Biosynthetic Pathways of Trichothecenes

From the first trichothecene skeleton, trichodiene, to the final product, T-2 toxin, through a series chemical modifications of oxidation, isomerization, cyclization, and esterification reactions, at least 16 know stable intermediates, which constitute the various members of the trichothecenes including the branch products DON and nivalenol, have been identified. The biochemical conversion scheme of these compounds are trichodiene, 2-hydroxytrichodiene, 12,13 epoxy-9,10-trichoene-2-ol, isotrichodiol, trichotriol, isotri-chodermol, isotrichodermin, 15-decalonectrin, calonectrin, 3,15-DAS, 3,4,15-triacetoxyscirpenol, 3-acetoxyscirpenol, 3-acetyl T-2 toxin, T-2 toxin. The DON is thought to have branched from 15-decalonectrin and the nivalenol is thought to have branched from 3,15-DAS (Brown et al. 2001; Proctor 2000). DON could be converted directly to nivalenol (Figure 2).

The initial step of the trichothecene biosynthesis involves the incorporation of three molecules of mevalonate into the trichothecene nucleus of trichothecin with the cyclization of farnesyl pyrophosphate to produce trichodiene. TRI5 is the first characterized pathway gene that encodes a trichodiene synthase involved in this initial step of trichothecene biosynthesis (Hohn and Beremand 1989). Hohn and Desjardins (1992) isolated a gene, named Tox5, encoding a trichodiene synthases for the synthesis of trichodiene from farnesyl pyrophosphate. The Tox5 gene (renamed TRI5) was characterized in great detail by Desjardins et al. (1987), Desjardins et al. (1993), Cane et al. (1995), and Hohn et al. (1993b). The predicted amino acid sequences of the TRI4 and TRI11 gene products were similar to those of cytochrome P-450 monooxygenases (Alexander et al. 1998; Hohn et al. 1995). TRI4 was found to catalyze the oxygenation of trichodiene at the C-2 position to yield 2-hydroxytrichocdiene (Hohn et al. 1995). The most significant oxidation step in trichothecene biosynthesis is provided by this cytochrome P-450 monooxygenase encoded by TRI4 to introduce the C-12, 13 epoxide. Alexander et al. (1998) demonstrated that the TRI11 is involved in the conversion of isotrichodermin to 15-decalonectrin by oxygenating the C-15 position of isotrichodermin. Studies by McCormick et al. (1999) indicated that the acetylation of isotrichodermin is catalyzed by TRI101. This gene may also play a role in self-protection against the toxic effect of trichothecene to the fungus by modulating trichothecene production (Muhitch et al. 2000).

By gene disruption experiment, the TRI3 gene was found to encode an acetyltransferase catalyzing the conversion of 15-decalonectrin to calonectrin via acetylation at the C-15 position (McCormick et al. 1996). Gene disruption and precursor feeding studies in F. sporotrichioides by Brown et al. (2001) demonstrated that TRI7 might function as C-4 acetylase or acetylesterase involved in the conversion from 3,15-DAS to 3,4,15-triacetoxyscirpenol. However, no functional enzyme encoded by TRI7 was predicted in F. graminearum due to the lack of initiation codon, six possible frameshifts, and early stop codon (Brown et al. 2001). Brown et al. (2001) demonstrated that disruption of the TRI8 gene failed to produce T-2 toxin instead accumulated 4,15-DAS. The chemical reaction was presumably involved in the 3-deacetylation of 3,4,15-DAS. Since the predicted amino acid sequence is homologous to lipase, the TRI8 might encode an esterase/oxygenase and be responsible for esterification of the C-8 oxygen for the conversion somewhere between 3,4,15-triacetoxyscirpenol and 3-acetyl T-2 toxin. TRI12 encode a toxin efflux pump (Alexander et al. 1999). TRI6 is a regulatory gene (Desjardins et al. 1993; Hohn et al. 1999; Keller and Hohn 1997; Matsumoto et al. 1999; Proctor et al. 1995b; Proctor 2000; Trapp et al. 1998). TRI10 encodes a polypeptide of 420 amino acids for another regulatory protein (Tag et al. 2001). The regulatory function of the TRI6 and TRI10 genes will be discussed in more detail in the next section. The enzymes responsible for some of the biosynthetic steps, such as the last step for the synthesis of T-2 toxin from 3-acetyl T-2 toxin by a deacetylase, have been proposed. However, the genes for those enzymes have not yet been identified (Brown et al. 2001). Most recently, two additional new genes, TRI13 (from Gibberrella zeae) and TRI14 (from F. sporotrichioides), involved in trichothecene biosynthesis, have been isolated and deposited in the GenBank database (Daren W. Brown et al., personal communication).

Synthesis of the structurally more complex macrocyclic trichothecenes such as roridin E, verrucrin, and baccharinoid B7 is most commonly by the genus Myrothecium. The macrocyclic trichothecenes exhibit about 10-fold more toxic than the Fusarium trichothecenes. Trapp et al. (1998) identified, in M. roridum, the homologues of the F. sporotrichioides TRI5 (MrTRI5), TRI4 (MrTRI4), and TRI6 (MrTRI6) genes which were located within a region of about 40 kb in M. roridum compared to only 8-kb in the F. sporotrichioides. The deduced amino acid sequence of the MrTRI5 product demonstrated a homology of 72% to 75% to trichodiene synthases from four Fusarium species. The predicted MrTRI6 gene product was twice as large as that of the FsTRI6 with low amino acid homologies except in the C-terminal region of MrTRI6 (78%) where the zinc-finger motifs reside.

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