Synthetic probes of natural product biosynthesis: implications for synthetic biology and drug discovery.

Lead Research Organisation: University of Warwick
Department Name: Chemistry

Abstract

Natural products represent a major source of powerful agents for the treatment of human, animal and plant diseases. Indeed the most potent antibiotics, anticancer agents, antiparasitic agents and pesticides in use today are natural substances produced in microorganisms and plants by dedicated sets of enzymes. Natural products owe their extraordinary bioactivity to their highly complex chemical structures, which still constitute a challenge for chemical synthesis. Intriguingly microorganisms and plants have the ability to produce these products from very simple precursors (e.g. acetate, amino acids and sugars) for their survival. Advances in research have increased our knowledge of the bacterial, fungal and plant enzymes and the genes responsible for their existence; however little is known about the chemical details of their biosynthesis. This is because the biosynthetic processes comprise of multiple transformations that take place in rapid succession and for which we currently have no generic and straightforward method of investigation. The limited amount of information obtained so far in relation to the number and the complexity of the existing natural product pathways prevent us from fully understanding and utilizing these pathways for improving the production of known products (via the metabolic engineering of microorganisms) and the generation of new ones (via synthetic biology approaches aimed at new drug discovery). At the present time these are extremely desirable goals in view of the many global challenges in health, environment, sustainability and energy that the scientific and the nonscientific communities face.

This research project aims at developing a novel and general methodology for the study of natural product biosynthesis based of the use of synthetic probes. These are chemically prepared compounds that mimic the basic precursors utilized in natural product formation and interfere in the pathways leading to the natural product assembly, 'catching' and retrieving the intermediate species from complex biological mixtures and throughout their processing. The pathways targeted in this research project are those leading to polyketide and nonribosomal peptide products, among which we count many commercially and industrially relevant products such as the blockbuster cardiovascular statin drugs and the antibiotics of last resort vancomycin and teicoplanin.
To develop this 'chemical' methodology, which is of general applicability and is capable of providing immediate answers to complex mechanistic issues, we have identified specific research objectives concerning (1) the development of synthetic probes of improved bioavailability for the study of polyketide biosynthesis 'in vivo' (meaning directly in live microorganisms); (2) the design, the development and the validation of putative probes of nonribosomal peptide biosynthesis, and (3) the application of the polyketide and nonribosomal peptide probes to elucidate poorly understood pathways leading to products of commercial and industrial interest.

The main outcomes of this research will be (a) powerful yet very practical tools capable of providing unique mechanistic insights into biological processes, whose beneficiaries include researchers in the broad area of biosciences; (b) new understanding of natural product biosynthesis, which will constitute the basis of for the biosynthetic engineering of new products.
The major beneficiary of this research outside the academic community will therefore be the biotechnology industry, who is going to utilize the information gained through our methodology to develop new products for the nation's health and for the growth of the UK economic competitiveness.

Technical Summary

Understanding the factors that control the programming of natural product biosynthetic enzymes is undeniably one of the greatest challenges and priorities in view of synthetic biology for drug discovery and production in microorganisms. One way to gain insights into the programming is to disclose the fine mechanistic details of the catalysis via the isolation of biosynthetic intermediates.

The overall aim of this project is to develop a chemical method for the investigation of polyketide and nonribosomal peptide biosyntheses based on the use of synthetic probes. These have been devised as nonhydrolysable analogues of the malonyl and the aminoacyl building blocks normally recruited in polyketide and nonribosomal peptide formation and compete with them for the natural product chain extension, capturing and off-loading premature biosynthetic species from the multifunctional enzymes.

In order to establish this chemical strategy as a leading method of investigation, we aim at: 1) developing the methodology for the investigation of polyketide biosynthesis in vivo, via second-generation probes of improved bioavailability and/or the aid of biochemical tools; 2) extending the methodology to other assembly lines, specifically to nonribosomal peptide synthetases (NRPSs), via nonhydrolysable aminoacyl cysteamine probes; 3) applying the methodology herein developed to elucidate poorly-understood biosynthetic pathways leading to commercially and industrially important natural products, such as thiolactomycin. These objectives will be accomplished through the combination of organic synthesis, in vivo feeding experiments, LC-HRMS and NMR analysis, molecular biology and protein chemistry tools. The outcome of this research will be powerful yet very practical probes that will provide unique insights into the sophisticated biosynthetic enzymes, laying the foundations for their novel exploitation in synthetic biology

Planned Impact

Beside the academic beneficiaries, our research will benefit:

1) the biotechnology commercial private sector, who will be the main user of the research outputs, both immediately and in the longer term.
The data acquired through our methodology will be indeed used by biotechnology companies for the rational re-engineering of microorganisms to improve the production of pharmaceuticals and agrochemicals currently in use, as well as to generate new ones of improved or different bioactivity.
At the present time these are extremely desirable goals in view of the many global challenges in health, environment, sustainability and energy that the scientific and the non-scientific communities face worldwide. After the completion of this project, and in particular of objective 3 which concerns the methodology application for the elucidation of a pharmaceutically relevant product, we will already have in our hands relevant and significant data for the beginning of a research programme addressing the generation of novel anticancer and anti-obesity agents via synthetic biology, with the realistic possibility of generating a library of novel compounds for testing within the next 10 years. This is just the initial example of how our methodology will have long-term impact and lead to many benefits for both the industrial and the public sector.

2) Nation's health. The public health sector (NHS) is currently facing a growing demand for the treatment of conditions associated to ageing population and obesity. By leading to the generation of new products for human health that can tackle these challenges, our methodology will ultimately contribute to the enhancement of the quality of life for a larger number of people at reduced costs for the public sector.

3) the UK economic competitiveness. By introducing and enhancing a new creative output for synthetic biology, our basic research will ultimately contribute to maintaining and leading UK innovation in biotechnology for animal health, agriculture, biomaterials and biofuels. This is particularly relevant in face of climate change and a growing demand for clean energy, for which UK research and technology need to provide novel and prompt solutions.

4) the staff working on the project. The researcher working full-time on this project will benefit of an extensive cross-disciplinary training which will include organic synthesis, microbiology, molecular biology, protein chemistry and analytical chemistry. The practical skills acquired by PDRA will be useful for his/her employment in both the academic and the industrial sectors at the end of the project. In addition the researcher will have the opportunity to develop key transferable skills such as work planning, project management and development, teamwork and public outreach, which are invaluable tools also for non-research based careers. The collective skills acquired throughout this project will give the researcher a very competitive edge which is indispensable in the current global job market.

Publications

10 25 50
 
Description Through this award we were able to:
1) develop second-generation probes for investigation of polyketide natural product biosynthesis in vivo: not only we generated probes of improved bioavailability capable of intercepting all steps in 'modular' assemblies, but we also proved that these tools can be efficiently utilised for the investigation of iterative enzymes (whose modus operandi is unpredictable a priori) in a variety of bacteria and fungi.
More importantly, we have preliminary shown that the unique information gained by the aid of chemical probes can lead to novel ways to 'redesign' biosynthetic pathways for the generation of 'unnatural' products- novel molecules that are still biosynthetically made but that differ in structure from the original natural products: these novel molecules may be of equal or superior value to the original products;

2) develop novel probes for the investigation of nonribosomal peptide biocatalysis in vivo. We successfully synthesised the original envisaged probes and improved them so that they can now be used as efficient tools for biosynthetic intermediate capture in the assembly of medically relevant nonribosomal peptides, such as the antitumor antibiotic echinomycin. With their proof of working established, these novel tools are now available to be further utilised for the investigation of challenging pathways leading to complex natural products of commercial and industrial importance;

3) apply the synthetic probe methodology herein developed to elucidate poorly-understood biosynthetic pathways. By utilising the chemical tools developed in this project, we were able to acquire preliminary chemical insights into the formation of thiolactomycin (a fatty acid synthase inhibitor and a promising lead for the development of novel antituberculotic and antimalarial agents) in its natural host. This venture (in collaboration with the Sun group, Wuhan, China, and the Leadlay group, Cambridge, UK) has established the basis for further work into the investigation of thiotetronate biosynthetic machineries.
Exploitation Route Most of the findings in this project have been published or will soon be. These will be taken forward by us and by others (e.g. academic and industrial groups working in the biotechnology field) to further unveil details of unknown biosynthetic pathways and devise novel ways to explore biocatalytic natural product machineries.
Sectors Agriculture, Food and Drink,Chemicals,Environment,Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description IAS Fellowship (Warwick)
Amount £90,000 (GBP)
Organisation University of Warwick 
Department Institute of Advanced Study
Sector Academic/University
Country United Kingdom
Start 01/2014 
End 12/2015
 
Description Marie Curie IEF
Amount € 222,000 (EUR)
Funding ID FP7-PEOPLE-2013 project number 628069 (MISELIAS) 
Organisation European Commission 
Department Seventh Framework Programme (FP7)
Sector Public
Country European Union (EU)
Start 03/2014 
End 03/2016
 
Title FT-ICR-MS 
Description use of FT-ICR-MS for the characterisation of small molecules and proteins involved in natural product biosynthetic pathways 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact mainly academic impact so far: data acquired for publication and future research 
 
Title nano-LC-HRMS methods 
Description methods for the advanced characterisation of biosynthetic intermediates from polyketide and nonribosomal peptide pathways 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact We are currently the only group able to extensively characterise biosynthetic intermediates from polyketide and nonribosomal peptide pathways via the use of chemical probes developed through this work. This unique expertise has allowed us to engage a number of collaborations with UK, European and Chinese groups, which will hopefully evolve into long-term partnerships. 
 
Title synthetic probes 
Description development of synthetic probes for the capture of biosynthetic intermediates and the generation of novel natural products in vitro and in vivo 
Type Of Material Technology assay or reagent 
Year Produced 2012 
Provided To Others? Yes  
Impact publications, manuscripts in preparation and interest from other research grous: we are currently sharing our tools with approximately 10 research groups based in the UK, Germany, Switzerland and China, and we expect to develop these early day collaborations into long-term partnerships 
 
Description COST CM1407 (NATCHEMDRUGS) 
Organisation European Cooperation in Science and Technology (COST)
Country Belgium 
Sector Public 
PI Contribution This partnership is a result of the BBSRC award. My research team provided training to a 6 month visiting researcher and also materials for biological testing to the COST CM1407 (NATCHEMDRUGS).
Collaborator Contribution Award to a researcher of another country to visit and carry out research in our labs for 6 months. Testing of chemical probes generated in our labs for different bioactivities through the COST network.
Impact The collaboration is multidisciplinary (organic synthesis, microbiology, anaytical chemistry, biological testing) One manuscript is in preparation arising from this collaboration.
Start Year 2015
 
Description chemical probes for nonribosomal peptide synthetases 
Organisation Monash University
Country Australia 
Sector Academic/University 
PI Contribution Provision of chemical probes; hosting and training of research team member
Collaborator Contribution Provision of plasmids and glycopeptide producing microbial strains; hosting and training of research team member
Impact This collaboration arised from the use of chemical probes devised and generated through this BBSRC award. Collaboration is multidisciplinary (synthetic chemistry, microbiology, in vitro enzymology, analytical chemistry). Outcomes are a joint manuscript currently in preparation on the chemical probing of glycopeptide assembly, and a current joint PhD award between our collaborator and our group.
Start Year 2015
 
Description chemical probing of polyketide biosynthesis in fungi 
Organisation Aalborg University
Country Denmark 
Sector Academic/University 
PI Contribution synthesis of chemical probes for dissecting polyketide biosynthetic pathways in fungi HR-LC-MS analyses of fungal extracts
Collaborator Contribution use of chemical probes in fungi; MS data analyses of fungal extracts
Impact This is a collaboration that started on the basis of chemical probed developed through this BBSRC award output: publication currently in preparation and ready for submission (Chemical probing of polyketide assembly in the plant pathogen Fusarium graminearum) multidisciplinary collaboration involving chemical synthesis and analytical chemistry (Warwick University), as well as fungal microbiology and genetic manipulation (Aalborg University)
Start Year 2017
 
Description genetic manipulation 1 
Organisation University of Cambridge
Department Department of Biochemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution preparation of substrates for in vitro and in vivo assays
Collaborator Contribution sharing of materials and protocols to prepare/utilise constructs for recombinant protein expression, generation of engineered bacteria
Impact Collaboration is multidisciplinary (involving synthetic chemistry, biochemistry, microbiology, molecular biology, analytical chemistry) Outcomes are the following research publications (in reverse chronological order): Unexpected enzyme-catalysed [4+2] cycloaddition and rearrangement in polyether antibiotic biosynthesis Nature Catalysis 2019-11 | journal-article DOI: 10.1038/s41929-019-0351-2 Chemical probing of thiotetronate bio-assembly Chemical Communications 2017 | journal-article DOI: 10.1039/C6CC09933E The polyketide backbone of thiolactomycin is assembled by an unusual iterative polyketide synthase Chemical Communications 2017 | journal-article DOI: 10.1039/C6CC09934C Insights into 6-Methylsalicylic Acid Bio-assembly by Using Chemical Probes Angewandte Chemie - International Edition 2016 | journal-article DOI: 10.1002/anie.201509038EID: 2-s2.0-84960886451 Chemical probes for the functionalization of polyketide intermediates Angewandte Chemie - International Edition 2014 | journal-article DOI: 10.1002/anie.201407448EID: 2-s2.0-84918814167
Start Year 2012
 
Description genetic manipulation 2 
Organisation Wuhan University
Country China 
Sector Academic/University 
PI Contribution synthesis of substrate/probes for biosynthetic investigations
Collaborator Contribution genetic manipulation of bacterial strains of interest
Impact collaboration is multidisciplinary (synthetic chemistry, genetic manipulation, analytical chemistry, microbiology) joint publications listed below, plus a further publication in preparation resulting from the use of engineered strains provided by Wuhan: Chemical probing of thiotetronate bio-assembly Chemical Communications 2017 | journal-article DOI: 10.1039/C6CC09933E The polyketide backbone of thiolactomycin is assembled by an unusual iterative polyketide synthase Chemical Communications 2017 | journal-article DOI: 10.1039/C6CC09934C A genomics-led approach to deciphering the mechanism of thiotetronate antibiotic biosynthesis Chemical Science 2016 | journal-article DOI: 10.1039/c5sc03059eEID: 2-s2.0-84951025893
Start Year 2012
 
Description iterative polyketide catalysis in bacteria 
Organisation Leibniz Association
Department Leibniz Institute for Natural Product Research and Infection Biology
Country Germany 
Sector Academic/University 
PI Contribution synthesis of chemical probes for the probing of micacocidin biosynthesis LC-MS analysis of microbial extracts sent from our collaborators
Collaborator Contribution testing of chemical probes on bacteria producing micacocidin
Impact OBC paper published in 2015 multidisciplinary collaboration (synthetic chemistry, microbiology, genetic manipulation, analytical chemistry, enzymology)
Start Year 2013
 
Description mass spectrometry 
Organisation University of Warwick
Country United Kingdom 
Sector Academic/University 
PI Contribution synthesis of small molecules probes and chemically modified pantetheine cofactors for carrier protein derivatisation
Collaborator Contribution FT-ICR-MS analysis of natural products and acyl carrier proteins
Impact Collaboration is multi-disciplinary (involving synthetic chemistry, biochemistry and mass spectrometry) Outcomes are 2 research papers and a book chapter resulting from the collaboration: A light-activated acyl carrier protein 'trap' for intermediate capture in type II iterative polyketide biocatalysis Chemistry - A European Journal 2019-10-09 | journal-article DOI: 10.1002/chem.201903662 Structural characterization of polyketides using high mass accuracy tandem mass spectrometry Analytical Chemistry 2012 | journal-article DOI: 10.1021/ac3022778EID: 2-s2.0-84869450232 High-Resolution Tandem Mass Spectrometry for Nonribosomal Peptide and Polyketide Analysis Natural Products Analysis: Instrumentation, Methods, and Applications 2014 | book DOI: 10.1002/9781118876015.ch12EID: 2-s2.0-84927574248
Start Year 2012
 
Description mass spectrometry 
Organisation University of Warwick
Country United Kingdom 
Sector Academic/University 
PI Contribution isolation of natural products and biosynthetic intermediates, synthetic mimics/analogues of natural products
Collaborator Contribution HR-MS analyses of our substrates (of synthetic and biosynthetic origin)
Impact data for publications and manuscripts currently in preparation the collaboration is multidisciplinary (organic chemistry, microbiology, enzymology, mass spectrometry)
Start Year 2012
 
Description organic synthesis 
Organisation University of Milan
Country Italy 
Sector Academic/University 
PI Contribution Hosting of visiting students/ researchers, training in microbiology and advanced MS analyses
Collaborator Contribution The project partner, met through COST Action 1407 (NATCHEMDRUGS), has provided visiting students/ researchers since 2015-16 that have contributed to the synthesis and the application of chemical probes object of this grant.
Impact Multi-disciplinary collaboration (organic chemistry, microbiology, analytical chemistry including small molecule advanced mass spectrometry) Outcomes: 1 publication (listed below) and one manuscript in preparation (not listed) Novel chemical probes for the investigation of nonribosomal peptide assembly Chemical Communications 2017 | journal-article DOI: 10.1039/C7CC02427D
Start Year 2015
 
Description NPRONET networking event/ presentation 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact The purpose of this presentation was to make aware the network event participants of the work developed through this BBSRC grant, particularly in relation to potential future collaborations with the biotechnology industry. Approximately 150 people from industry and academia participated at this event; the project presentation lead to further interactions with peers colleagues in the field and the meeting of industrial participants who manifested interest in future work developments.
Year(s) Of Engagement Activity 2015
 
Description outreach 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact explaining visiting students my research sparked a lot of questions and interest in natural products and our work

after interacting with a number of students and explaining my research, a number of them came back to visit our Department for a second time and placed an application for one of our courses
Year(s) Of Engagement Activity 2012,2013,2014,2015
 
Description school visit (Cambridge) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact talk sparked student interest in our work- questions asked/ received after the talk and via email

after my talk, this school reported higher interest in our Department and specifically in Chemical Biology studies (much higher number of undergraduate applications received)
Year(s) Of Engagement Activity 2013