In vitro reconstitution of the 6-deoxyerythronolide B synthase : insights into the mechanism and engineering of polyketide synthases
Abstract/Contents
- Abstract
- Polyketides are a large class of natural product metabolites with incredible structural diversity and remarkable medicinal utility. While existing polyketides have met a wide variety of therapeutic needs, society will continue to have a steady demand for new medicinal polyketides. Our hope is that by studying the detailed biochemistry involved in the biosynthesis of polyketides, we will be able to develop informed strategies for rationally engineering new therapeutic molecules. Polyketides are assembled by enzymatic complexes known as polyketide synthases (PKSs), which can vary widely in their overall protein architecture. Bacteria are known to harbor enormous, multimodular PKSs that build their polyketides by sequential chemical transformations in a manner that is reminiscent of the automobile assembly line. The modular architecture of these enzymes makes them attractive candidates for protein engineering and gives hope to the idea of generating novel polyketides through combinatorial biosynthesis. Arguably the most-well characterized multimodular PKS is the 6-deoxyerythronolide B synthase (DEBS) which produces the macrocyclic core, 6-deoxyerythronolide B (6-dEB), to the erythromycin family of antibiotics. Notwithstanding over 20 years of research, DEBS (or any other assembly line PKS) has never been completely reconstituted in vitro using purified protein components. In this dissertation, we present first the full, in vitro reconstitution and biochemical analysis of the full DEBS assembly line. This reconstituted system has enabled three unprecedented studies into the mechanism and engineering of PKSs. First, we have analyzed the full DEBS assembly line from a kinetic perspective, demonstrating that the entire complex features a turnover frequency of approximately 1 min-1, which is in good agreement with in vivo productivity estimates. This analysis also demonstrated that the full assembly line has a degree of relaxed substrate specificity for which we report the structural characterization of a chemical analog of the 6-dEB. In addition, we have used the reconstituted system to test a key hypothesis relating to the assembly line processing of polyketide intermediates by the DEBS complex. In particular, our results indicate that a module within DEBS can carry no more than one growing polyketide chain at any point during its catalytic cycle. Finally, as an extension of the reconstituted system, we have developed and tested protein engineering strategies for the construction of non-natural (or "hybrid") bimodular PKSs, giving insights into the design considerations for building novel PKS assembly lines. We hope that this work will inspire the continued study of these remarkable biocatalysts as a gateway to developing new polyketides.
Description
| Type of resource | text |
|---|---|
| Form | electronic; electronic resource; remote |
| Extent | 1 online resource. |
| Publication date | 2015 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Associated with | Lowry, Brian Robert |
|---|---|
| Associated with | Stanford University, Department of Chemical Engineering. |
| Primary advisor | Khosla, Chaitan, 1964- |
| Thesis advisor | Khosla, Chaitan, 1964- |
| Thesis advisor | Dunn, Alexander Robert |
| Thesis advisor | Sattely, Elizabeth |
| Advisor | Dunn, Alexander Robert |
| Advisor | Sattely, Elizabeth |
Subjects
| Genre | Theses |
|---|
Bibliographic information
| Statement of responsibility | Brian Robert Lowry. |
|---|---|
| Note | Submitted to the Department of Chemical Engineering. |
| Thesis | Thesis (Ph.D.)--Stanford University, 2015. |
| Location | electronic resource |
Access conditions
- Copyright
- © 2015 by Brian Robert Lowry
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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