Dissecting and Harnessing Carrier Protein Interactions in Fungal Megasynth(et)ases
Lead Research Organisation:
University of Warwick
Department Name: Chemistry
Abstract
Fungal polyketide synthases (PKS) are giant multi-domain proteins responsible for the biosynthesis of a vast number of biologically active natural products. Whilst many find important applications as medicinal or agrochemical agents, some are toxins that are harmful to both human health and agriculture. The precise assembly of these complex compounds relies on the highly programmed protein-protein interactions (PPIs) between a small carrier protein (CP) domain, to which biosynthetic intermediates are covalently tethered, and the individual catalytic domains within the PKSs, ensuring that the overall process is efficient and maintains product fidelity. Despite the importance of these interactions, they remain poorly understood, primarily due to their ephemeral nature, requiring a combination of techniques to be studied effectively. Uncovering the molecular factors governing programming is the greatest remaining problem in our understanding of fungal PKSs, and represents a huge obstacle to rewiring these enzymes towards user-designed molecules.
This project aims to combine cutting-edge mass spectrometry, structural biology and biochemical techniques to elucidate the molecular details of CP-dependent interactions underpinning fungal PKS machinery. The research will initially focus on characterising PPIs in two similar PKS systems; one involved in the biosynthesis of cyclosporin (clinically used immunosuppressant), the other responsible for the construction of lovastatin (clinically used cholesterol-lowering agent). These PKSs differ by the positioning of a single catalytic domain, and therefore represent excellent model systems to establish the common principles underlying CP-dependent interactions. Taken together, this body of work will significantly deepen our understanding of the roles played by PPIs in fungal PKSs. It will also form a knowledge-base to begin exploiting such interactions to construct engineered systems capable of producing novel natural product analogues.
This project aims to combine cutting-edge mass spectrometry, structural biology and biochemical techniques to elucidate the molecular details of CP-dependent interactions underpinning fungal PKS machinery. The research will initially focus on characterising PPIs in two similar PKS systems; one involved in the biosynthesis of cyclosporin (clinically used immunosuppressant), the other responsible for the construction of lovastatin (clinically used cholesterol-lowering agent). These PKSs differ by the positioning of a single catalytic domain, and therefore represent excellent model systems to establish the common principles underlying CP-dependent interactions. Taken together, this body of work will significantly deepen our understanding of the roles played by PPIs in fungal PKSs. It will also form a knowledge-base to begin exploiting such interactions to construct engineered systems capable of producing novel natural product analogues.
Publications
Jenner M
(2023)
Elucidating the molecular programming of a nonlinear non-ribosomal peptide synthetase responsible for fungal siderophore biosynthesis.
in Nature communications
| Description | Achievements have been made in key areas of this project - as outlined below. 1). We have dissected the condensing region of a highly reducing polyketide synthase (hrPKS) and its cognate acyl carrier protein (ACP). The activity of these proteins has been fully reconstituted of activity in vitro, allowing initial interaction mapping using a combination of scanning alanine mutagenesis and biochemical assays. 2). We have produced a hrPKS(delta-ACP) protein that can be overexpressed in Saccaromyces cerevisae and purified to homogeneity. This has set the stage for mechanism-based crosslinking experiments and structure determination (with Prof. Martin Schmeing, McGill University - Project Partner), work that is currently ongoing. 3). Labelled ACPs from several hrPKS systems have been produced and initial NMR spectra obtained. Full assignment of structures and titration experiments with catalytic domains are ongoing to probe interactions and dynamics. 4). We have engineered a hrPKS catalytic domain to accept alternative extender units, allowing production of commercially useful analogues of fungal polyketides. |
| Exploitation Route | We are providing unique structural and functional insights into complex biosynthetic enzymes, which will allow us (and others) to perform rational engineering towards user-designed molecules. |
| Sectors | Agriculture Food and Drink Chemicals Healthcare Pharmaceuticals and Medical Biotechnology |