Engineering phase II detoxification of mycotoxins in yeast

Plant pathogenic fungi of the genus Fusarium cause diseases of important crop plants (e.g. wheat, barley, rye, oat, maize). In addition to the yield reductions, the high economical importance of Fusarium diseases is due to the toxic fungal metabolites accumulating in the grain. After toxicological evaluation and intake studies, EU wide regulatory measures are in preparation for Fusarium mycotoxins. The protein biosynthesis inhibitor deoxynivalenol (DON) and the estrogenic metabolite zearalenone (ZON) are the most prevalent mycotoxins in Europe, and pose a potential threat to human and animal health. Detoxification mechanisms based on formation of mycotoxin-conjugates such as glucuronides and sulfates exist in plant and animal cells, and most likely also in the toxin producing fungus. In this project mycotoxin inactivating glucuronosyltransferases (UGTs) and sulfotransferases (SULTs) should be identified, and the conjugation products structurally characterized. The goal of this project is to genetically engineer baker`s yeast so that it can functionally express UGTs and SULT genes. The detoxification activity of an expressed gene should be detectable by a simple phenotypic readout. In case of deoxynivalenol growth on toxin containing plates will be used, while in case of ZON the altered response of estrogen-responsive reporter genes will be monitored. The identities and molecular structures of the mycotoxin conjugates will be investigated by selective LC/MS/MS techniques. The problem to be solved is that yeast lacks the ability to synthesize the cofactor UDP-glucuronic acid (GlcUA), and that mammalian glucuronosyl-transferases are located in the lumen of the endoplasmatic reticulum. Therefore yeast strains have to be engineered which can produce GlcUA and efficiently transport it to the target organelle. Such strains, and others efficiently providing the sulfotransferase co-substrate PAPS ("active sulfate") will then be used as hosts for heterologous expression of UGT and SULT genes, respectively. Also candidate SULT and UGT genes identified in the sequenced Fusarium graminearum genome will be characterized. In addition, we plan to utilize yeast expressing appropriate UGT and SULT genes as bioreactors for the production of mycotoxin-conjugates, which are needed as reference materials for the development of analytical techniques to detect and quantify toxin-metabolites. If functional expression of foreign detoxification genes can be achieved, this would have implications far beyond the mycotoxin aspect. A set of yeast strains expressing individual detoxification enzymes would be a useful tool for drug metabolism studies in general.