Manipulating posttranslational modification in natural product biosynthesis

Fatty acids, polyketides and nonribosomal peptides are three classes of natural products that play critical roles in human health, disease and therapy. Investigations that lead to a broader understanding of their biosynthesis may one day allow scientists to engineer the biomanufacture of novel compounds with tailored chemistries and biological activities. A unifying characteristic between these classes of compounds is the posttranslational modification of their synthase enzymes with a 4'-phosphopantetheinyl (4'-PP) arm from a coenzyme A (CoA) donor, installed by phosphopantetheinyl transferase (PPTase). We have previously described a method to chemoenzymatically install modified 4'-PP arms to heterologously expressed synthase enzymes through the use of CoA analogues that have been alkylated at the thiol terminus with fluorescent and affinity probes. We have since sought to extend this technique to the isolation of synthases from producer organisms, but this application was complicated by the efficiency of endogenous modification. This dissertation focuses on evaluating the use of PPTase inhibitors to enable the extension of our technique to these organisms.We have previously described a method to chemoenzymatically install modified 4'-PP arms to heterologously expressed synthase enzymes through the use of CoA analogues that have been alkylated at the thiol terminus with fluorescent and affinity probes. We have since sought to extend this technique to the isolation of synthases from producer organisms, but this application was complicated by the efficiency of endogenous modification. This dissertation focuses on evaluating the use of PPTase inhibitors to enable the extension of our technique to these organisms. Chapter 1 provides an introduction to this work, and Chapter 2 reviews the current literature regarding the site-specific labeling of proteins. Chapter 3 details the development of a homogenous resonance energy transfer assay for PPTase. Chapter 4 for confirms endogenous phosphopantetheinylation as the source of complication, and evaluates known PPTase inhibitor architectures for this application; revealing the limited spectrum of activity of these compounds. As such, this dissertation also reports efforts to identify a new inhibitor scaffolds by the screening of large chemical libraries. Studies toward this include Chapter 5, describing optimization of the assay process described in Chapter 3 for this purpose. Chapter 6 details the preparation of reporters to support this large scale robotic screen. Chapter 7 applies the results of Chapters 5 and 6 through an automated high throughput screen of over 311,000 compounds, and details efforts to nominate a lead structure for further optimization to develop a PPTase chemical prob