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Towards Characterization of the Polyketide Synthase Gene Cluster Responsible for the Production of Gephyronic Acid
Gephyronic acid is a secondary metabolite exhibiting potent anti-cancer and antibiotic activities. This polyketide natural product was first isolated at the HZI (Helmholtz-Zentrum fÌ_r Infektionsforschung) by Sasse, Hfle, and Reichenbach from the myxobacterium Archangium gephyra in 1995. It was reported to exist as a separable mixture of open keto-alcohol and closed hemiketal forms, but only the gross structure was identified.
Gephyronic acid has been shown to inhibit the growth of yeast and mold in the micromolar range (MIC 1-25 μg/mL); it also exhibits a cytostatic effect on mammalian cell cultures (human cervix, human leukemia, human breast) in the nanomolar range (IC50 10-60 ng/mL). An in vitro translation assay demonstrated that gephyronic acid is a specific inhibitor of eukaryotic protein synthesis (IC50 66-80 ng/mL).
Our recent total synthesis and structural reassignment of gephyronic acid incorporates a synthetic route that provides a means by which to prepare gephyronic acid efficiently and selectively and has provided significant quantities, thus facilitating further biological evaluation towards identifying gephyronic acids specific mode of action in cancer cells. The newly proposed structure for gephyronic acid also provides further insight in to the structural relationship between related eukaryotic protein synthesis inhibitors tedanolide, myriaporone, and pederin. Thus, gephyronic acid may represent a potential pharmacophoric link between structurally distinct classes of biologically active polyketides with related modes of action.
Moreover, we sought to identify and characterize the polyketide synthase (PKS) gene cluster responsible for gephyronic acid production in the producing organism. Our proposed biosynthetic pathway, has been validated via genetic analysis through complementary use of whole-genome sequencing and the use of a cosmid library derived from the myxobacteria strain Cystobacter violaceus strain Cb vi76, another known producer of gephyronic acid. The gene cluster identified spans 52 kb and encodes 5 type I polyketide synthases (gphFGHIJ) and post-PKS modifying enzymes including an O-methyltransferase (gphA) and cytochrome P450 (gphK). The gephyronic acid assembly line also includes several unique features, such as methyl branch incorporation from S-adenosyl-L-methionine (SAM) by the action of SAM-dependent methyltransferases and the presence of a unique loading domain. Targeted gene inactivation of key genes resulted in deactivation of the biosynthetic pathway, providing direct evidence for its isolation and characterization. Identification of the gephyronic acid gene cluster is the key step in a heterologous expression system, which will provide an alternative source of the natural product and a fermentation system for precursor directed biosynthesis of gephyronic acid analogues.
History
Date Modified
2017-06-02Defense Date
2012-08-27Research Director(s)
Richard E. TaylorCommittee Members
Richard E. Taylor Jeff Schorey Holly GoodsonDegree
- Doctor of Philosophy
Degree Level
- Doctoral Dissertation
Language
- English
Alternate Identifier
etd-10192012-085824Publisher
University of Notre DameProgram Name
- Chemistry and Biochemistry