Thesis
Escherichia coli Nissle 1917 secondary metabolism: aryl polyene biosynthesis and phosphopantetheinyl transferase crosstalk
Master of Science (M.S.), Drexel University
Jun 2018
DOI:
https://doi.org/10.17918/nnje-sv46
Abstract
Escherichia coli Nissle 1917 (EcN) is a probiotic Gram-negative bacterium that is used to treat inflammatory bowel diseases. Although used clinically, it is poorly understood why this bacterium is probiotic and other E. coli strains are not; this may be due to EcN's increased biosynthetic potential. We hypothesized that an understanding of EcN secondary metabolism could explain some of its probiotic properties. Besides siderophores and colibactin, we identified and characterized for the first time an aryl polyene (APE) in EcN. Through construction of an in-frame deletion of the biosynthetic gene cluster (BGC) we verified the cluster responsible for APE production. To our surprise this cluster produces four molecules with the same mass, which we tentatively assigned to four APE isoforms. Supplementation studies with different benzoic acids revealed details of the biosynthetic mechanism, and functional assays showed that APEs have antioxidant activity. Next, we focused on an enzyme family that activates BGCs, which is necessary for secondary metabolite production. The genome of EcN encodes for three activating Sfp-type phosphopantetheinyl transferases (PPTases) of which we systematically constructed single, double, and a triple mutant(s). Using liquid chromatography mass spectrometry (LCMS) we showed that APE production requires PPTases AcpT and EntD, but not ClbA. Using our mutants and LCMS, we showed an intricate network of PPTases and BGCs, and we define the PPTase-BGC crosstalk relationship in EcN. This work begins to explain the complex biosynthetic potential of EcN, which has implications for further medically relevant applications.
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Details
- Title
- Escherichia coli Nissle 1917 secondary metabolism
- Creators
- Courtney Virginia Fesko - DU
- Contributors
- Patrick J. Loll (Advisor) - Drexel University (1970-)
- Awarding Institution
- Drexel University
- Degree Awarded
- Master of Science (M.S.)
- Publisher
- Drexel University; Philadelphia, Pennsylvania
- Number of pages
- x, 61 pages
- Resource Type
- Thesis
- Language
- English
- Academic Unit
- Biochemistry and Molecular Biology; College of Medicine; Drexel University
- Other Identifier
- 8850; 991014632180204721