Date of Award

Winter 12-15-2018

Author's School

Graduate School of Arts and Sciences

Author's Department


Degree Name

Doctor of Philosophy (PhD)

Degree Type



Secondary metabolites are molecules produced by bacteria that grant a competitive advantage in

their native environment. Many life-saving antibiotics are directly taken from or inspired by these

biologically active and structurally diverse molecules. In addition to the compounds, the

biosynthetic enzymes that produce these structurally complex molecules are of interest, due to

their 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 by

plant associated species of Pseudomonas fluorescens. We present a detailed genetic and

biochemical characterization of the entire obi biosynthetic pathway which includes the

biosynthesis of the non-proteinogenic amino acid β-OH-p-NO2-homophenylalanine, produced en

route to obi. The biosynthesis is completed on the non-ribosomal peptide synthetase ObiF, which

contains a rare and essential serine to cysteine mutation in the type I thioesterase domain active

site. Our biosynthetic studies culminate with the in vitro reconstitution of five enzymes that enable

total chemoenzymatic synthesis of the -lactone obi. In addition, we show that homologous gene

clusters are present in environmental bacteria and human pathogens. We report the utilization of

biosynthetic enzymes in a chemoenzymatic synthesis of obi analogs and the protein crystal

structure of ObiF, which provides structural evidence to the mechanism of strained ring formation.


English (en)

Chair and Committee

Timothy A. Wencewicz

Committee Members

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


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