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Biosynthetic Studies of Resorcylic Acid Lactones, Hypothemycin, Radicicol, and Dehydrocurvularin Open Access


Other title
Organic Chemistry
Type of item
Degree grantor
University of Alberta
Author or creator
Gao, Zhizeng
Supervisor and department
Vederas, John C (Chemistry)
Examining committee member and department
Bundle, David R (Chemistry)
Campbell, Robert E (Chemistry)
Bressler, David C (Agrichultural, Food & Nutritional Science)
Boddy, Christopher N (Chemistry, University of Ottawa)
Lucy, Charles A (Chemistry)
Department of Chemistry

Date accepted
Graduation date
Doctor of Philosophy
Degree level
Fungal polyketides, a vast source for pharmaceutical industries, are biosynthesized by multifunctional iterative polyketide synthases (PKS). The biosynthesis employs complex programming rules, which are currently unresolved. To decode the programming rules, the biosynthesis of three polyketides, hypothemycin, radicicol and dehydrocurvularin were investigated via heterologous reconstitution of corresponding PKSs in Saccharomyces cerevisiae strain BJ5464-NpgA. The biosyntheses of hypothemycin and radicicol share significant similarity. They both employ a pair of highly reducing PKS (HRPKS) and non-reducing PKS (NRPKS): the HRPKSs (Hpm8 and Rdc5) construct the reduced portions of the PKS-product backbones, whereas the NRPKSs (Hpm3 and Rdc1) synthesize the aromatic portions. To determine the distribution of reactions between HRPKS and NRPKS in the two biosynthetic systems, putative biosynthetic intermediates (27 for hypothemycin and 149 for radicicol) were synthesized as their S-N-acetylcysteamine (SNAC) thioesters. We found that hypothemycin biosynthesis employs a 6+3 distribution of reactions, but radicicol biosynthesis uses a 5+4 distribution of reactions. The substrate-dependent stereospecificity of Hpm8 ketoreductase (KR) was investigated by incubating Hpm8 with a series of β-keto SNAC thioesters. The KR domain stereospecificity was found to be dependent only on substrate chain lengths; the KR domain reduced the β-keto groups of diketides (4 carbon) to L-hydroxyl groups, but reduced all other β-keto intermediates to D-hydroxyl groups. This unprecedented, switchable stereospecificity suggests that iterative PKSs are more complex than previous thought, and that the substrate-PKS interactions play a significant role in iterative PKS programming. To fully investigate the substrate-HRPKS interactions, a series of 13C-labeled, partially assembled precursors were synthesized and assayed with Hpm8 and Hpm3. Our data suggest that Hpm8 prefers substrates resembling biosynthetic intermediates after the reductive modifications. The putative gene cluster of dehydrocurvularin was uncovered via genomic sequencing of producing strain Alternaria cinerariae. A pair of HRPKS (Dhc3) and NRPKS (Dhc5) was identified, analogous to hypothemycin and radicicol biosynthesis. Both PKS genes were cloned into expression plasmids, and the gene products were successfully expressed and purified from BJ5464-NpgA. In vivo reconstitution of DHC production in BJ5464-NpgA containing genes encoding Dhc3 and Dhc5 was not successful. Other cryptic enzymes may be involved in the biosynthesis of DHC.
Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.
Citation for previous publication
Zhou, H.; Qiao, K.; Gao, Z.; Meehan, M. J.; Li, J. W.; Zhao, X.; Dorrestein, P. C.; Vederas, J. C.; Tang, Y. J. Am. Chem. Soc. 2010, 132, 4530-4531.Zhou, H.; Gao, Z.; Qiao, K.; Wang, J.; Vederas, J. C.; Tang, Y. Nat. Chem. Biol. 2012, 8, 331-333.Gao, Z.; Wang, J.; Norquay, A. K.; Qiao, K.; Tang, Y.; Vederas, J. C. J. Am. Chem. Soc. 2013, 135, 1735-1738.Zhou, H.; Qiao, K.; Gao, Z.; Vederas, J. C.; Tang, Y. J. Biol. Chem. 2010, 285, 41412-41421.

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