Verification of TRI3 Acetylation of Trichodermol to Trichodermin in the Plant Endophyte Trichoderma taxi

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Trichodermin, a trichothecene first isolated inTrichodermaspecies, is a sesquiterpenoid antibiotic that exhibits significant inhibitory activity to the growth of many pathogenic fungi such asCandida albicans,Rhizoctonia solani, andBotrytis cinereaby 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 inTrichodermahas been identified, and most of the involved genes have been functionally characterized. An exception isTRI3, which encodes a putative acetyltransferase. Here, we report the identification of a gene cluster that contains seven genes expectedly involved in trichodermin biosynthesis (TRI3,TRI4,TRI6,TRI10,TRI11,TRI12, andTRI14) in the trichodermin-producing endophytic fungusTrichoderma taxi. As inTrichoderma brevicompactum,TRI5is not included in the cluster. Functional analysis provides evidence that TRI3 acetylates trichodermol, the immediate precursor, to trichodermin. Disruption ofTRI3gene eliminated the inhibition toR. solanibyT. taxiculture filtrates and significantly reduced the production of trichodermin but not of trichodermol. Both the inhibitory activity and the trichodermin production were restored when nativeTRI3gene was reintroduced into the disruption mutant. Furthermore, a His-tag-purified TRI3 protein, expressed inEscherichia coli, was able to convert trichodermol to trichodermin in the presence of acetyl-CoA. The disruption ofTRI3also resulted in lowered expression of both the upstream biosynthesisTRIgenes and the regulator genes. Our data demonstrate thatT. taxi TRI3encodes 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.