The principal investigator and his industrial collaborators have characterized all of the genes involved in the biosynthesis of the deoxysugars that are present in avermectin (L-oleandrose), doxorubicin (L-daunosamine), an erythromycin A (L-mycarose and D-desosamine). Many of the enzymes in the biosynthetic pathways to these four deoxysugars have a close functional relationship, which stems from a high sequence similarity of the genes and deduced proteins. Consequently, the principal investigator proposes that novel combinations of these genes will likely result in the formation of new natural product analogs, and aims to test this hypothesis. In addition, the principal investigator plans to examine selected novel analogs of erythronolide B produced by Kosan Biosciences, Inc., as substrates for the same enzymes in an attempt to make new antibacterial agents. Precedents from the principal investigator's work during the previous grant period and those from other groups suggest that novel glycosides will arise from these efforts, even thoug there is no way to predict how many of them will have significant biological activity. The long-range goal of this research is to develop approaches to new antibiotics through the creation and manipulation of genetically engineered bacteria. If successful, the proposed work will likely motivate others to seek different kinds of new natural product drugs by the same approach, thereby justifying the research even if new antibiotics are not discovered. During the proposed grant period, the following specific aims will be pursued in the order listed: (1) Elucidate the properties of the enzymes involved in the biosynthesis of oleandrose, daunosamine, mycarose, and desosamine to the extent necessary for combinatorial biosynthesis. (2) Develop ways to express all of their genes in a Streptomyces sp. and/or E. coli so as to confer the ability to glycosylate the known avermectin, daunorubicin, and erythromycin aglycones. (3) By expressing novel combinations of the deoxysugar biosynthesis genes, explore the possibility of making new analogs of erythromycin and related macrolides and of novel erythronolide B analogs prepared at Kosan Biosciences. (4) Attempt to alter the substrate specificity of the deoxysugar glycosyl transferases by DNA shuffling and other methods if the native enzymes are found not to have a broad enough substrate specificity. (5) Assay the resulting novel glycosidic products for antibacterial activity.
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