The Clustering of Aflatoxin Biosynthetic Pathway Genes

Significant progress has been made in the last decade in deciphering aflatoxin biosynthetic pathway (Bennett 1981; 1987; 1986; 1994; Bhatnagar et al. 1988; 1991; 1992; 1993; 1998; Bhatnagar and Cleveland 1990; Cary et al. 2000a,b; Chang et al. 1992; 1993; 1995a,b; 2000b; Cleveland and Bhatnagar 1992; 2002; Dutton 1988; Minto and Townsend 1997; Papa 1982; 1984; Payne 1998; Payne and Brown 1998; Silva and Townsend 1996; Silva et al. 1996; Townsend 1997; Trail et al. 1995a; 1995b; Yu et al. 1993; 1995a; 1997; 1998; 2000a,b). As many as 15 structurally defined aflatoxin intermediates have been identified in the aflatoxin/ST biosynthetic pathway starting with the acetate, the polyketide precursors. At least 23 enzymatic steps involved in the aflatoxin biosynthesis have been characterized or proposed (Figure 1).

It has been long proposed that the aflatoxin pathway genes may be clustered with a common regulator (Cleveland and Bhatnagar 1991). The first experimental evidence showing the potential clustering of aflatoxin pathway genes was, however, demonstrated by Trail et al. (1995b) when they found that the nor-1 and ver-1 genes were linked in a cosmid clone (NorA) with the regulatory gene aflR and a putative aflatoxin pathway gene, uvm8 (now named fas-1) in between. This observation provided evidence that at least the early stages of the pathway may be linked. By mapping overlapping cosmid clones in A. parasiticus and A. flavus (Yu et al. 1995a), a linkage of genes involved in aflatoxin formation, from early stage nor-1 gene to later stage omtA, was established indicating that the entire aflatoxin biosynthetic pathway genes may be clustered. This observation and subsequent discoveries of pathway genes led to a consensus cluster map consisting of at least nine aflatoxin pathway genes pksA, nor-1, uvm8 (now named fas-1), aflR, ver-1, omtA and three additional open reading frames (ORFs) with unknown function (ord-1, now named avnA; ord-2, now named ordA; and aad, now named norA) (Yu et al. 1995a). The concept of aflatoxin pathway gene cluster greatly accelerated the rate of gene discovery (Cary et al. 1996; Chang et al. 1995a; 2000b; Chang and Yu 2002; Feng and Leonard 1995; Mahanti et al. 1996; Silva et al. 1996; Silva and Townsend 1996; Yu et al. 1997; 1998; 2000a,b,c). It was identified that at least 23 genes (Cary et al. 1996; Chang et al. 1992; 1993; 1995a; 2000b; Chang and Yu 2002; Cleveland et al. 1997; Meyers et al. 1998; Motomura et al. 1999; Prieto and Woloshuk 1997; Silva et al. 1996; Silva and Townsend 1996; Yu et al. 1993; 1995a; 1997; 1998; 2000a,b) or ORFs involved in aflatoxin biosynthesis including the regulatory genes, aflR and aflJ, were clustered together within approximately 80 kb DNA regions in the A. parasiticus and A. flavus genomes respectively. Most of these genes on the aflatoxin pathway gene cluster have been cloned and characterized. The cloning of cypX and moxY genes (Yu et al. 2000a), the ordB gene (potentially for an oxidoreductase, Yu, unpublished), and the sugar utilization gene cluster (Yu et al. 2000c) defined one end of the aflatoxin pathway gene cluster (Figure 1, panel A). The norB, cypA and aflT might possibly mark the other end of

Figure 1 Proposed and generally accepted pathway for aflatoxin B1, B2, G1 and G2 biosynthesis and the corresponding genes and their enzymes are presented. The aflatoxin biosynthetic pathway gene cluster in A. parasiticus and A. flavus (panel A), sterigmatocystin biosynthetic pathway gene cluster in A. nidulans (panel B), and the non-functional, partially duplicated aflatoxin gene cluster in A. parasiticus (panel C) are shown. The gene names are labeled on the side of the cluster. Arrows indicate the direction of gene transcription. The homologous genes between the sterigmatocystin pathway gene cluster in A. nidulans and aflatoxin pathway gene cluster in A. parasiticus and A. flavus is connected by line. The four sugar utilization genes linked to the aflatoxin pathway gene cluster are on the bottom of panel A. Abbreviations for the intermediates are: norsolorinic acid (NOR), averantin (AVN), 5'-hydroxyaverantin (HAVN), averufanin (AVNN), averufin (AVF), versiconal hemiacetal acetate (VHA), versiconal (VAL), versicolorin B (VER B), versicolorin A (VER A), demethylsterigmatocystin (DMST), sterigmatocystin (ST), O-methylsterigmatocystin (OMST), aflatoxin B1 (AFB1), aflatoxin G1 (AFG1), DMDHST, dihydrosterigmatocystin (DHST), dihydro-O-methylsterigmatocystin (DHOMST), aflatoxin B2 (AFB2), and aflatoxin G2 (AFG2), Methyltransferase (M-transferase).

this cluster (Yu et al., unpublished; Chang et al., unpublished; Figure 1, panel A). Using a similar strategy, Brown et al. (1996b) defined ST biosynthetic pathway gene cluster consisting of 25 coregulated transcripts in A. nidulans. The ST gene cluster contains probably a complete set of ST genes within approximately 60-kb DNA fragment (Brown et al. 1996b). The aflatoxin pathway gene clusters between A.flavus and A. parasiticus are identical in term of the sequential order of the genes, the sizes of these genes and the direction of gene transcription. The nucleotide and amino acid sequences of the aflatoxin pathway genes are also highly conserved (> 95%) between A. parasiticus and A. flavus (Yu et al. 1995a). Analyzing the corresponding genes and their enzymes for a specific pathway conversion step between A. flavus/ A. parasiticus for aflatoxin synthesis and A. nidulans for ST synthesis, it has been shown a high degree of similarity with respect to the genes, the enzyme encoded by these genes and their enzyme function. However, the order of these genes on the chromosome between the two clusters are totally scrambled (Figure 1, panel A & B).

In A. parasiticus, duplication of aflatoxin genes ver-1 and aflR was first suggested (Mehigh et al., unpublished; Liang and Linz, unpublished) and reported by Liang et al. (1996). This partial duplicated aflatoxin gene cluster consisting of seven duplicated genes, aflR2, aflJ2, adhA2, estA2, norA2, ver1B, omtB2, has been cloned and characterized by Chang and Yu (2002). The genes within this partial duplicated cluster, due possibly to the chromosome location (Chiou et al. 2002; Yu et al., unpublished), were found to be unfunctional under normal conditions even though no apparent defects are identified in some of these genes (aflR2, aflJ2, adhA2, estA2).

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