Characterization of a Fusarium 2-Gene Cluster Involved in Trichothecene C-8 Modification

Abstract

The Fusarium trichothecenes T-2 toxin and deoxynivalenol (DON) are potent inhibitors of eukaryotic protein synthesis and are a significant agricultural problem. Three coregulated loci are required for T-2 toxin synthesis by Fusarium sporotrichioides. The core-trichothecene gene cluster consists of 12 genes (Tri3-Tri14) while the second locus consists of a single gene (Tri101). The third locus was recently partially described and encodes 1-2 biosynthetic enzymes and a putative regulatory gene. Here, we describe a detailed characterization of this locus. Located adjacent to Tri1 is Tri16, which is required for esterification of the C-8 hydroxyl. A putative regulatory gene, also adjacent to Tri1, is not required for T-2 toxin synthesis. The genomic sequence of Fusarium graminearum (a DON producer) contains a putative functional Tri1 and a nonfunctional Tri16. The presence of the Tri16 pseudogene is consistent with the chemical structure of DON, which has a C-8 keto group rather than the C-8 ester of T-2 toxin.

Identification of Two Aflatrem Biosynthesis Gene Loci in Aspergillus flavus and Metabolic Engineering of Penicillium paxilli To Elucidate Their Function

“…the genes for trichothecene biosynthesis are split between three coregulated loci including a core cluster of 10 or 11 genes (3), a two-gene minicluster (4), and a third locus with a single trichothecene gene (Tri101) (15). Genes in the outlying miniclusters appear to have been recruited for trichothecene biosynthesis more recently than those in the core cluster, and Tri101 appears to have evolved separately, suggesting that these genes have never been located at a single locus (4,16). The physical arrangement of atm genes in A. flavus and A. oryzae is likely to have arisen by fragmentation of a single ancestral cluster.…”

Functional demarcation of the Fusarium core trichothecene gene cluster

“…TRI6 is a DNA-binding regulatory protein that acts as a positive regulator of trichothecene biosynthesis . All previously characterized TRI structural genes, located within and outside the core cluster, have TRI6 binding sites in their promoter regions (Brown et al, 2001(Brown et al, , 2003McCormick et al, 1999). Second, Northern blot analysis using hybridization probes to all 12 ORFs indicate that they do not exhibit the same pattern of expression as genes located within (e.g., TRI5) or outside (e.g., TRI1 and TRI16) the trichothecene gene cluster.…”

“…For example, the Gibberella fujikuroi gibberellin biosynthetic genes P450-1 and P450-4 encode monooxygenases that catalyze 4 and 3 biosynthetic steps, respectively (Helliwell et al, 2001;Tudzynski et al, 2002). TRI11, TRI13, and TRI1 are unlikely multifunctional candidates because gene deletion studies clearly indicated that their proteins catalyze single steps later in the pathway (Alexander et al, 1998;Brown et al, 2003;Hohn et al, 1995). Interestingly, TRI4 transcripts are 8 times more abundant than the three other trichothecene P450 monooxygenases (Table 5) in the F. sporotrichioides EST library.…”

“…It has been noted that both of these steps may proceed non-enzymatically (McCormick et al, 1990). If genes are required for any of these five un-assigned biochemical steps, they may be identified by co-expression studies using ESTs; an approach used successfully used to find the genes required for C-8 modification (Brown et al, 2003;Meek et al, 2003).…”

“…The final 3 trichothecene genes are located elsewhere in the genome. TRI101, encoding a C-3 acetyltransferase (Kimura et al, 1998), is flanked by two house-keeping genes while TRI1, encoding a C-8 oxygenase (Brown et al, 2003;Meek et al, 2003), and TRI16, encoding a C-8 acyltransferase (Brown et al, 2003;Peplow et al, 2003) are adjacent to each other and are flanked by non-trichothecene related genes (Brown et al, 2003).…”