Elucidating and Engineering Legonmycin Biosynthesis: a framework for heterobicyclic biotransformation
Lead Research Organisation:
University of Aberdeen
Department Name: Chemistry
Abstract
Many currently used drugs that we rely on for the treatment of cancer, bacterial infection, immune disorders and viral infections are either natural products or are derived from natural products. As such natural products still remain the major source of lead compounds for the development of new drugs and diagnostic molecules. However, natural products are often complex in terms of structure and composition making them difficult for synthetic chemists to access and thus develop further. In contrast bacteria can possess an amazing inventory of complex chemistry that organic chemists only dream of. In making new molecules, a key challenge is often to identify plausible new scaffolds or motifs (also known as chemical diversity) which is at the heart of drug discovery. We are going to study two bacterial enzymes that have the capability to convert linear peptide into heterobicyclic molecules under mild reactive conditions. These heterobicyclic ring systems are a common motif in biologically important compounds but there does not have any good way of making them by synthetic chemistry. The use of the bacterial enzymes to accomplish chemical tasks is well established in food industry and the benefits in terms of sustainable manufacturing are well document. However, the application of enzymatic process/ industrial biotechnology processes are still under developed the fine chemical, pharmaceutical and agricultural industries. By working out the catalytic capabilities of the two enzymes we will gain the ability to develop their industrial biotechnological potential. In doing this, we will be able to make novel building blocks (also known as scaffolds) and, access new biologically active compounds.
Technical Summary
We propose to investigate the capabilities of two novel enzymes, the thioesterase LgnD-Te and FAD-dependent monooxygenase LgnC from the soil bacterium Streptomyces sp. MA37, responsible for the formation of heterobicycles. These heterobicyclic rings are critical components of many biologically important and industrially relevant molecules. We have made significant progress in understanding the chemical mechanism of LgnC, which is the first step towards harnessing the industrial potential this enzyme. We intend to continue this work and elucidate its mechanism in detail through site direct mutagenesis studies to locate the active site. We will also probe the capacity of this enzyme using modified substrates with structural diversity. From our preliminary result we have begun to understand the mechanism of the thioesterase LgnD-Te and our data indicates the formation of a heterobicyclic ring using a surrogate synthetic substrate. We will advance our initial mechanistic work by using substrates with different stereogenic centres and through site direct mutagenesis to locate its active site. Substrate tolerance is a key feature for a successful industrial biocatalyst and we will synthesize peptide thioesters equipped with different chemical handles (i.e. bromine/chlorine/hydroxyl/amine) and probe the substrate flexibility of LgnD-Te. Furthermore, we will identify new homologues of LgnC and LgnD-Te that possess enhanced kinetic profiles and broader substrate tolerance through genome mining and synthetic gene expression. This will not only give us exquisite control of the biochemical experiments but lead to more diverse chemical scaffolds being accessible thus broadening the molecule library generated for bioactivity screening.
Planned Impact
Impact will be delivered by fulfilling the research program's key objective which is to provide stakeholders in various sectors with new bioactive compounds and knowledge in industrial biocatalysis.
The project will deliver impact across four main areas.
People: The program will deliver multidisciplinary training in the chemical and biological sciences to two PDRAs. The UK has identified synthetic biology and industrial biotechnology as key deficits in scientists' training for the future workforce and this program will address that need. The project is at the cutting edge of industrial biotechnology and it will provide excellent training for the PDRAs if they intend to continue to work in this research field (in industry or academia). The PDRAs will also have access to a variety of staff training courses run by both the University of Aberdeen and the University of Durham, which are designed to enhance a wide variety of transferable and career based skills. All of the aforementioned points will ensure that the recruited PDRAs have enhanced job prospects upon completion of the program and this will help to enhance the UK's scientific skill base. The PI's (Deng) lab regularly hosts undergraduates from the UK or Europe as summer placement students. Both Deng and Jaspars are very active in outreach programmes that engage with school children. If funded we will offer summer placements (1-2 per year) to local children to experience research at the chemical-biology interface. We also intend to create a range of practical biochemical experiments suitable for University undergraduates in the first instance at Aberdeen but that will be made open-access via the PI's webpage. The Co-I (Cobb) regularly gives educational lectures to teachers as part of a program of University engagement with Secondary School Education. Interaction with local high schools will also help to enthuse the next generation of scientists. In this area the Co-I (Cobb) has prior experience having led various sixth form projects as part of the annual North East Schools Industry Partnership scheme (NESIP) held in the Durham University Chemistry Department. Students from secondary schools in the North-East of England, accompanied by their teachers, spend a full week undertaking research projects.
Society: The work will generate new biologically active molecules and moreover allow their generation in useful quantities using sustainable manufacturing methods (i.e. biocatalysts). This means they can be used in drug development programmes and in screening campaigns for the identification of new agents such as anti-biotics. The generation of new antimicrobial agents has the potential to reduce the burden of infection both in the National Health Service (NHS), and, more directly in everyday life. Delivering in these areas will in turn have a direct and considerable impact on the health of the UK population.
Economic: The generation of novel IP will help to develop new markets for commercial exploitation which help UK PLC to remain internationally competitive in the areas of chemical-biology and industrial biocatalysis. As part of the work program we will utilise existing links and aim to establish new collaborations with industry to maximise the commercial exploitation of the research. We would expect to transfer technology through service agreements (supply novel compounds) or by technology licensing. We will consider founding our own spin out company as the project develops.
Knowledge: On a fundamental level the work program will deliver exciting new insights into the, biosynthesis of HAs and specifically the mode of action of the novel enzymes, LgnD-Te and LgnC, This new knowledge will be of benefit to researchers both in academia and in industry who are engaged in enhancing biotransformation to access novel molecular scaffolds for biological screening.
The project will deliver impact across four main areas.
People: The program will deliver multidisciplinary training in the chemical and biological sciences to two PDRAs. The UK has identified synthetic biology and industrial biotechnology as key deficits in scientists' training for the future workforce and this program will address that need. The project is at the cutting edge of industrial biotechnology and it will provide excellent training for the PDRAs if they intend to continue to work in this research field (in industry or academia). The PDRAs will also have access to a variety of staff training courses run by both the University of Aberdeen and the University of Durham, which are designed to enhance a wide variety of transferable and career based skills. All of the aforementioned points will ensure that the recruited PDRAs have enhanced job prospects upon completion of the program and this will help to enhance the UK's scientific skill base. The PI's (Deng) lab regularly hosts undergraduates from the UK or Europe as summer placement students. Both Deng and Jaspars are very active in outreach programmes that engage with school children. If funded we will offer summer placements (1-2 per year) to local children to experience research at the chemical-biology interface. We also intend to create a range of practical biochemical experiments suitable for University undergraduates in the first instance at Aberdeen but that will be made open-access via the PI's webpage. The Co-I (Cobb) regularly gives educational lectures to teachers as part of a program of University engagement with Secondary School Education. Interaction with local high schools will also help to enthuse the next generation of scientists. In this area the Co-I (Cobb) has prior experience having led various sixth form projects as part of the annual North East Schools Industry Partnership scheme (NESIP) held in the Durham University Chemistry Department. Students from secondary schools in the North-East of England, accompanied by their teachers, spend a full week undertaking research projects.
Society: The work will generate new biologically active molecules and moreover allow their generation in useful quantities using sustainable manufacturing methods (i.e. biocatalysts). This means they can be used in drug development programmes and in screening campaigns for the identification of new agents such as anti-biotics. The generation of new antimicrobial agents has the potential to reduce the burden of infection both in the National Health Service (NHS), and, more directly in everyday life. Delivering in these areas will in turn have a direct and considerable impact on the health of the UK population.
Economic: The generation of novel IP will help to develop new markets for commercial exploitation which help UK PLC to remain internationally competitive in the areas of chemical-biology and industrial biocatalysis. As part of the work program we will utilise existing links and aim to establish new collaborations with industry to maximise the commercial exploitation of the research. We would expect to transfer technology through service agreements (supply novel compounds) or by technology licensing. We will consider founding our own spin out company as the project develops.
Knowledge: On a fundamental level the work program will deliver exciting new insights into the, biosynthesis of HAs and specifically the mode of action of the novel enzymes, LgnD-Te and LgnC, This new knowledge will be of benefit to researchers both in academia and in industry who are engaged in enhancing biotransformation to access novel molecular scaffolds for biological screening.
Publications
Fang Q
(2021)
Genomic scanning enabling discovery of a new antibacterial bicyclic carbamate-containing alkaloid
in Synthetic and Systems Biotechnology
Maglangit F
(2020)
Characterization of the promiscuous N -acyl CoA transferase, LgoC, in legonoxamine biosynthesis
in Organic & Biomolecular Chemistry
Maglangit F
(2021)
Bacterial pathogens: threat or treat (a review on bioactive natural products from bacterial pathogens).
in Natural product reports
Valera A
(2022)
Characterization of a class II ketol-acid reductoisomerase from Mycobacterium tuberculosis.
in RSC advances
Wang S
(2022)
A ribosomally synthesised and post-translationally modified peptide containing a ß-enamino acid and a macrocyclic motif.
in Nature communications
Wang S
(2024)
Dehydroamino acid residues in bioactive natural products
in Natural Product Reports
Wang S
(2020)
Discovery and Biosynthetic Investigation of a New Antibacterial Dehydrated Non-Ribosomal Tripeptide
in Angewandte Chemie
Wang S
(2021)
Discovery and Biosynthetic Investigation of a New Antibacterial Dehydrated Non-Ribosomal Tripeptide.
in Angewandte Chemie (International ed. in English)
Wang S
(2021)
Peculiarities of promiscuous l-threonine transaldolases for enantioselective synthesis of ß-hydroxy-a-amino acids
in Applied Microbiology and Biotechnology
Wang S
(2022)
Aminoacyl chain translocation catalysed by a type II thioesterase domain in an unusual non-ribosomal peptide synthetase.
in Nature communications
Wang S
(2020)
The X-factor: Enhanced ß-oxidation on intracellular triacylglycerols enabling overproduction of polyketide drug-like molecules in microorganisms.
in Synthetic and systems biotechnology
Wu L
(2020)
Fluorine biocatalysis
in Current Opinion in Chemical Biology
Wu L
(2020)
An unusual metal-bound 4-fluorothreonine transaldolase from Streptomyces sp. MA37 catalyses promiscuous transaldol reactions.
in Applied microbiology and biotechnology
Description | 1. A new type of thioesterase enzymes was found to facilitate key enzymatic reactions through protein-protein interaction. The relevant results were published in a recent Nature Communication paper. 2. identification of key biosynthetic intermediates through chemical probing and LC-MS analysis. The relevant results were published in a recent Nature Communication paper. 3. Identification of key enzymes that catalyse dehydration reactions, which led us to study another interesting biosynthetic pathway of a non-ribosomal peptide, resulting in a publication in Angew Chemie Int Ed. 4. various synthetic proline analogues (provided by Durham collaborators) and threonine/serine have been tested and generated compound library with diverse functionality. Due to the Covid impact, this part of research progress was slower than what we expected. We are still in the process of gathering the results for future publication. 5. developing a new collaboration with researchers in Warwick using native MS to analyse the new type of protein-protein interactions. The relevant results were published in a recent Nature Communication paper. 6. developing a new collaboration with researchers in Newcastle using density functional theory to calculate the plausible stereochemistry of reactive biosynthetic intermediates. However, due to the negative impact of the pandemic, the funding was ended while the experimental data were still on the process of collecting. The outcomes/publications are significantly delayed. |
Exploitation Route | The successful outcome of the project is to develop unique natural product like molecules that have the capacity to be developed into a new therapeutics and agrochemicals. By the end of the project the aim is to have sound, proof of principal data that can be used to further develop the biotechnology developed and the compounds prepared. Ingenza, a biotechnology company based in Edinburgh, showed considerable interest in this biotechnology. |
Sectors | Chemicals Manufacturing including Industrial Biotechology Pharmaceuticals and Medical Biotechnology |
URL | https://doi.org/10.1038/s41467-021-27512-0. |
Description | Dissection of the cyclization mechanism underlying the assembly of the bacterial tricyclic carbazole natural products |
Amount | £12,000 (GBP) |
Funding ID | IEC\NSFC\170617 |
Organisation | The Royal Society |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 03/2017 |
End | 04/2020 |
Description | NPRONET |
Amount | £10,000 (GBP) |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 06/2017 |
End | 01/2018 |
Title | A bioactive ribosomally synthesised peptide with unusual structural features |
Description | enzymes that make unusual unsaturated beta-enamide in a peptide and mediate a new cyclized moiety. |
Type Of Material | Biological samples |
Year Produced | 2022 |
Provided To Others? | Yes |
Impact | Will offer the bioengineering/synthetic biology community new tools to incorporate unsaturated beta-amino acid and new cyclic moieties into their corresponding peptide systems. |
URL | https://doi.org/10.1038/s41467-022-32774-3 |
Title | A new group of dual dehydration/condensation enzyme domains in the biosynthetic pathways of non-ribosomal peptides |
Description | A new group of dual dehydration/condensation enzyme domains in the NRPS biosynthetic pathways was discovered. |
Type Of Material | Biological samples |
Year Produced | 2022 |
Provided To Others? | Yes |
Impact | such discoveries will provide a useful tool for NRPS bioengineering community to incorporate this cassette into their corresponding system and provide functionalised moieties in structurally complex peptides for further diversifications. |
URL | https://doi.org/10.1038/s41467-021-27512-0 |
Title | A new type of Type II thioesterases in the biosynthetic pathways of non-ribosomal peptides |
Description | This enzyme plays an essential role of shuttling substrates from one enzyme domain to another in order to maintain the biosynthetic fidelity. this new mechanism of aminoacyl chain translocation was found to be widely distributed in bacterial non-ribosomal peptide biosynthesis, |
Type Of Material | Biological samples |
Year Produced | 2022 |
Provided To Others? | Yes |
Impact | Our finding changes the conventional view of Type II thioesterases that the main role of this group of enzymes in the biosynthesis only catalyse removal of aberrant intermediates. this finding can be used as a guide for future genome mining to discover new drug-like molecules from nature or can be developed as a toolbox for synthetic biology to generate bioactive peptidyl molecules with structural diversity. |
URL | https://doi.org/10.1038/s41467-021-27512-0 |
Title | a new group of condensation domains in non-ribosomal peptide synthetases that catalyse dehydration reaction to yield dehydroamino acid residues |
Description | The finding changes the conventional view of condensation domains in the field that this enzyme domain solely acts as a gatekeeper for downstream biosynthesis. We found a new group of condensation domains can catalyse beta-elimination reaction to yield dehydroamino acid residues during the biosynthesis, thus increasing the structural diversity of the resultant bioactive peptidyl molecules. |
Type Of Material | Biological samples |
Year Produced | 2021 |
Provided To Others? | Yes |
Impact | Our finding potentially provided a toolbox for synthetic biology to incorporate unusual amino acid residues in other pathways of drug-like non-ribosomal peptides. The presence of such dehydroamino acid residues in peptides can further offer a bioorthogonal handle for diversification for peptide-based compound library generation. |
URL | https://doi.org/10.1002/anie.202012902 |
Title | monooxygenase |
Description | We discovered a series of monooxygenases from different sources that catalyse a similar biochemical transformation which can be used as biocatalysts. |
Type Of Material | Biological samples |
Year Produced | 2018 |
Provided To Others? | Yes |
Impact | Not yet |
Title | non-ribosomal peptide synthetases |
Description | Identification and overexpression of a series of non-ribosomal peptide synthetase homologues for characterization. |
Type Of Material | Biological samples |
Year Produced | 2019 |
Provided To Others? | No |
Impact | We will test these homologues with the aim of finding the enzymes with the best kinetics and wider substrate specificity for potential application as new biocatalysts for the synthesis of new bicyclic alkaloids. |
Description | Chemo-enzymatic synthesis |
Organisation | Durham University |
Department | Department of Chemistry |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | This is a collaborative project between Dr Hai Deng , University of Aberdeen and Dr Steven Cobb, Durham University. Aberdeen contribution is mainly focused on the characterization of the targeted enzymes and application of the enzymes of interest for new compound synthesis. |
Collaborator Contribution | Durham contribution is mainly focused on the chemical synthesis of small molecule substrate analogues for the enzymes of interest. |
Impact | Not yet but we are accumulating preliminary data right now for future publications. |
Start Year | 2017 |
Description | Warwick |
Organisation | University of Warwick |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | This is a collaborative project between Dr Hai Deng , University of Aberdeen and Dr Matthew Jenn, Department of Chemistry, University of Warwick. Aberdeen contribution is mainly focused on the characterization of the targeted enzymes and application of the enzymes of interest for new compound synthesis. |
Collaborator Contribution | Warwick contribution is mainly focused on protein-protein interactions of the enzymes of interest through native MS analysis. |
Impact | Not yet due to the pandemic although we are accumulating preliminary data for future publication and application. |
Start Year | 2020 |
Description | One-day symposium with Chinese visitors and collaborators |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Postgraduate students |
Results and Impact | The Chinese collaborators visited the research teams in Aberdeen, in July, 2020 under the funding support of NSFC-RS Newton Mobility scheme. The team hosted a symposium for the visitors and showcased the preliminary results and technologies from both awards. After the symposium, Chinese collaborators and the team decided to the next research activities and next scientific publications. |
Year(s) Of Engagement Activity | 2020 |
Description | Oral presentation in annual Perkin meeing |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Postgraduate students |
Results and Impact | Gave an oral presentation in annual Perkin meeting of Scotland. |
Year(s) Of Engagement Activity | 2020 |
Description | Research visit to Chinese collaborators in Wuhan in 2019 |
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 | Visit Chinese collaborators in the School of Pharmaceutical Sciences, Wuhan University and Renji Hospital, the School of Medical Sciences, Shanghai JiaoTong University, China, and discuss potential publication writings under the funding support of NSFC-RS newton mobility scheme, 13-19, April, 2019. |
Year(s) Of Engagement Activity | 2019 |
Description | School visit |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Schools |
Results and Impact | Performed a "flashes and bangs" show for secondary school aged children focussing on the concepts of energy changes during chemical reactions. |
Year(s) Of Engagement Activity | 2019,2020 |
Description | School visit |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Schools |
Results and Impact | St Joseph Primary School organised "Science Day" June, 2019 and invited HD to participate in this event. HD briefly introduced the principles of biochemistry and presented a colorimetric experiment including a series of enzymatic reactions to aspire school pupiles and general audience. The teachers told HD that it appeared that there have been increased interest in the school in biochemistry/chemistry. |
Year(s) Of Engagement Activity | 2019 |
Description | engagement training |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Supporters |
Results and Impact | 3 day course aimed at enhancing public engagement skills and at designing an event to be held in Oban during West Highland Yachting Week in July where various aspects of current ocean based research will be showcased. |
Year(s) Of Engagement Activity | 2018,2019 |
Description | outreach event |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Public/other audiences |
Results and Impact | During 2 days of the West Highland Yachting Week in Oban, the events designed during the training programme were rolled out for the general public to try. |
Year(s) Of Engagement Activity | 2019 |