Abstract
Trichodermin, a trichothecene first isolated in Trichoderma species, is a sesquiterpenoid antibiotic that exhibits significant inhibitory activity to the growth of many pathogenic fungi such as Candida albicans, Rhizoctonia solani, and Botrytis cinerea 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 Trichoderma has been identified, and most of the involved genes have been functionally characterized. An exception is TRI3, 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, and TRI14) in the trichodermin-producing endophytic fungus Trichoderma taxi. As in Trichoderma brevicompactum, TRI5 is not included in the cluster. Functional analysis provides evidence that TRI3 acetylates trichodermol, the immediate precursor, to trichodermin. Disruption of TRI3 gene eliminated the inhibition to R. solani by T. taxi 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 TRI3 gene was reintroduced into the disruption mutant. Furthermore, a His-tag-purified TRI3 protein, expressed in Escherichia coli, was able to convert trichodermol to trichodermin in the presence of acetyl-CoA. The disruption of TRI3 also resulted in lowered expression of both the upstream biosynthesis TRI genes and the regulator genes. Our data demonstrate that T. taxi TRI3 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.
Highlights
Trichothecenes are a group of naturally occurring sesquiterpenoids capable of inhibiting the function of eukaryotic ribosomes, thereby causing hemorrhagic lesions, dermatitis, and immunological problems to people and animals (Pace et al, 1998; McLaughlin et al, 2009; McCormick et al, 2011)
The results reveal that TRI clusters in T. taxi are significantly similar to those in T. arundinaceum and T. brevicompactum and that TtTRI3 encodes an acetyltransferase that performs the step by acetylating trichodermol at C-4 to trichodermin
Further BLASTp search found that the contig encodes several other TRI orthologues, namely, TtTRI4, TtTRI6, TtTRI10, TtTRI11, TtTRI12, and TtTRI14, and that they were located in immediate vicinity on the contig
Summary
Trichothecenes are a group of naturally occurring sesquiterpenoids capable of inhibiting the function of eukaryotic ribosomes, thereby causing hemorrhagic lesions, dermatitis, and immunological problems to people and animals (Pace et al, 1998; McLaughlin et al, 2009; McCormick et al, 2011). Trichodermin was first isolated from a Trichoderma species as an antifungal antibiotic that is active against a variety of pathogenic fungi including Candida albicans (Godtfredsen and Vangedal, 1965) This sesquiterpenoid metabolite is known to be produced by only two Trichoderma species (Trichoderma brevicompactum and Trichoderma arundinaceum; Kubicek and Druzhinina, 2015; Nielsen et al, 2005) and exhibits significant inhibition to several human pathogenic Candida spp. and a nosocomial strain of the filamentous fungus Aspergillus fumigatus (Tijerino et al, 2011). Trichodermin has drawn attentions from cancer biologists because it can induce cell apoptosis through mitochondrial dysfunction and endoplasmic reticulum stress in human chondrosarcoma cells and by mitotic arrest and DNA damage in human p53-mutated pancreatic cancer cells (Su et al, 2013; Chien et al, 2016) These new findings suggest that trichodermin is a potential therapeutic agent worthy of further development to a clinical trial candidate for treating cancer. A thorough understanding of the biosynthetic pathway leading to trichodermin is essential
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