Verification of Acetylation of Trichodermol to Trichodermin in the Plant Endophyte .

Trichodermin, a trichothecene first isolated in species, is a sesquiterpenoid antibiotic that exhibits significant inhibitory activity to the growth of many pathogenic fungi such as , , and by inhibiting the peptidyl transferase involved in eukaryotic protein synthesis. Trichodermin has also been shown to selectively induce cell apoptosis in several cancer cell lines and thus can act as a potential lead compound for developing anticancer therapeutics. The biosynthetic pathway of trichodermin in has been identified, and most of the involved genes have been functionally characterized. An exception is , which encodes a putative acetyltransferase. Here, we report the identification of a gene cluster that contains seven genes expectedly involved in trichodermin biosynthesis (, , , , , , and ) in the trichodermin-producing endophytic fungus . As in , is not included in the cluster. Functional analysis provides evidence that TRI3 acetylates trichodermol, the immediate precursor, to trichodermin. Disruption of gene eliminated the inhibition to by culture filtrates and significantly reduced the production of trichodermin but not of trichodermol. Both the inhibitory activity and the trichodermin production were restored when native gene was reintroduced into the disruption mutant. Furthermore, a His-tag-purified TRI3 protein, expressed in , was able to convert trichodermol to trichodermin in the presence of acetyl-CoA. The disruption of also resulted in lowered expression of both the upstream biosynthesis genes and the regulator genes. Our data demonstrate that encodes an acetyltransferase that catalyzes the esterification of the C-4 oxygen atom on trichodermol and thus plays an essential role in trichodermin biosynthesis in this fungus.