Elucidating and engineering natural product biosynthesis in actinomycetes

Lead Research Organisation: John Innes Centre
Department Name: Contracts Office

Abstract

My research encompasses the discovery, characterisation and engineering of pathways that make drug-like molecules in actinomycete bacteria. I am particularly interested in bioactive natural products that also possess unusual structural features. We employ a "gene to product" approach, which requires a wide variety of techniques, such as the computational analysis of bacterial genomes, the genetic manipulation of gene clusters and the in vitro analysis of pathway proteins.
Two major routes to peptide natural products have evolved: non-ribosomal peptide synthetases (NRPSs) and ribosomally synthesized and post-translationally modified peptides (RiPPs). We study the biosynthesis of complex metabolites produced by both types of pathway. NRPSs are massive multi-domain assembly line proteins and are responsible for the biosynthesis of the glycopeptide antibiotics. These hugely complex molecules are used as drugs of last resort to treat aggressive methicillin-resistant Staphylococcus aureus (MRSA) infections. Through a variety of collaborations, I am investigating the regulation, activity and biosynthesis of these molecules. In contrast to NRPSs, RiPPs originate from ribosomal precursor peptides. Bottromycin is a clinically promising and structurally unique RiPP, and is active towards multi-drug resistant bacteria, such as MRSA. Its structurally novelty makes it a promising lead compound in the fight against infection. Following the discovery of the bottromycin pathway in Streptomyces scabies, we are now characterising the enzymatic steps of this pathway to determine how structural complexity is introduced into this compound. Additional work on this pathway includes mutagenesis to produce novel derivatives and an analysis of the regulatory factors that control bottromycin biosynthesis. This research will inform future investigations into other novel RiPP pathways.

Publications

10 25 50
 
Description This research grant has helped support research across a variety of projects relating to the biosynthesis of bacterial natural products. This includes understanding how potent antibiotics are made, including bottromycin (http://onlinelibrary.wiley.com/doi/10.1002/anie.201604304/abstract), teicoplanin (https://pubs.acs.org/doi/10.1021/acschembio.6b00018 , https://link.springer.com/article/10.1007%2Fs00253-014-5969-z), actinonin and matlystatin (https://www.nature.com/articles/s41467-017-01975-6). Additional work has focused on the discovery of biosynthetic pathways to new antibacterial and anticancer compounds, including ristocetin (http://aac.asm.org/content/58/10/5687.long) and thioviridamide-like molecules (http://pubs.acs.org/doi/10.1021/acschembio.7b00677). This reflects a significant number of diverse natural products with potent biological activities, and this work provides information on how these molecules are made, including some very unusual enzymatic transformations. More fundamentally, we have been able to develop methodology that is assisting with the chemical analysis of natural products.
Exploitation Route We have identified a wide array of interesting biosynthetic pathways that will be of significant interest to the natural products community, as well as researchers in biocatalysis and microbiology. This could include structural characterisation of enzymes, employment of enzymes as biocatalysts for challenging chemical reactions, analysis of the mechanism of action of some of these natural products, or the exploitation of the methodology that we report on other areas of natural products research.
Sectors Agriculture, Food and Drink,Chemicals,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description Methodology supported by this grant led to the establishment of a collaboration with a local company (Celbius Ltd) to assess whether sonication can aid in natural product yield and/or ease of extraction. This was supported by an NPRONET Proof of Concept grant (code POC005_2) and was reported at an NPRONET meeting (Manchester, Oct 2016) to a mixed audience of academia and industry. Another collaboration (joint with Jake Malone, John Innes Centre) was established with a local company (VCS Potatoes Ltd) to assess the ability of soil-dwelling bacteria to control important plant diseases. This work has led to new methodology to assess bacterial soil populations and their ability to suppress plant diseases, as well as the establishment of further biocontrol projects with other companies (led by Jake Malone). This work was reported to industry members at Science for Innovation Showcase event at the John Innes Centre, where we subsequently discussed opportunities for large-scale field trials with a variety of interested parties.
First Year Of Impact 2017
Sector Agriculture, Food and Drink,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
Impact Types Economic

 
Description Contribution to SAW Antibiotics book
Geographic Reach National 
Policy Influence Type Influenced training of practitioners or researchers
URL http://www.sawtrust.org/buy-the-books/saw-antibiotics/
 
Description NPRONET Proof of Concept funding
Amount £54,342 (GBP)
Funding ID NPRONET POC005_2 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 01/2016 
End 05/2016
 
Description NRP Science Links Seed Corn funding
Amount £15,232 (GBP)
Organisation Norwich Research Park 
Sector Private
Country United Kingdom
Start 04/2015 
End 10/2015
 
Description Norwich Research Park BBSRC DTP PhD Studentship
Amount £95,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2016 
End 09/2020
 
Description Royal Society Enhancement Award
Amount £170,640 (GBP)
Funding ID RGF\EA\181083 
Organisation The Royal Society 
Sector Academic/University
Country United Kingdom
Start 10/2018 
End 03/2021
 
Description Royal Society Enhancement Award
Amount £99,859 (GBP)
Funding ID RGF/EA/180154 
Organisation The Royal Society 
Sector Academic/University
Country United Kingdom
Start 12/2017 
End 03/2021
 
Description Royal Society University Research Fellowships Renewal
Amount £363,410 (GBP)
Funding ID URF\R\180007 
Organisation The Royal Society 
Sector Academic/University
Country United Kingdom
Start 10/2018 
End 09/2021
 
Description UK China AMR Call (Newton Fund)
Amount £490,718 (GBP)
Funding ID MR/P007570/1 
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 07/2016 
End 06/2019
 
Title RIPPER genome mining 
Description RiPPER is a command line computational tool that assists in the identification of biosynthetic gene clusters and associated precursor peptides for RiPPs, a large and important class of natural product. 
Type Of Material Improvements to research infrastructure 
Year Produced 2018 
Provided To Others? Yes  
Impact This has assisted in research projects in my group and I have had contact with researchers in other groups who have benefited from the tool and the results we have reported from its use. It assisted in the identification of a new family of natural products, the thiovarsolins, which were characterised by my group. The identification of these natural products and the development of the tool were reported in a BioRxiv pre-print (https://www.biorxiv.org/content/10.1101/494286v1) and have just been accepted for publication in Nucleic Acids Research. 
URL https://github.com/streptomyces/ripper
 
Description Agrobacterium pathway discovery 
Organisation University of Zagreb
Country Croatia 
Sector Academic/University 
PI Contribution 1. Host laboratory for research visit (enzymology, chemical synthesis, mass spectrometry) 2. Input into design of experiments and analysis of data.
Collaborator Contribution 1. Started project and designed experiments. 2. Prepared materials (enzymes, substrates) for project.
Impact Multidisciplinary: microbiology, biochemistry, chemistry
Start Year 2014
 
Description Calabria thioviridamides 
Organisation University of Calabria
Country Italy 
Sector Academic/University 
PI Contribution We established a project to discovery new thioviridamide-like molecules (TLMs) by the use of a genome mining method. This involved pathway identification, strain fermentation, pathway cloning and mutagenesis, and then purification and chemical analysis of the products of these pathways.
Collaborator Contribution The group of Anna Rita Cappello determined the biological activity of our purified compounds against bacteria, fungi and human cell lines.
Impact Publication: https://pubs.acs.org/doi/10.1021/acschembio.7b00677 This collaboration is multi-disciplinary. We carry out microbiology, genetics and chemistry and the partners carry out cell biology assays.
Start Year 2016
 
Description Glycopeptide discovery and activity 
Organisation University of Cambridge
Country United Kingdom 
Sector Academic/University 
PI Contribution 1. Isolation and identification of ristocetin from an Amycolatopsis producing strain. 2. Analysis of the ristocetin gene cluster. 3. Analysis of the vancomycin-zinc interaction alongside an interactions D-Ala-D-Ala and D-Ala-D-Lac using NMR and ITC. 4. Design of experiments and input into manuscript preparation.
Collaborator Contribution 1. Project design. 2. Mutagenesis of actinomycetes for glycopeptide discovery and activity analysis. 3. Transcriptional analysis and bioassays with Streptomyces coelicolor. 4. Manuscript writing.
Impact PubmedID: 25022591 PubmedID: 26797186 Multidisciplinary: chemistry, biochemistry, microbiology
Start Year 2013
 
Description HIPS Bottromycin Biosynthesis 
Organisation Helmholtz Association of German Research Centres
Department Helmholtz Institute for Pharmaceutical Research, Saarbrucken
Country Germany 
Sector Academic/University 
PI Contribution Using tandem mass spectrometry, we characterised enzymatic transformations to the bottromycin precursor peptide. In addition, we generated mutant forms of the bottromycin producing organism (Streptomyces scabies) to determine the importance of a number of enzyme residues for catalysis. This was assessed by looking at the metabolites produced by these mutants using LC-MS.
Collaborator Contribution They expressed and purified enzymes involved in bottromycin biosynthesis, and then carried out enzymatic assays. The products of these assays were then sent to us for analysis. They also carried out mutagenesis of these enzymes, which guided our mutations in Streptomyces scabies.
Impact Publication: https://pubs.acs.org/doi/10.1021/jacs.7b09898
Start Year 2017
 
Description Teicoplanin biosynthesis 
Organisation Helmholtz Association of German Research Centres
Department Helmholtz Institute for Pharmaceutical Research, Saarbrucken
Country Germany 
Sector Academic/University 
PI Contribution 1. Analysis of mutants in the teicoplanin biosynthetic pathway. 2. Manuscript writing. 3. Input into project design.
Collaborator Contribution 1. Genetic modification of teicoplanin gene cluster. 2. Transcriptional analysis of teicoplanin gene cluster. 2. Manuscript writing. 3. Project design.
Impact PubmedID: 25104028 PubmedID: 27285718 Multidisciplinary: microbiology, analytical chemistry, molecular biology.
Start Year 2013
 
Description Teicoplanin biosynthesis 
Organisation University of Lviv
Department Department of genetics and biotechnology
Country Ukraine 
Sector Academic/University 
PI Contribution 1. Analysis of mutants in the teicoplanin biosynthetic pathway. 2. Manuscript writing. 3. Input into project design.
Collaborator Contribution 1. Genetic modification of teicoplanin gene cluster. 2. Transcriptional analysis of teicoplanin gene cluster. 2. Manuscript writing. 3. Project design.
Impact PubmedID: 25104028 PubmedID: 27285718 Multidisciplinary: microbiology, analytical chemistry, molecular biology.
Start Year 2013
 
Description University of León Collaboration 
Organisation Fundacion MEDINA
Country Spain 
Sector Charity/Non Profit 
PI Contribution This collaboration was started to identify the natural products that were responsible for antifungal activity in Streptomyces clavuligerus following introduction of a regulatory gene into this strain. We also mapped the global changes to metabolism in this strain too using LC-MS. This led to the identification of a wide variety of tunicamycins, where a few specific versions of this molecule exhibited antifungal activity. We helped write the resulting research paper.
Collaborator Contribution The partners led this research project, where they genetically manipulated Streptomyces clavuligerus and assessed changes to gene expression using microarrays. They also assessed the changes in production of other natural products known to be produced by Streptomyces clavuligerus , including the beta-lactam antibiotic cephamycin and the beta-lactamase inhibitor clavulanic acid. They led the writing of the research paper.
Impact A paper has just been accepted in Frontiers in Microbiology that reports this work: https://www.frontiersin.org/articles/10.3389/fmicb.2019.00580/abstract
Start Year 2016
 
Description University of León Collaboration 
Organisation University of Leon
Country Spain 
Sector Academic/University 
PI Contribution This collaboration was started to identify the natural products that were responsible for antifungal activity in Streptomyces clavuligerus following introduction of a regulatory gene into this strain. We also mapped the global changes to metabolism in this strain too using LC-MS. This led to the identification of a wide variety of tunicamycins, where a few specific versions of this molecule exhibited antifungal activity. We helped write the resulting research paper.
Collaborator Contribution The partners led this research project, where they genetically manipulated Streptomyces clavuligerus and assessed changes to gene expression using microarrays. They also assessed the changes in production of other natural products known to be produced by Streptomyces clavuligerus , including the beta-lactam antibiotic cephamycin and the beta-lactamase inhibitor clavulanic acid. They led the writing of the research paper.
Impact A paper has just been accepted in Frontiers in Microbiology that reports this work: https://www.frontiersin.org/articles/10.3389/fmicb.2019.00580/abstract
Start Year 2016
 
Description University of Tübingen Actinonin Biosynthesis 
Organisation Eberhard Karls University of Tubingen
Country Germany 
Sector Academic/University 
PI Contribution We sequenced the genome of the actinonin producing organism to identify its biosynthetic gene cluster, as well as identifying the gene cluster of the related compound matlystatin in a different organism. We carried out chemical feeding experiments to determine the biosynthesis of actinonin and also carried out feeding experiments to make novel matlystatin derivatives.
Collaborator Contribution They independently identified the same gene clusters as ourselves. Following this, they cloning the matlystatin gene cluster and investigated its biosynthesis by mutating most genes in the pathway. They also carried out different feeding experiments to ourselves to support biosynthetic proposals.
Impact Publication: http://europepmc.org/abstract/med/29213087
Start Year 2017
 
Description SAW (Science, Art and Writing) Antibiotics Project at City of Norwich School. 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Workshop with year 7 schoolchildren at a local school. I presented on microbes, antibiotics and disease, and the students then carried out creative writing and art projects based around the science. This was a full day project carried out alongside an artist, a writer, the schoolteacher and an outreach coordinator. The students prepared some fantastic poems and artwork.
Year(s) Of Engagement Activity 2015
 
Description School visit from Ipswich High School 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact A small group of sixth-form students visited the research institute. I provided a tour of the labs and gave a presentation on antibiotics and Streptomyces bacteria. Following the visit, a student contacted me to obtain information on antibiotics for a school project.
Year(s) Of Engagement Activity 2015
 
Description Antibiotic Hunters Exhibit at the Great British Biosciences Festival (London) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact An exhibit to communicate the research we're doing to discover new antibiotics. This was a four-day festival, with VIPs on the first day, schools on the second (1500 pupils) and the public on the third and fourth (5000 in total). The exhibit was manned from researchers from a variety of groups in our department and a huge amount of interaction was made with the public. In connection with this, we prepared an animation to accompany the exhibit, which describes our research (linked in URL).
Year(s) Of Engagement Activity 2014
URL https://www.youtube.com/watch?v=4tdK9VX5gcY
 
Description Antibiotic Hunters Stand at Science in Norwich Day 
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 Exhibit at a science fair in the centre of Norwich. Comprehensive feedback from the public was obtained by an independent reviewer to inform future outreach activities. Example feedback provided below:
- Very many participants had a positive experience at the stand; none recorded a negative one.
- Many responses noted the enthusiasm, knowledge, friendliness and approachability of all volunteers staffing the stand.
- The relative simplicity of the materials at the stand, and the focus and clarity of the message, helped make the stand accessible.
Year(s) Of Engagement Activity 2014
 
Description Antibiotic Hunters stand at the Big Bang Science Fair (Birmingham) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact Outreach exhibit at a large national science fair attended by thousands of schoolchildren, as well as interested members of the public. Lots of interactions with schools and lots of requests for information about the research. Provided an opportunity to determine the level of understanding of this area of science in schoolchildren.
Year(s) Of Engagement Activity 2015
 
Description Blog post for science website 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Interview with the scientific website Sparrho on the discovery and development of antibiotics
Year(s) Of Engagement Activity 2015
URL http://blog.sparrho.com/post/121265105627/the-medicine-makers
 
Description Invited talk at Helmholtz Institute for Pharmaceutical Research Saarland 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Invited talk entitled "Deciphering the biosynthesis of peptidic natural products" given at the Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Germany
Year(s) Of Engagement Activity 2016
 
Description Invited talk at the University of East Anglia (BIO Open Lecture) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Postgraduate students
Results and Impact Invited talk entitled "Deciphering the biosynthesis of peptide antibiotics" given at the University of East Anglia (BIO Open Lecture)
Year(s) Of Engagement Activity 2016
 
Description Meeting with Science Museum about an exhibition on antibiotics 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact A meeting with Science Museum staff about content for a new exhibit on antimicrobial resistance. Information leaflets were prepared, along with information about the wide array of AMR research taking place across the Norwich Research Park. UEA later became formally involved in this "Superbugs" exhibition.
Year(s) Of Engagement Activity 2016
URL https://www.sciencemuseum.org.uk/see-and-do/superbugs-fight-our-lives
 
Description School visit (INTO Norwich) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Schools
Results and Impact One hour presentation entitled "Looking for drugs made by bugs" presented to international students participating in the Newton Programme at INTO Norwich. Purpose was to communicate my research to A-level students interested in science. Following the presentation, the teacher reported: "The students have been very keen to discuss points you raised in class (we are studying antibiotics this week) and were excited to see the links between the knowledge they have gained through their AS studies and your research."
Year(s) Of Engagement Activity 2014
 
Description Science Museum Superbugs Late Event 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact "Antibiotic Hunters" exhibit at a Science Museum Superbug Late Event. This was an adult-only event held in the evening at the Science Museum in London and attended by thousands of people. The exhibit was organised jointly between members of my research group and members of the Wilkinson group (also at JIC).
Year(s) Of Engagement Activity 2018
 
Description Year 10 Science Camp Talk 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact A talk on natural product discovery to attendees of the year 10 Science Camp at the John Innes Centre. This was followed by questions on antibiotic and anticancer compound discovery.
Year(s) Of Engagement Activity 2018
URL https://www.jic.ac.uk/training-careers/work-experience/year-10-science-camp/