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<p>Fungal head blight (FHB) and its associated trichothecene mycotoxins are major source of crop losses in N. America. They not only reduce the yield, but also generate grain that is unsuitable for milling, brewing, or livestock. Thus far there is no satisfactory way to control FHB or reduce the toxicity of contaminated grain. The goal of this proposal is to develop improved enzymes for the inactivation and degradation of fungal mycotoxins associated with FHB. The current enzymatic methods for reducing the toxicity of trichothecenes focus on modification by either acetylation or glycosidation. Although these show some promise for controlling FHB they suffer from the fundamental weakness that the modification is reversible so that the toxin is not truly inactivated. This problem would be resolved in part if enzymes could be identified that irreversibly inactivate the trichothecenes. The obvious target for irreversible chemical modification is their 12,13 epoxide group which is necessary for substantial toxicity. Excitingly, bacterial isolates have been discovered that contain several unknown activities that lead to destruction of the epoxide moiety in trichothecenes. The focus here is identification and optimization of the enzymes responsible for degradation of fungal mycotoxins as a method for controlling the consequences of FHB.The goal of this study is to identify enzymes that deepoxidate the trichothecenes implicated in FHB. Thus the specific aims are:1. to identify the enzymes responsible for the deepoxidation activity in the six bacterial species that have been shown to exhibit this activity.2.to determine the biochemical strategy by which the toxins are inactivated.3.to optimize the enzymatic activity by biological selection and structural design.</p>
<p>The bacterial enzymes responsible for deepoxidation will be identified in a yeast screen that has been developed that is sensitive to low levels of mycotoxin. The genomic DNA for six aerobic bacterial species that have been demonstrated to deepoxidate trichothecenes has been provided by Dr. Zhou Ting at Agriculture and Agri-Food, Guelph, Ontario, Canada. It is planned to search for the source of the deepoxidase activity by screening genomic fragment plasmid libraries in S. cerevisiae. This search for activity has to be performed in a eukaryotic organism because trichothecenes do not inhibit bacterial growth. S. cerevisiae is the best system in which to perform this screen because of powerful array of genetic tools that provide a facile way to connecting biology to function. This is a proven approach for finding resistance genes.Once the enzymes have been identified, they will be optimized by biological selection and structural design. The genes identified in the initial screen will serve as a template for the generation of an improved enzyme. The initial goal will be to increase the enzymatic activity of the enzyme(s) and broaden the specificity. This will be an iterative process. The first step will be to create a mutant library from which improved variants can be selected with enhanced activity. A wide variety of methods have been developed to create mutant libraries including error prone PCR (epPCR), chemical mutagenesis, mutator strains, and site-saturation mutagenesis. Each of these methods has its advantages. In the first instance epPCR will be used to generate primarily single point mutations with a combined Taq and Mutazyme based protocol to generate unbiased libraries. These will be selected for improved activity. The main concern is whether sufficient sequence space can be explored to find a better gene, however there are numerous examples where this approach has been successful. Strong candidates will be passed iteratively through cycles of mutagenesis and selection seeking to find the best combination of mutations.Three dimensional structures of candidate enzymes will be determined to form the foundation for a rational optimization of the enzyme activity.</p>