Abstract

Secondary metabolites are molecules produced by bacteria that grant a competitive advantage intheir native environment. Many life-saving antibiotics are directly taken from or inspired by thesebiologically active and structurally diverse molecules. In addition to the compounds, thebiosynthetic enzymes that produce these structurally complex molecules are of interest, due totheir ability to assemble these molecules in an efficient and stereochemically controlled manner.We report the biosynthetic gene cluster of the antibiotic obafluorin (obi), a β-lactone produced byplant associated species of Pseudomonas fluorescens. We present a detailed genetic andbiochemical characterization of the entire obi biosynthetic pathway which includes thebiosynthesis of the non-proteinogenic amino acid β-OH-p-NO2-homophenylalanine, produced enroute to obi. The biosynthesis is completed on the non-ribosomal peptide synthetase ObiF, whichcontains a rare and essential serine to cysteine mutation in the type I thioesterase domain activesite. Our biosynthetic studies culminate with the in vitro reconstitution of five enzymes that enabletotal chemoenzymatic synthesis of the -lactone obi. In addition, we show that homologous geneclusters are present in environmental bacteria and human pathogens. We report the utilization ofbiosynthetic enzymes in a chemoenzymatic synthesis of obi analogs and the protein crystalstructure of ObiF, which provides structural evidence to the mechanism of strained ring formation.

Committee Chair

Timothy A. Wencewicz

Committee Members

John-Stephen Taylor, Kevin Moeller, Robert Blankenship, Joseph Jez,

Comments

Permanent URL: https://doi.org/10.7936/933q-pt67

Degree

Doctor of Philosophy (PhD)

Author's Department

Chemistry

Author's School

Graduate School of Arts and Sciences

Document Type

Dissertation

Date of Award

Winter 12-15-2018

Language

English (en)

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Available for download on Tuesday, December 19, 2028

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Chemistry Commons

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