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Studies on the biosynthesis of the antifungal polycyclic tetramate macrolactam HSAF and the AMPK-activating mycoepoxydiene
In search for antifungal compounds with novel chemistry and a new mode of action, we isolated a heat stable antifungal factor (HSAF) from Lysobacter enzymogenes C3. HSAF was determined to be dihydromaltophilin, a polycyclic tetramate macrolactam with novel antifungal mechanism. Our ultimate goal is to develop this type of compounds as new antifungal drug leads. As a key step toward this goal, we have investigated the mechanism for HSAF biosynthesis, its regulation and genetic engineering. Firstly, we constructed two NRPS adenylation domain swapping mutants (replacing ornithine adenylation domain with lysine adenylation domain) with expectation to generate the analogue of HSAF with one additional methylene group. The expected compound was not detected in the resulted strain, possibly due to the miscommunication between domains within the single-modular PKS-NRPS, the key enzyme for the construction of the HSAF skeleton. To explore the enzymes involving in the post-PKS-NRPS biosynthesis of HSAF, a zinc-dependent oxidoreductase (OX4) and a FAD-dependent oxidoreductase (OX3) were deleted. Both mutants abolished the production of HSAF. Then we isolated a cosmid that bears the complete HSAF biosynthetic gene cluster, and transferred the cosmid into Streptomyces for heterologous expression. Metabolite analysis indicates that HSAF and its analogues were produced in the transformed Streptomyces strains. This result demonstrates that a one-module PKS can synthesize two separate polyketide chains, an unprecedented mechanism for bacterial polyketide biosynthesis. In probing the regulatory mechanism of HSAF biosynthesis, we identified a cAMP receptor like protein (Clp) and demonstrated that a cis-acting element within its promoter region was essential for the regulation. In the fourth project, a cosmid bearing the complete gene cluster for mycoepoxydiene biosynthesis was transformed into Fusarium verticilloides for heterologous expression. Mycoepoxydiene was isolated from a fungal endophyte of a mangrove plant with unusual structure features and a strong AMPK-activating activity. The metabolites analysis of the Fusarium verticilloides mutants led to identification of fusaric acid, a mycotoxin with significance in food safety and agriculture.
Xie, Yunxuan, "Studies on the biosynthesis of the antifungal polycyclic tetramate macrolactam HSAF and the AMPK-activating mycoepoxydiene" (2013). ETD collection for University of Nebraska - Lincoln. AAI3603580.