A Role in 15-Deacetylcalonectrin Acetylation in the Non-Enzymatic Cyclization of an Earlier Bicyclic Intermediate in Fusarium Trichothecene Biosynthesis.

The trichothecene biosynthesis in Fusarium begins with the cyclization of farnesyl pyrophosphate to trichodiene, followed by subsequent oxygenation to isotrichotriol. This initial bicyclic intermediate is further cyclized to isotrichodermol (ITDmol), a tricyclic precursor with a toxic trichothecene skeleton. Although the first cyclization and subsequent oxygenation are catalyzed by enzymes encoded by Tri5 and Tri4, the second cyclization occurs non-enzymatically. Following ITDmol formation, the enzymes encoded by Tri101, Tri11, Tri3, and Tri1 catalyze 3-O-acetylation, 15-hydroxylation, 15-O-acetylation, and A-ring oxygenation, respectively. In this study, we extensively analyzed the metabolites of the corresponding pathway-blocked mutants of Fusarium graminearum. The disruption of these Tri genes, except Tri3, led to the accumulation of tricyclic trichothecenes as the main products: ITDmol due to Tri101 disruption; a mixture of isotrichodermin (ITD), 7-hydroxyisotrichodermin (7-HIT), and 8-hydroxyisotrichodermin (8-HIT) due to Tri11 disruption; and a mixture of calonectrin and 3-deacetylcalonectrin due to Tri1 disruption. However, the ΔFgtri3 mutant accumulated substantial amounts of bicyclic metabolites, isotrichotriol and trichotriol, in addition to tricyclic 15-deacetylcalonectrin (15-deCAL). The ΔFgtri5ΔFgtri3 double gene disruptant transformed ITD into 7-HIT, 8-HIT, and 15-deCAL. The deletion of FgTri3 and overexpression of Tri6 and Tri10 trichothecene regulatory genes did not result in the accumulation of 15-deCAL in the transgenic strain. Thus, the absence of Tri3p and/or the presence of a small amount of 15-deCAL adversely affected the non-enzymatic second cyclization and C-15 hydroxylation steps.

Attempting to Create a Pathway to 15-Deacetylcalonectrin with Limited Accumulation in Cultures of Fusarium Tri3 Mutants: Insight into Trichothecene Biosynthesis Machinery.

The compound 15-deacetylcalonectrin (15-deCAL) is a common pathway intermediate in the biosynthesis of Fusarium trichothecenes. This tricyclic intermediate is metabolized to calonectrin (CAL) by trichothecene 15-O-acetyltransferase encoded by Tri3. Unlike other trichothecene pathway Tri gene mutants, the Δtri3 mutant produces lower amounts of the knocked-out enzyme's substrate 15-deCAL, and instead, accumulates higher quantities of earlier bicyclic intermediate and shunt metabolites. Furthermore, evolutionary studies suggest that Tri3 may play a role in shaping the chemotypes of trichothecene-producing Fusarium strains. To better understand the functional role of Tri3p in biosynthesis and evolution, we aimed to develop a method to produce 15-deCAL by using transgenic Fusarium graminearum strains derived from a trichothecene overproducer. Unfortunately, introducing mutant Tri3, encoding a catalytically impaired but structurally intact acetylase, did not improve the low 15-deCAL production level of the ΔFgtri3 deletion strain, and the bicyclic products continued to accumulate as the major metabolites of the active-site mutant. These findings are discussed in light of the enzyme responsible for 15-deCAL production in trichothecene biosynthesis machinery. To efficiently produce 15-deCAL, we tested an alternative strategy of using a CAL-overproducing transformant. By feeding a crude CAL extract to a Fusarium commune strain that was isolated in this study and capable of specifically deacetylating C-15 acetyl, 15-deCAL was efficiently recovered. The substrate produced in this manner can be used for kinetic investigations of this enzyme and its possible role in chemotype diversification.