasiticus and an A. flavus mutant strain and demonstrated by expression and substrate feeding in yeast system that this gene is responsible for the conversion of OMST to AFB1 and AFG1, and DHOMST to AFB2 and AFG2. In this study, the critical amino acids for the enzymatic activity and heme-binding motif were identified by site-directed mutagenesis as well. Yu et al. (1998) also demonstrated that additional enzyme(s) is required for the G-group (AFG1 and AFG2) aflatoxin synthesis. However, the enzyme(s) and corresponding gene(s) have not been isolated as yet. It should be noted that the functions of three additional genes in the aflatoxin pathway gene cluster, cypA(Yu, unpublished), cypX (cypX — stcB) and moxY (moxY — stcW) encoding cyto-chrome P-450 monooxygenases and monooxygenase (Yu et al. 2000a), have not yet been assigned. Keller et al. (2000) examined several characterized cytochrome P-450 mono-oxygenases and proposed the functions for some of the identified genes or ORFs encoding monooxygenases in aflatoxin/ST synthesis. However, there is a possibility that these genes might be involved in the G-group toxin formation.

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