Title: Bioprospecting for TRI101 in Fusarium: Searching for a Better Enzyme to Detoxify Deoxynivalenol (DON) Authors
|Khatibi, P - VIRGINIA POLYTECHNIC INS|
|Schmale Iii, D - VIRGINIA POLYTECHNIC INS|
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: November 10, 2008
Publication Date: December 2, 2008
Citation: Khatibi, P.A., Mccormick, S.P., Alexander, N.J., Schmale Iii, D.G. 2008. Bioprospecting for TRI101 in Fusarium: Searching for a Better Enzyme to Detoxify Deoxynivalenol (DON) [abstract]. Proceedings of the 2008 National Fusarium Head Blight Forum. Poster No. 52. p. 112. Technical Abstract: The mycotoxin deoxynivalenol (DON) is a common contaminant of wheat and barley in the United States. New strategies to mitigate the threat of DON need to be developed and implemented. Previous research has shown the value of an enzyme (TRI101) to modify DON and reduce its toxicity. Recent work by Garvey et al. (2008) highlighted differences in the activity of TRI101 from two different species of Fusarium (F. graminearum and F. sporotrichioides), but little is known about the relative activity of TRI101 enzymes produced by other species of Fusarium. We cloned TRI101 from four different species of Fusarium: F. sporotrichioides, F. graminearum, F. oxysporum, and F. fujikuroi. Pairwise comparisons of genetic identity between TRI101 sequences ranged from 65% (FgTRI101 and FjTRI101) to 85% (FoTRI101 and FjTRI101). To increase the transfer of mycotoxin in and out of the yeast cells for our expression studies, we also cloned TRI12 (a trichothecene efflux pump) from F. sporotrichioides. Both genes were cloned into the yeast expression vectors pYes2.1 (TRI101) and pESC-LEU (TRI12), and the resulting vectors were co-transformed into the yeast strain RW2802. Transformed strains of RW2802 expressing TRI101 and/or TRI12 were fed DON at a concentration of 10ppm for 4 days at 28C. Fungal secondary metabolites were extracted, and DON and 3-acetyl-deoxynivalenol (3-ADON) were quantified using GC/MS. All of the TRI101 genes tested were able to acetylate DON in vitro, and the ratio of [3-ADON]/[DON] ranged from 0.77 (FoTRI101) to 10.44 (FsTRI101). Our results suggest that other species of Fusarium (even those that do not produce DON) may contain functional TRI101 genes, some with the potential to ‘outperform’ those evaluated in the present study. We are currently developing an Agrobacterium transformation vector to move these TRI101 genes into hulless barley lines. We plan to monitor potential decreases in DON in both raw grain and dried distiller’s grains with solubles (a byproduct of ethanol fermentation and a significant source of feed for domestic animals) following ethanol production using our genetically-engineered lines. TRI101 has tremendous potential to enhance food safety in the United States in the near future.