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Date of Award
Doctor of Philosophy (PhD)
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.
Chair and Committee
Timothy A. Wencewicz
John-Stephen Taylor, Kevin Moeller, Robert Blankenship, Joseph Jez,
Schaffer, Jason Earl, "Biosynthesis and Biological Activity of Nonribosomal Peptide Beta-Lactones" (2018). Arts & Sciences Electronic Theses and Dissertations. 1713.
Available for download on Tuesday, December 19, 2028