Abstract
Trichothecene mycotoxins such as deoxynivalenol, 4,15-diacetoxyscirpenol, and T-2 toxin, are potent protein synthesis inhibitors for eukaryotic organisms. The 3-O-acetyl derivatives of these toxins were shown to reduce their in vitro activity significantly as assessed by assays using a rabbit reticulocyte translation system. The results suggested that the introduction of an O-acetyl group at the C-3 position in the biosynthetic pathway works as a resistance mechanism for Fusarium species that produce t-type trichothecenes (trichothecenes synthesized via the precursor trichotriol). A gene responsible for the 3-O-acetylation reaction, Tri101, has been successfully cloned from a Fusarium graminearum cDNA library that was designed to be expressed in Schizosaccharomyces pombe. Fission yeast transformants were selected for their ability to grow in the presence of T-2 toxin, and this strategy allowed isolation of 25 resistant clones, all of which contained a cDNA for Tri101. This is the first drug-inactivating O-acetyltransferase gene derived from antibiotic-producing organisms. The open reading frame of Tri101 codes for a polypeptide of 451 amino acid residues, which shows no similarity to any other proteins reported so far. TRI101 from recombinant Escherichia coli catalyzes O-acetylation of the trichothecene ring specifically at the C-3 position in an acetyl-CoA-dependent manner. By using the Tri101 cDNA as a probe, two least overlapping cosmid clones that cover a region of 70 kilobase pairs have been isolated from the genome of F. graminearum. Other trichothecene biosynthetic genes, Tri4, Tri5, and Tri6, were not clustered in the region covered by these cosmid clones. These new cosmid clones are considered to be located in other parts of the large biosynthetic gene cluster and might be useful for the study of trichothecene biosynthesis.
Highlights
Trichothecene mycotoxins such as deoxynivalenol, 4,15-diacetoxyscirpenol, and T-2 toxin, are potent protein synthesis inhibitors for eukaryotic organisms
The results suggested that the introduction of an Oacetyl group at the C-3 position in the biosynthetic pathway works as a resistance mechanism for Fusarium species that produce t-type trichothecenes
The acetyl substituents of trichothecenes were quite unstable under alkaline conditions, and even in buffer at pH 7.5, a considerable amount of deacetylated products were generated during an overnight incubation period
Summary
Microorganisms and Chemicals—The deoxynivalenol (DON) producing strain of Fusarium graminearum F15 was cultured in liquid. After double digestion with NotI and SalI, the synthesized cDNA was ligated into the expression cloning vector pcDSP21 [15] and transformed to E. coli DH5. Acetyltransferase assays were initiated by the addition of 100 l of the crude enzyme fraction to a reaction mixture containing 3.66 ml of 10 mM Tris-HCl (pH 7.5), 200 l of trichothecenes in 50% ethanol (5 mg/ml), and 40 l of acetyl-CoA in water (100 mM). Each cosmid clone containing Tri101 was completely digested with NotI, an enzyme that cut off the insert DNA from the vector, and partially digested with EcoRI, ClaI, and KpnI. Analysis of the Trichothecene Biosynthetic Gene Cluster by PCR— PCR was performed to check if a portion of Tri4 [17], Tri5 [18], and Tri6 [19] could be amplified from the cosmid clones containing Tri101. PCR products were cloned directly into TA cloning vector pCRII (Invitrogen), which were confirmed to contain the corresponding genes by partial nucleotide sequence analyses
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