Mycotoxins are toxic secondary metabolites produced by fungi that are a threat to the health of humans and domestic animals. The most important mycotoxin in the U.S. is deoxynivalenol (DON), which causes symptoms such as vomiting, feed refusal, and weight loss in farm animals. The fungus Fusarium graminearum produces DON in staple crops such as wheat, barley, and corn. It is estimated that the economic losses associated with DON contamination alone exceed $650 million per year in the U.S. New strategies are needed to mitigate DON and improve food safety in the U.S. The overall goal of my research is to discover and employ microorganisms and enzymes to detoxify DON. The specific objectives are to: (1) discover and characterize microorganisms that detoxify DON, (2) use a cell free protein synthesis (CFPS) system to study enzymes that modify DON, (3) engineer yeast to detoxify DON with a metabolic engineering strategy, and (4) deliver a high school unit to teach high school students about mycotoxins in food. In Objective 1, two mixed cultures were identified from environmental samples that converted DON into the less toxic 3-keto-deoxynivalenol (3-keto-DON). In Objective 2, a CFPS system was used to express three known acetyltransferase genes to convert DON to 3-acetyl-DON (3-A-DON). In Objective 3, we identified a potential DON transporter from a library of randomly amplified fragments from the genomes of mixed cultures of microbes isolated from the environment. In Objective 4, we developed and delivered a unique high school unit to educate high school students about potential mycotoxins in food and feed products. The work presented here represents new and improved methods for mitigating mycotoxin contamination in the United States. / Ph. D. / Some fungi produce dangerous toxins called mycotoxins that contaminate food and feed and cause adverse affects when consumed. The mycotoxin deoxynivalenol (DON) contaminates staple crops such as wheat, barley, and corn and when consumed by domesticated animals it can cause weight loss, feed refusal, vomiting, and even death. The goal of this research is to detoxify DON using miroorganisms such as bacteria or fungi as well as enzymes. The specific objectives are to: (1) discover and characterize microorganisms that detoxify DON, (2) utilize a cell free protein synthesis (CFPS) system to detoxify DON using known acetyltransferase genes, (3) engineer yeast to detoxify DON with a metabolic engineering strategy, and (4) deliver a high school unit to teach high school students about mycotoxins in food and strategies to mitigate them. For objective one, microorganisms were collected from plant and soil samples and incubated in solution containing 100 ppm DON. Two mixed cultures were discovered to convert DON to another metabolite, 3-keto-DON that is considered less toxic. In objective two, a cell free protein synthesis (CFPS) system was used to establish its functionality as a tool to screen for enzymes that will detoxify DON. Known acetyltransferase genes were expressed in the CFPS and DON was converted to the metabolite 3-acetyl-DON. The mixed cultures discovered in objective one were then utilized in objective three to determine what enzymes were responsible for the conversion of DON to 3-keto-DON. Objective four was established to shed light about the dangers of mycotoxins and how growers and scientists test for mycotoxins in food and feed.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/77928 |
| Date | 06 June 2017 |
| Creators | Wilson, Nina Marie |
| Contributors | Plant Pathology, Physiology, and Weed Science, Schmale, David G. III, Senger, Ryan S., Feng, Xueyang, Griffey, Carl A. |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Detected Language | English |
| Type | Dissertation |
| Format | ETD, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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