Exploring protein-protein interactions and vectorial substrate processing in modular polyketide synthases
Abstract/Contents
- Abstract
- One of the largest threats to the achievements of modern medicine is the spread of antimicrobial resistance, which degrades the efficacy of many vital therapeutics. If not addressed, antimicrobial resistance threatens to cause a post-antibiotic era, in which minor infections become life-threatening crises. In addition to curbing the spread of antimicrobial resistance through systems level interventions, it is vital that new antimicrobial compounds are developed. Polyketides are an important class of natural products that have found wide use as therapeutics, including antibiotics, antifungals, and cholesterol lowering agents. The discovery and engineering of novel polyketides represents one route to regenerating our stock of life-saving therapeutics. A subset of polyketides is synthesized by modular polyketide synthases (PKSs), in which separate modules add simple extender units to a growing polyketide. After an extender unit is added, each module houses additional catalytic machinery to modify the recently added portion before passing its product on to the next module. By combining multiple modules to create large synthases, impressively complex biomolecules can be synthesized from simple substrates. Their modular architecture has made these synthases attractive targets for engineering and combinatorial biosynthesis to make novel polyketides. PKSs display exquisite control over the stereocenters in the resulting polyketide. The ketoreductase (KR) tailoring domain is often responsible for setting two stereocenters at each module and is largely responsible for the stereochemistry of the final product. We characterized the specificity of a panel of KRs for their native and enantiomeric acyl substrate, either presented as acyl-CoAs or tethered to acyl carrier proteins (ACPs) that normally shuttle nascent polyketides between different tailoring domains. Our results show the contribution of KR-ACP interactions towards catalysis and the general preference of KRs for their native substrates and ACPs. This work will guide future attempts to engineer KRs and access the vast polyketide stereochemical space that remains unexplored. A longstanding goal in protein engineering has been the recombination of different PKS modules to form hybrid or chimeric synthases that create novel polyketides. Previous work has yielded functional hybrid synthases albeit with total productivity reduced by orders of magnitude. We undertook a rigorous in vitro analysis of a subset of these hybrid synthases to more accurately measure their productivity and identify any bottlenecks. Polyketide production correlated well with translocation of a nascent polyketide from a donor module to a non-native acceptor module, suggesting that the translocation reaction is a major limit on the productivity of hybrid PKSs. Attempts to alleviate this limit by engineering an epitope of the donor module's ACP confirmed our hypothesis and led to increased polyketide production by a hybrid synthase. Finally, by using the knowledge gleaned from our studies of the KR-ACP interactions, we engineered a donor module with a substituted KR, resulting in a donor module that produced a diastereomer of its native polyketide. A direct comparison of this native and KR-substituted donor module illustrated that protein-protein interactions, rather than protein-substrate interactions, play a more dominant role in polyketide translocation and overall productivity in hybrid PKSs. A fundamental question regarding PKSs is how they maintain unidirectional biosynthesis, in which each module carries out exactly one round of catalysis on each starter unit. Recent investigations have revealed a turnstile mechanism by which PKSs prevent transacylation from an upstream module until the product from the previous catalytic cycle is offloaded from the ACP. Experiments with chemoenzymatic loading of the ACP revealed that the condensation reaction is required to activate the turnstile mechanism. While this mechanism clearly plays a large, if not the sole, role in mediating vectorial chain processing in PKSs, there are many open questions regarding its function. First, we measured the stability of the turnstile mechanism and confirmed that a module was reloaded after offloading of the acyl-ACP to a downstream module. Next, we developed two independent assays that allowed us to show that the carbon dioxide released from the condensation reaction is not involved in maintaining the turnstile mechanism. Limited proteolysis of an open module or with an active turnstile showed no obvious differences, suggesting that the turnstile mechanism is not the result of a global change in module architecture. Finally, while the turnstile mechanism prevents intermodular reloading, PKSs also prevent intramodular reloading or backtransfer; we used site-directed mutagenesis to test the independence of these mechanisms. Although these results were not conclusive, they suggest that the turnstile and backtransfer prevention mechanisms may be independent. In summary, understanding polyketide biosynthesis is an important problem in enzymology with broad significance in medicine. This dissertation focuses on answering major questions regarding the function and engineering of PKSs. Through increased understanding of the fundamentals that control these enzymes, we may one day realize the goal of synthesizing new therapeutic polyketides.
Description
| Type of resource | text |
|---|---|
| Form | electronic; electronic resource; remote |
| Extent | 1 online resource. |
| Publication date | 2017 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Associated with | Ostrowski, Matthew Phillip |
|---|---|
| Associated with | Stanford University, Department of Chemical Engineering. |
| Primary advisor | Khosla, Chaitan, 1964- |
| Thesis advisor | Khosla, Chaitan, 1964- |
| Thesis advisor | Sattely, Elizabeth |
| Thesis advisor | Smolke, Christina D |
| Advisor | Sattely, Elizabeth |
| Advisor | Smolke, Christina D |
Subjects
| Genre | Theses |
|---|
Bibliographic information
| Statement of responsibility | Matthew Phillip Ostrowski. |
|---|---|
| Note | Submitted to the Department of Chemical Engineering. |
| Thesis | Thesis (Ph.D.)--Stanford University, 2017. |
| Location | electronic resource |
Access conditions
- Copyright
- © 2017 by Matthew Phillip Ostrowski
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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