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<P>We expect to gain several valuable resources and knowledge from this research. First a strain of yeast that can degrade OTA and will be utilized by winemakers around the world. Second this will be the first time the enzymes that degrade OTA in vivo will be identified. Third we may identify novel methods of detoxification of Ochratoxin-A. Fourth we may identify mechanisms of resistance to Ochratoxin-A that could lead to novel therapeutics for Balkan Endemic Nephropathy. Fifth, understanding mechanisms of resistance will also give us a better understanding of the mechanism of OTA-induced toxicity. Upon achievement of these deliverables we will publish our results in a peer-reviewed journal, and disseminate this information via appropriate scientific and technical conferences. Additionally, in experiments beyond the scope of this grant proposal, we intend to investigate whether wines produced from must containing OTA using yeast with high-OTA detoxification activity are safe for human consumption. Should these be safe, we intend to patent these high OTA-detoxification activity yeast strains and then license them to large yeast manufacturing and distribution organizations such as Lallemand, Red Star, White Labs, and Wyeast. Discoveries of mechanisms of toxicity and resistance to OTA will be discussed with scientists and physicians specializing in diseases believed to be caused by OTA such as Balkan Endemic Nephropathy and collaborations may be pursued. </P>
<P>NON-TECHNICAL SUMMARY: The long-term goal of the proposed project is to study the mechanism of Ochratoxin-A toxicity, and create an inexpensive, simple, and powerful method of detoxifying Ochratoxin-A in wine-musts. Ochratoxin-A is one of the most common mycotoxins present in foods and beverages and is a threat to human health. It has been found in human plasma and milk and is classified as a possible carcinogen. It is produced by fungi such as Aspergillus carbonarius that grow on fruits and grains in warm environments throughout the world. With projected increases in global temperature, more winemaking regions are predicted to become hospitable environments to these Ochratoxin-A-producing fungi, which would cause an increase in the frequency and intensity of Ochratoxin-A contamination in wines. The current methods used for removal of Ochratoxin-A from wines are inefficient, or unusable in a commercial winery. We propose a novel method to detoxify Ochratoxin-A-contaminated wines through directed-evolution of a strain of the wine-yeast Saccharomyces cerevisiae to select for robust OTA-detoxification activity. We believe that this is an ideal solution to an important human health problem. In the course of developing this strain of yeast, we will also gain insight into the mechanism of Ochratoxin-A induced toxicity, resistance, and detoxification. </P>
<P>APPROACH: The proposed research will employ a three-tiered screening approach to identify OTA-detoxifying strains of S. cerevisiae from a chemically mutagenized pool and/or established non-GMO mutant libraries. Subsequently selected strains will be characterized using three assays in parallel: chemically in terms of their OTA-detoxifying capabilities, genetically by identification of the causal mutation, and functionally by the chemical profile resulting from their fermentation of OTA contaminated wine-must. </P>