Bioprospecting for trichothecene 3-O-acetyltransferases in the fungal genus Fusarium yields functional enzymes with different abilities to modify the mycotoxin deoxynivalenol

Abstract

The trichothecene mycotoxin deoxynivalenol (DON) is a common contaminant of small grains, such as wheat and barley, in the United States. New strategies to mitigate the threat of DON need to be developed and implemented. TRI101 and TRI201 are trichothecene 3-O-acetyltransferases that are able to modify DON and reduce its toxicity. Recent work has highlighted differences in the activities of TRI101 from two different species of Fusarium (F. graminearum and F. sporotrichioides), but little is known about the relative activities of TRI101/TRI201 enzymes produced by other species of Fusarium. We cloned TRI101 or TRI201 genes from seven different species of Fusarium and found genetic identity between sequences ranging from 66% to 98%. In vitro feeding studies using transformed yeast showed that all of the TRI101/TRI201 enzymes tested were able to acetylate DON; conversion of DON to 3-acetyl-deoxynivalenol (3ADON) ranged from 50.5% to 100.0%, depending on the Fusarium species from which the gene originated. A time course assay showed that the rate of acetylation varied from species to species, with the gene from F. sporotrichioides having the lowest rate. Steady-state kinetic assays using seven purified enzymes produced catalytic efficiencies for DON acetylation ranging from 6.8 × 10(4) M(-1)·s(-1) to 4.7 × 10(6) M(-1)·s(-1). Thermostability measurements for the seven orthologs ranged from 37.1°C to 43.2°C. Extended sequence analysis of portions of TRI101/TRI201 from 31 species of Fusarium (including known trichothecene producers and nonproducers) suggested that other members of the genus may contain functional TRI101/TRI201 genes, some with the potential to outperform those evaluated in the present study.

FIG. 1.

Phylogenetic tree based on a total of 688 bp of sequence internal to the 3′ end of TRI101/TRI201. Sequences included in the tree are from a total of 54 strains of Fusarium: 33 strains from the Leslie collection (see Table S2 in the supplemental material), four strains from the Schmale collection, and 17 strains from GenBank (see Table S1 in the supplemental material). Strain numbers or GenBank accession numbers follow the underscores. *, species whose full-length TRI101/TRI201 gene products were analyzed in this study for their ability to detoxify DON. Numbers at branch points represent bootstrap values.

FIG. 2.

Western blot of TRI101/TRI201 enzymes from F. pseudograminearum (Fps2TRI101), F. graminearum (Fg1TRI101), F. crookwellense (Fcr2TRI101), F. culmorum (Fcu2TRI201), F. fujikuroi (Ff1TRI201), F. sporotrichioides (Fs1TRI101), and F. oxysporum (Fo1TRI201) (1 and 2 indicate that the strain is from the Schmale or Leslie collection, respectively). Protein extracts from yeast cultures were collected following 24 h of expression. Untransformed yeast strain RW2802 represents the negative control. Ten microliters of each protein extract was loaded onto a 12% acrylamide-SDS-PAGE gel and run at 150 V for 1 h. Precision plus protein dual color standard was used to determine protein size. Ten microliters of TRI101 from F. sporotrichioides and TRI201 from F. fujikuroi, both purified from E. coli, were loaded as positive controls at known amounts of 1,000 ng, 250 ng, and 31.25 ng. The Western blot was probed with rabbit anti-FsTRI101 primary antibody and detected with alkaline phosphatase-conjugated anti-rabbit antibody.