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
Zdouc M
(2024)
MIBiG 4.0: advancing biosynthetic gene cluster curation through global collaboration
in Nucleic Acids Research
| 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 |
| Description | A Mass Spectrometer for Structural Biology and Top-down Protein Identification |
| Amount | £1,274,659 (GBP) |
| Funding ID | BB/Z515693/1 |
| Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
| Sector | Public |
| Country | United Kingdom |
| Start | 07/2024 |
| End | 07/2025 |
| Description | Chemoenzymatic synthesis of novel siderophore scaffolds |
| Amount | £95,000 (GBP) |
| Funding ID | 2883889 |
| Organisation | Engineering and Physical Sciences Research Council (EPSRC) |
| Sector | Public |
| Country | United Kingdom |
| Start | 09/2023 |
| End | 03/2027 |
| Description | Enabling new characterisation methods for dynamic systems through the upgrade of 700 MHz solution NMR spectrometer |
| Amount | £799,374 (GBP) |
| Funding ID | BB/W020297/1 |
| Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
| Sector | Public |
| Country | United Kingdom |
| Start | 07/2022 |
| End | 07/2023 |
| Description | International Institutional Awards Tranche 2 Warwick |
| Amount | £62,500 (GBP) |
| Funding ID | BB/Z514548/1 |
| Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
| Sector | Public |
| Country | United Kingdom |
| Start | 04/2024 |
| End | 02/2026 |
| Description | Mapping Protein-Protein Interactions in Fungal Megasynth(et)ases |
| Amount | £95,000 (GBP) |
| Funding ID | 2587430 |
| Organisation | Engineering and Physical Sciences Research Council (EPSRC) |
| Sector | Public |
| Country | United Kingdom |
| Start | 08/2021 |
| End | 03/2025 |
| Description | PhD Project to be confirmed on completion of the training year |
| Amount | £125,000 (GBP) |
| Funding ID | 2887572 |
| Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
| Sector | Public |
| Country | United Kingdom |
| Start | 09/2023 |
| End | 09/2027 |
| Description | Interrogating unusual programming steps in fungal PKSs and NRPSs |
| Organisation | University of Western Australia |
| Country | Australia |
| Sector | Academic/University |
| PI Contribution | Application of intact / native mass spectrometry methods to study unusual programming steps in fungal PKS / NRPS enzymes, and chemical synthesis of substrates. Jenner lab hosted a PhD student from UWA in March 2024, forming basis for collaboration. |
| Collaborator Contribution | Collaborators provided protein expression constructs of PKS / NRPS pathways. |
| Impact | Further funding: BBSRC International Institutional Award (Tranche 2) - £10,000 |
| Start Year | 2022 |
| Description | MIBiG 4.0: Gobal Collaborative BGC Annotation Project |
| Organisation | Wageningen University & Research |
| Country | Netherlands |
| Sector | Academic/University |
| PI Contribution | Served as part of a global curation / annotation effort on the Minimum Information about a Biosynthetic Gene cluster (MIBiG) database (https://mibig.secondarymetabolites.org). Jenner was appointed to role of 'Interest Group Coordinator' for Polyketides based on expertise, which involved managing multiple users during annotation sessions. |
| Collaborator Contribution | Collaborators (WUR) established the MIBiG database in 2015 and set-up / organised the annotation sessions. |
| Impact | Publication: M. Zdouc, K. Blin et. al. MIBiG 4.0: Advancing biosynthetic gene cluster curation through global collaboration. Nucleic Acids Res., 2024, DOI: 10.1093/nar/gkae1115. Key Outcome: Curation of a highly-detailed database for biosynthetic gene clusters - https://mibig.secondarymetabolites.org/. |
| Start Year | 2023 |
| Description | Understanding fungal siderophore biosynthesis |
| Organisation | University of California, Los Angeles (UCLA) |
| Country | United States |
| Sector | Academic/University |
| PI Contribution | We have used intact protein mass spectrometry to visualise large (>100 kDa) protein fragments from NRPS enzymes responsible for producing siderophores in fungi. Monitoring the location / size of (amino)acyl chains attached to the NRPS allowed the molecular programming of these systems to be uncovered, and has set the stage for future exploitation of these pathways towards new scaffolds. |
| Collaborator Contribution | Collaborators provided proteins and substrates for assays / measurements. |
| Impact | Publication: M. Jenner, Y. Hai, H.H. Nguyen, M. Passmore, W. Skyrud, J. Kim, N.K. Garg, W. Zhang, R.R. Ogorzalek Loo and Y. Tang. Elucidating the molecular programming of a nonlinear nonribosomal peptide synthetase responsible for fungal siderophore biosynthesis. Nature Communications, 2023, 14, e2832 (DOI: https://doi.org/10.1038/s41467-023-38484-8) 'Behind the Paper' article (DOI: https://communities.springernature.com/posts/knowing-fe-knowing-you) Key Outcome: Development and application of mass spectrometry-based methods for unpicking location of biosynthetic intermediates. This collaboration is multi-disciplinary, involving molecular biologists, protein biochemists, synthetic and analytical chemists. Further funding: EPSRC DTP studentship (2883889) |
| Start Year | 2021 |
| Description | Award Lecture - RSC Felix Franks Biotechnology Medal |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Postgraduate students |
| Results and Impact | Delivered RSC Felix Franks Biotechnology Medal award lecture at RSC Directing Biosynthesis VII. |
| Year(s) Of Engagement Activity | 2024 |
| URL | https://www.rsc.org/events/detail/76071/directing-biosynthesis-vii |
| Description | Chemistry and Biology of Natural Products Symposium |
| Form Of Engagement Activity | A formal working group, expert panel or dialogue |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Postgraduate students |
| Results and Impact | Serve as co-organiser / chair (with Dr. Fabrizio Alberti, Warwick) of the annual Chemistry and Biology of Natural Products (CBNP) Symposium. This meeting is particularly aimed at postgraduate students, PDRAs and early career academics, but open to any academic/industrialist working in any areas of natural product chemistry and biology. Whilst typically hosted at Warwick, in 2022 and 2024 the meeting was paired with the flagship RSC Directing Biosynthesis conference in Edinburgh and Birmingham, respectively. |
| Year(s) Of Engagement Activity | 2022,2023,2024 |
| URL | https://warwick.ac.uk/fac/sci/wcibb/meetings/chemistryandbiologysymposium |
| Description | Invited Seminar - Durham University |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | Local |
| Primary Audience | Postgraduate students |
| Results and Impact | Invited seminar to researchers in fields of Chemistry / Biology. |
| Year(s) Of Engagement Activity | 2023 |
| Description | Invited Seminar - King's College London |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | Local |
| Primary Audience | Postgraduate students |
| Results and Impact | Following announcement of the FLF award, I was invited to deliver a seminar to researchers in fields of Chemistry / Biology. |
| Year(s) Of Engagement Activity | 2022 |
| Description | Invited Seminar - Strathclyde University |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | Local |
| Primary Audience | Postgraduate students |
| Results and Impact | Invited seminar to researchers in fields of Chemistry / Biology. |
| Year(s) Of Engagement Activity | 2024 |
| Description | RSC Biotechnology Group |
| Form Of Engagement Activity | A formal working group, expert panel or dialogue |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Postgraduate students |
| Results and Impact | Serving committee member (2024 - present) of RSC Biotechnology Group. Involves attendance at committee meetings (in person / online) and typically organising 1-day meetings / symposia on specialist topics under the 'Biotechnology' theme. |
| Year(s) Of Engagement Activity | 2024,2025 |
| URL | https://www.rsc.org/membership-and-community/connect-with-others/through-interests/interest-groups/b... |