Elucidating and engineering bottromycin biosynthesis

Lead Research Organisation: John Innes Centre
Department Name: Molecular Microbiology

Abstract

One of the greatest threats to public health in the 21st century is the rise of multi-drug resistant bacterial infections, which has been caused by a shortage in new types of antibiotics, as well as the improper use of antibiotics in medicine and agriculture. This has prompted the World Health Organisation to warn that "the need for action to avert a developing global crisis in health care is increasingly urgent" and the UK's Chief Medical Officer, Prof. Dame Sally Davies, to declare that "we are also not developing new drugs fast enough". One incredibly rich resource for antibiotics are microorganisms that live in soil, and the majority of clinically used antibiotics come from these bacteria. These bacteria have evolved the ability to produce natural products with excellent antibacterial activities, as the ability to kill surrounding bacteria is a big advantage when competing for nutrients.

One compound I am currently researching is an antibiotic called bottromycin. The molecule has some highly unusual structural features and is a very active antibiotic towards dangerous infections like the "superbug" MRSA (methicillin-resistant Staphylococcus aureus), which kills tens of thousands of people worldwide each year. Bottromycin has actually been known for many years but problems with its stability and availability have prevented it from being used clinically. However, its entirely novel structure and mode of action make it highly promising antibiotic of the future. An understanding of bottromycin biosynthesis would provide the information necessary for the pathway to be modified to alter the structure of this antibiotic and increase the amount that can be made.

Natural products are produced by the action of a series of enzymes (proteins), which are encoded by genes (DNA) in the bacterial genome. I have previously identified the genes required for bottromycin production and will now focus on the details of each biochemical step. I will also develop methods to make new bottromycin-like compounds. From a purely scientific perspective, the biochemical steps in this pathway are highly unusual, and an understanding of the enzyme mechanisms will increase our understanding of enzyme function. The experimental techniques involved in this work include the fermentation of bacterial cultures, the purification of enzymes and the analysis of biochemical reactions using mass spectrometry, which determines the mass of a compound and can provide important structural information.

Technical Summary

Ribosomally synthesised and post-translationally modified peptides (RiPPs) are a class of natural products that have not yet been exploited clinically but do possess significant clinical promise. For example, bottromycin is a structurally unique peptide that possesses potent antibacterial activity towards life-threatening infections, such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE). It functions by blocking aminoacyl-tRNA binding to the A-site of the 50S ribosome, thus inhibiting bacterial protein synthesis. I have previously identified the biosynthetic gene cluster for bottromycin in Streptomyces scabies, a pathogen that is the causative agent of potato scab and demonstrated that bottromycin derives from a ribosomal precursor peptide. Uniquely, bottromycin originates from the N-terminus of a precursor peptide and it is proposed that a "follower" peptide has an analogous role to leader peptides in other RiPP pathways.

Bottromycin possesses some highly unusual structural features such as an unprecedented macrocyclic amidine and rare Beta-methylated amino acids. This research programme will biochemically characterise the enzyme(s) responsible for macrocycle formation and utilise this protein as an in vitro tool for the assembly of bottromycin like cyclic peptides. A mixture of techniques will be employed, including in vitro enzymology and in vivo co-expression with the precursor peptide. The pathway will also be engineered to generate novel bottromycins. The transcriptional regulation of the pathway will be investigated to inform the rational reprogramming of the pathway to enhance bottromycin yield.

Planned Impact

The research programme described in this proposal will increase the understanding of how complex drug-like compounds are constructed from ribosomal precursors. Methods will be developed to rapidly modify an important biosynthetic pathway. The urgent requirement to combat multi-drug resistant bacteria, and the vital role natural products have in a variety of other therapeutic areas, means that the research will benefit a number of parties across academia, industry and government, as well as the wider public.

The multidisciplinary nature of this research means that post-doctoral researchers working on the project itself will develop a wide array of skills in the fields of microbiology, analytical chemistry, enzymology and molecular biology that are highly relevant for working in the biotechnology or pharmaceutical sectors. This will help support a strong knowledge-based UK economy, and assist in maintaining the UK's position as a world-leader in the biological sciences. In addition to the positive intellectual benefits this has, success in these fields generates significant wealth for the United Kingdom through the establishment of biotechnology companies and the creation of high-value pharmaceutical products.

It is feasible that a library of promising antimicrobial compounds will be generated, which could then be studied for their clinical and commercial potential. Routes include establishing a new spin-out company or a partnership/licensing agreement with a biotechnology company or a large pharmaceutical company. The Department of Molecular Microbiology at the John Innes Centre has strong recent track record in the establishment of companies based on commercially promising academic research, such as Novacta Biosystems and Procarta Biosystems.

The discovery of enzymes with novel functions will be of interest to members of the chemical industry that use enzymes for the production of fine chemicals and pharmaceutical ingredients. The generation of complex chemicals using biosynthesis or biocatalysis represents a much more environmentally benign approach to chemical manufacturing. Significant investment is being made in biocatalysis by large UK-based pharmaceutical companies such as GlaxoSmithKline at Stevenage and Dr. Reddy's Technology Centre in Cambridge. Biocatalysis assists in reducing the carbon footprint of chemical manufacture, so provides a widespread environmental benefit.

In the longer term, the identification of novel drug-like compounds and improved methods for ribosomal peptide pathway engineering will have a number of wider beneficiaries. This research will provide medical researchers and the pharmaceutical industry with new chemical scaffolds for therapeutic trials. Ultimately, this research could lead to improvements in the treatment of multi-drug resistant bacterial infections, and thus benefit the health of the general public. The development of novel antibiotics benefits the NHS, where a new antibiotic would increase the efficacy of infection treatment strategies and thus save money.

Finally, the research programme aligns with BBSRC strategic research priority 2: Bioenergy and Industrial Biotechnology, and will therefore be valuable to policy makers. More specifically, elucidating and engineering the pathway of a clinically-promising natural product fits with the BBSRC high priority of the "molecular and cellular basis of key biosynthetic processes and their regulation".

Publications

10 25 50
 
Description This grant concerns the biosynthesis of the antibiotic bottromycin, which has activity against clinically-relevant multidrug resistant bacteria. Using cutting edge mass spectrometry-based methods combined with genetic modification of the bottromycin pathway, we have identified key steps in this biosynthetic pathway. In particular, we can confidently predict the timing and role of each enzymatic step in this pathway. This methodology should be widely applicable to the analysis of other biosynthetic pathways, and the findings may help us modify the bottromycin pathway to make more effective forms of this antibiotic. This has been reported in a recent publication (https://www.ncbi.nlm.nih.gov/pubmed/27374993). We have also developed synthetic biology methodology to modify the bottromycin biosynthetic gene cluster using a yeast-based method, which was published in 2018 (http://pubs.acs.org/doi/10.1021/acssynbio.8b00038).

We also established a collaboration with the Koehnke group (Helmholtz Centre for Infection Research, Saarbrucken, Germany) to combine our expertise to confirm the function of individual enzymes in the pathway. Research has been published in collaboration with the Koehnke group on two of the most unusual steps in the pathway (http://pubs.acs.org/doi/10.1021/jacs.7b09898). We have used our expertise in bottromycin biosynthesis to identify pathways to other novel ribosomal peptide natural products, which has led to the discovery of new compounds with anti-cancer activity (http://pubs.acs.org/doi/10.1021/acschembio.7b00677). This also led to the discovery of further novel natural products that are modified by the same class of protein as bottromycin (manuscript accepted by Nucleic Acids Research), as well as a genome mining tool that can aid in the identification of natural products (RiPPER, https://github.com/streptomyces/ripper).

2020 update:
We have recently had a manuscript accepted for publication in Frontiers in Microbiology that characterises the regulation of bottromycin biosynthesis, where we show that an unusual internal transcription start site and a cluster-situated modulator are involved in regulating bottromycin production. Much of this work was carried out during the grant funding period.
Exploitation Route The identification of key steps in this pathway allows others to focus on these steps for their own research. For example, to inform the structural characterisation of enzymes involved in novel steps in this pathway, as well as discovering pathways to new natural products with novel structures and/or bioactivities. Our use and development of mass spectrometry networking methods for the analysis of a biosynthetic pathway will be applicable to other researchers in this area. Our genomics-led discovery of a novel ribosomally synthesised and post-translationally modified peptides (RiPPs) identified thioalbamide as a compound with potent inhibitory activity towards cancer cells. This is currently being investigated by collaborating cancer biologists for its utility in treating cancers. The development of the genome mining tool RiPPER will enable other researchers to search for novel RiPP pathways, while our studies of bottromycin regulation and yeast-based modification of bottromycin biosynthesis provides methodology for other researchers to rationally modify other biosynthetic pathways.

Much of this work has been discussed in public via a variety of routes (schools, media, open days, industry talks) where it provides a clear example of how bacteria make molecules that have the potential to function as antibiotics or anti-cancer agents. The PDRA employed on this project intends on using a lot of what he has learned in mass spectrometry, chemistry and biosynthetic analysis to start his own research programme.
Sectors Chemicals,Education,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
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 Royal Society Enhancement Award
Amount £99,859 (GBP)
Funding ID RGF/EA/180154 
Organisation The Royal Society 
Sector Charity/Non Profit
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 Charity/Non Profit
Country United Kingdom
Start 10/2018 
End 09/2021
 
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 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 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 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 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 Interview with science writer (Kat Arney) about antibiotic discovery and combating antimicrobial resistance. 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Media (as a channel to the public)
Results and Impact Interview with science writer (Kat Arney) about antibiotic discovery and combating antimicrobial resistance. In particular a focus on the re-purposing of old antibiotics for treating multi-drug resistant infections. This was in relation to an in-depth article she is preparing on AMR and antibiotic discovery, which was later published in the Daily Mail.
Year(s) Of Engagement Activity 2018
URL https://www.dailymail.co.uk/health/article-5623417/Could-antibiotics-mens-beards-soil-weapons-battle...
 
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 Invited talk at the University of Leeds 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact Invited talk for the seminar series in the School of Molecular and Cellular Biology, University of Leeds. Participation in a discussion about my research with a group of MSc students.
Year(s) Of Engagement Activity 2018
 
Description JIC 50 Open Day 
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 Public/other audiences
Results and Impact Outreach stand for Department of Molecular Microbiology as part of an Open Day in relation to the 50th anniversary of the John Innes Centre in Norwich.
Year(s) Of Engagement Activity 2017
URL https://www.jic.ac.uk/news-and-events/blog-copy/2017/09/open-day/
 
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 Presentation at Science for Innovation Showcase Event 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Provided presentation entitled "Discovery and biosynthesis of bacterial peptides with antibacterial and anticancer activities" to an audience of industry and research council representatives invited to the John Innes Centre. Participated in a follow-up panel discussion.
Year(s) Of Engagement Activity 2018
 
Description Presentation at Xiamen University (China) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Invited presentation given to faculty, post-docs and students at Xiamen University in China.
Year(s) Of Engagement Activity 2019
 
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 Talk given at the 1st International RiPPS Conference 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Talk given on natural product discovery at the 1st International RiPPS Conference, held in Granada, Spain
Year(s) Of Engagement Activity 2019
URL https://www.rippsconference.org/
 
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/