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Biosynthesis of 3-Acetyldeoxynivalenol and Sambucinol

Instead of deprotonation at C12 as observed for trichodiene biosynthesis ..

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The Trichothecenes and Their Biosynthesis | SpringerLink

The x-ray crystal structure of recombinant trichodiene synthase from Fusarium sporotrichioides has been determined to 2.5-A resolution, both unliganded and complexed with inorganic pyrophosphate. This reaction product coordinates to three Mg(2+) ions near the mouth of the active site cleft. A comparison of the liganded and unliganded structures reveals a ligand-induced conformational change that closes the mouth of the active site cleft. Binding of the substrate farnesyl diphosphate similarly may trigger this conformational change, which would facilitate catalysis by protecting reactive carbocationic intermediates in the cyclization cascade. Trichodiene synthase also shares significant structural similarity with other sesquiterpene synthases despite a lack of significant sequence identity. This similarity indicates divergence from a common ancestor early in the evolution of terpene biosynthesis.

The trichothecenes are a group of naturally-occurring sesquiterpenoid epoxides ..

The x-ray crystal structure of recombinant trichodiene synthase from Fusarium sporotrichioides has been determined to 2.5-A resolution, both unliganded and complexed with inorganic pyrophosphate. This reaction product coordinates to three Mg(2+) ions near the mouth of the active site cleft. A comparison of the liganded and unliganded structures reveals a ligand-induced conformational change that closes the mouth of the active site cleft. Binding of the substrate farnesyl diphosphate similarly may trigger this conformational change, which would facilitate catalysis by protecting reactive carbocationic intermediates in the cyclization cascade. Trichodiene synthase also shares significant structural similarity with other sesquiterpene synthases despite a lack of significant sequence identity. This similarity indicates divergence from a common ancestor early in the evolution of terpene biosynthesis.

Bioscience, Biotechnology, and Biochemistry - …

Trichodiene presence determined by a dynamic headspace method can serve as an indicator for undergoing trichothecene toxins biosynthesis.

The presence of a C-15 hydroxyl group is characteristic of most Fusarium trichothecenes and all of the macrocyclic trichothecenes. Recently, it was reported that microsomal fractions from Fusarium culmorum can convert isotrichodermin to 15-decalonectrin, 7α-hydroxyisotrichodermin, and 8α-hydroxyisotrichodermin (). The reaction conditions used in these experiments were consistent with the established requirements for cytochrome P-450 monooxygenases; however, the oxygenation of isotrichodermin did not appear to be inhibited by known P-450 inhibitors such as carbon monoxide and cyanide. On the basis of these results, it was concluded that cytochromes P-450 were not likely to be involved in the later oxygenation steps of trichothecene biosynthesis. Our identification of TRI11 as the C-15 hydroxylase in F. sporotrichioides indicates that cytochromes P-450 do catalyze some late pathway steps. Other evidence supporting the participation of cytochromes P-450 after the isotrichodermin step in the pathway include the observation that all of the oxygens linked directly to the trichodiene carbon skeleton are derived from molecular oxygen (). The apparent failure of known P-450 inhibitors to affect the cell-free oxygenation of isotrichodermin may reflect differences in the sensitivity of these enzymes to the inhibitors or the need for different experimental conditions to demonstrate their effectiveness. Identification of TRI11 as a cytochrome P-450 involved in trichothecene biosynthesis further emphasizes the importance of this group of enzymes in mycotoxin biosynthetic pathways.

Trichothecenes are a large family of sesquiterpenoid secondary metabolites of species (, ) and other molds. They are major mycotoxins that can cause serious problems when consumed contaminated cereal grains. In the past 20 years, an outline of the trichothecene biosynthetic pathway has been established based on the results of precursor feeding experiments and blocked mutant analyses. Following the isolation of the pathway gene encoding the first committed enzyme trichodiene synthase, 10 biosynthesis genes ( genes; two regulatory genes, seven pathway genes, and one transporter gene) were functionally identified in the gene cluster. At least three pathway genes, (separated alone), and and (located in the two-gene cluster), were found outside of the gene cluster. In this review, we summarize the current understanding of the pathways of biosynthesis, the functions of cloned genes, and the evolution of genes, focusing on species.

Improved synthesis of (+/-)-trichodiene--a volatile …

Trichodiene presence determined by a dynamic headspace method can serve as an indicator for undergoing trichothecene toxins biosynthesis.

We examined the functions of Tatri4, Tatri5, and Tatri11 by expressing each gene independently in yeast. For Tatri5 expression, transformed yeast cells were analyzed without the addition of a trichothecene biosynthetic intermediate, because the substrate for the Fusarium Tri5 enzyme is farnesyl pyrophosphate, which is an intermediate of primary metabolism in yeast. Extraction and GCMS analysis of pYETri5-transformed yeast cultures following galactose induction revealed the presence of trichodiene (). This is the same product of the reaction catalyzed by the Fusarium Tri5 enzyme and the first intermediate in the trichothecene biosynthetic pathway. The presence of trichodiene was observed in four independent yeast transformants carrying pYETri5. These results indicate that the TaTri5 enzyme catalyzes cyclization of farnesyl pyrophosphate to form trichodiene.

The genus Trichoderma includes species that have been evaluated extensively as biological control agents (, ). Strains of Trichoderma can produce extracellular enzymes and antibiotics but also may compete with fungal pathogens for space and nutrients through rhizosphere competence (). Some species of Trichoderma can produce the trichothecenes trichodermin and harzianum A (HA). These metabolites are similar to the Fusarium trichothecenes T-2 toxin, NIV, and DON in that they have the core tricyclic 12,13-epoxytrichothec-9-ene (EPT) structure. They are also similar to T-2 and NIV in that they have an oxygen atom attached to carbon atom 4 (C-4). Although their structures are well characterized, biosynthesis of HA and trichodermin by Trichoderma has been subjected to little genetic analysis. However, orthologues of TRI5 have been identified in a trichodermin-producing strain (IBT 40841) of Trichoderma brevicompactum () and a harzianum A-producing strain (ATCC 90237) of Trichoderma arundinaceum (, ). The identification of the TRI5 orthologues in the two Trichoderma species indicates that the first committed reactions in trichothecene biosynthesis in Fusarium and Trichoderma are the same (). However, the absence of the C-3, C-8, and C-15 oxygen atoms in trichodermin and HA indicates that there are multiple differences in trichothecene biosynthesis in the two genera. These differences probably are reflected in the presence/absence/divergence of functional orthologs of some trichothecene biosynthetic genes in the two genera. For example, in Fusarium, the TRI4-encoded cytochrome P450 monooxygenase catalyzes oxygenation of trichodiene at four carbons, C-2, C-3, C-11, and C-13 (). In contrast, the Myrothecium TRI4-encoded monooxygenase catalyzes oxygenation of trichodiene at only three carbons, C-2, C-11, and C-13 (). This difference in patterns of oxygenation is consistent with the presence of a C-3 oxygen in Fusarium trichothecenes and its absence in Myrothecium trichothecenes. The absence of a C-3 oxygen in trichodermin and HA suggests that the Trichoderma Tri4 orthologue is more similar in function to the Myrothecium than the Fusarium orthologue. The absence of C-8 and C-15 oxygen atoms in trichodermin and HA also suggests that trichothecene-producing Trichoderma strains lack orthologues of the Fusarium trichothecene C-8 and C-15 oxygenase genes TRI1 and TRI11, respectively. In contrast, the presence of the C-4 oxygen atom in trichodermin and HA suggests that T. brevicompactum and T. arundinaceum have an orthologue of the Fusarium trichothecene C-4 oxygenase gene TRI13.

08/03/2017 · Analysis of the interaction of trichodiene synthase 5 (TRI5) with natural and natural-like inhibitors of trichothecene biosynthesis
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For Tatri4 expression, cultures of pYETri4-transformed yeast were induced with galactose and then fed trichodiene, the substrate of the Tri4 enzymes in Fusarium and Myrothecium. Extraction and GCMS analysis of the resulting cultures revealed the presence of the trichothecene biosynthetic intermediate isotrichodiol (). This result was confirmed in eight yeast transformants. The function of TaTri4 was also assessed by introducing a Tatri4 expression cassette, on plasmid pSPT4-4, into the trichothecene-nonproducing fungus F. verticillioides. The addition of trichodiene to cultures of eight F. verticillioides transformants carrying the expression cassette also resulted in the formation of isotrichodiol ().

Molecular and Genetic Studies of Fusarium …

The biosynthesis of the Fusarium trichothecenes T-2 toxin (T-2), nivalenol (NIV), and deoxynivalenol (DON) has been subjected to extensive biochemical and genetic analyses (reviewed in references and ). These studies have elucidated a complex biosynthetic pathway that begins with the cyclization of farnesyl pyrophosphate (FPP) to form trichodiene, which then undergoes a series of oxygenation, isomerization, cyclization, and esterification reactions to form T-2 toxin, nivalenol, or deoxynivalenol. Most of the Fusarium genes directly involved in synthesis of the toxins are positioned at a locus designated the core trichothecene biosynthetic gene (TRI) cluster (). In the two most thoroughly examined species, Fusarium graminearum and Fusarium sporotrichioides, the cluster can include seven genes (TRI8, TRI7, TRI3, TRI4, TRI5, TRI11, TRI13) encoding enzymes that catalyze 10 trichothecene biosynthetic reactions, two genes (TRI6 and TRI10) encoding transcriptional regulators, one gene (TRI12) encoding a transport protein, and two genes (TRI9 and TRI14) with uncertain functions. Some Fusarium spp. have two additional TRI loci located on chromosomes other than the one on which the core cluster is located (, ). One locus is a cluster that contains two genes (TRI1 and TRI16), which encode biosynthetic enzymes, and the other locus contains a single gene (TRI101) that also encodes a biosynthetic enzyme. In some Fusarium species, however, TRI1 and TRI101 are located in the core TRI cluster (). Myrothecium roridum has a 40-kb cluster that includes orthologs of TRI4, TRI5, and TRI6 (). Whether other TRI orthologs are present in this cluster is not known.

MetaCyc superpathway of trichothecene biosynthesis

The difference in gene content of the clusters indicates that genes have been gained and/or lost during evolution of Trichoderma and Fusarium. Perhaps the most notable difference is the absence of a tri5/TRI5 orthologue in the Trichoderma cluster. Whether the Trichoderma tri5 and tri clusters are physically linked on the same chromosome or located on different chromosomes has not been determined. Results from the current study and those from previous studies () indicate that the Trichoderma and Fusarium tri/TRI5 orthologues encode trichodiene synthase, the enzyme that catalyzes the first committed step in trichothecene biosynthesis. Thus, tri5/TRI5 is essential for trichothecene production in both fungi. The location of the tri5/TRI5 orthologue outside the tri cluster in Trichoderma but inside the cluster in Fusarium and Myrothecium (; R. H. Proctor, unpublished data) suggests that the ancestral tri/TRI cluster included a tri5/TRI5 gene but that the gene moved out of the cluster to another genomic location during evolution of Trichoderma. One hypothesis for the function of secondary metabolite biosynthetic gene clusters is that they facilitate vertical and horizontal inheritance of all genes directly involved in the biosynthesis of a secondary metabolite (). A second hypothesis for the function of secondary metabolite biosynthetic gene clusters is that they facilitate coordinated expression of all genes directly involved in biosynthesis of a metabolite (). The location of tri5 outside the Trichoderma tri cluster runs counter to both hypotheses in that tri5 would not necessarily be inherited with the rest of the cluster and its expression is correlated with other tri genes and with trichothecene production by Trichoderma (S. Gutiérrez, unpublished data).

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