Structure and mechanism of a trans-acyltransferase polyketide synthase

Lead Research Organisation: University of Bristol
Department Name: Biochemistry

Abstract

Polyketides are among the most important compounds known to man. They perform many vital roles in nature acting as hormones, toxins, flavours, smells and pigments. These compounds are also the basis of numerous medically important drugs used to treat cancer, lower cholesterol, suppress the immune system and fight infection. Sales of polyketide based medicines total over £30 billion each year and there is enormous worldwide interest in identifying new polyketides and making new and improved versions of existing ones. Polyketides are made within microorganisms by clusters of proteins called polyketide synthases, usually shortened to PKSs. PKSs function like miniature factory assembly lines within cells. Each different protein within the assembly line is responsible for building or modifying a specific part of the carbon skeleton of the polyketide product. There are considerable differences in the structures and activities of different polyketides made by different PKSs from different microorganisms. This is despite the fact that all of these compounds are produced from the same initial chemical building blocks. Understanding how these differences are achieved relies on an in-depth knowledge of how PKSs work, essentially, what do each of the proteins in the assembly line do, how do they do it and how are they arranged relative to each other? This research project will focus on a new family of PKSs which generate highly unusual products used to treat a range of different diseases. By examining the structures of the components of the PKS in fine detail using a technique called X-ray crystallography, we will try to work out how each of different parts of the PKS works and how they fit together and interact with each other. These experiments will not only allow us to decipher how these systems make their important products, but will also provide us with a blue-print that we can use to construct new PKSs which make new medicines.

Technical Summary

Modular polyketide synthases, non-ribosomal peptides synthases or hybrids thereof are giant (upto 5 MDa) multi-functional enzymes that assemble complex natural product scaffolds in a highly programmed linear fashion. Within the synthase complex, individual enzymatic domains are organised into associated extension modules, with each module responsible for the elongation of the product chain by a single functional unit. Extension modules are often elaborated to house multiple catalytic domains which act in concert with the minimal chain extension machinery to further modify the growing product chain. Recent work focused on the characterisation of modular systems from 'exotic' bacterial taxa has resulted in the identification of a new group of multi-modular synthases that do not adhere to the established colinearity rules of the polyketide biosynthetic pathway. These novel systems, termed trans-AT synthases, have evolved independently from other modular systems and comprise disparate enzymatic activities from apparently unrelated metabolic pathways, which have converged to yield a single functional synthase. The aim of this project is to provide a molecular description of this novel enzymatic machine. To achieve this goal we will deconstruct the individual components of the model trans-AT system bacillaene synthase and by marrying X-ray crystallography with in vitro kinetic, thermodynamic and spectroscopic methods, and organic synthesis, provide a detailed description of the trans-AT biosynthetic framework at the domain, module and multi-module level.

Planned Impact

Around two thirds of all drugs in current clinical use are derived from or based upon molecules isolated from natural sources. This accounts for a multi-billion pound global market comprising antibiotics, antifungals, antiparasitics, anticancer agents and the cholesterol lowering statins. In 2009 four of the top twenty best selling pharmacological agents world-wide were molecules based on natural product scaffolds, with the statin derived cholesterol lowering agent Lipitor ranked number one. As the feasibility of high-throughput screening methodologies for target based drug development is increasingly being brought into question, there in now renewed interest in the identification and isolation of new natural product based medicines. This renewed interest has resulted in the re-establishment of many natural product drug discovery pipelines in the pharmaceutical industry and companies dedicated to the development of new therapeutic agents from natural sources are in operation in the UK, France, Spain, Germany, Switzerland, USA, China and Japan. Among new natural product targets the polyketides, non-ribosomal peptides and hybrids thereof remain one of if not the most attractive molecules for clinical development, with many polyketide and non-ribosomal peptide derived compounds in the latter stages of clinical trials. The enormous potential to modify and manipulate the polyketide and non-ribosomal peptide biosynthetic pathway offers a route to both the rational generation of tailor-made product analogues and the high-throughput generation of large compound libraries without the cost, complexity and environmental impact of synthetic chemical approaches. For this goal to be realised however, a detailed understanding of the synthetic process is essential. The work outlined within this application will provide this information, bridging the gap that currently exists between gene sequence and product chemistry. Our studies will not only offer insight into one of nature's most complex biosynthetic assemblies impacting significantly on both the academic and industrial biosciences community, but also provide a structural framework for the manipulation of synthase systems for the production of 'next-generation' natural product derived therapeutic agents. Such output will inevitably impact on the healthcare and well-being of the populous and the economic competitiveness of the UK. This program will offer those involved (PDRAs, students, etc.) experience and training at the chemistry-biology interface, providing them with the skills to succeed in a future career in academia or industry. The research output from this program will be reported to the wider scientific community through publication in leading peer reviewed journals, presentation at national and international conferences and at meetings with industrial and academic collaborators. Appropriate training will be given to the PDRAs in the preparation of papers, posters and oral presentations to ensure that, alongside their scientific knowledge and skills, they are developing a portfolio of widely transferable skills. Further, significant opportunities exist for the presentation of research findings (by the PI and PDRA) to a more general audience through public engagement activities organised by the PIs department (school talks, Bristol sciences festival, etc.) and through the PIs links with the Royal Society eg. the Summer 2010 Science Festival. Such activities will ensure that as broad an audience as is feasible will be informed of our ongoing research.

Publications

10 25 50
 
Description Polyketides are a structurally and functionally diverse family of bioactive natural products that are the basis of, or inspiration for, many of our most important pharmaceuticals and agrochemicals. This project focuses on the multi-component biosynthetic mega-enzymes, termed polyketide synthases (PKSs), which are responsible for the assembly of polyketides in microorganisms. We have been specifically examining the so-called trans-AT PKSs, which exhibit unusual and potentially exploitable enzymology. The purpose of this research is to establish general rules and mechanisms regarding trans-AT PKS biosynthesis that may be exploited to direct the production of non-natural polyketides with improved bioactivities.
During this project structural and functional characterization of the prototypical trans-AT PKS bacillaene synthase has been performed. This research has (i) established the molecular basis of beta-methyl branch formation in this system and explored the substrate tolerances of the enzymes responsible; (ii) provided a detailed molecular description of the trans-acting loading and reductive domains from this system and primed further studies to generate engineered loading domains with variant substrate selectivities; (iii) explored the enzymology of the trans-acting proof-reading enzyme PksD and demonstrated its ability to proof-read non-cognate acyl-carrier proteins in vitro and in vivo; (iv) allowed us to produce a range of multi-modular constructs from the bacillaene system for detailed in vitro characterization. We have been able to expand the scope of the project through collaboration with groups in Bristol and Birmingham, and explored equivalent biosynthetic steps in the mupirocin and kalamanticin trans-AT PKSs. We have demonstrated that although some trans-AT PKS enzymology is context dependent, it is possible to establish general rules that can be used predictively to determine trans-AT PKS function from amino acid sequence. It has also allowed us to identify new pressing research questions that relate to trans-AT PKS architecture and function. For example, can methyl-branch forming enzymes be freely exchanged between different trans-AT PKS? What are the determinants of ACP selectivity by trans-acting acyltransferases? How are three different successive branch insertions (an exomethylene, a secondary methyl, and an olefinic methyl) introduced in trans-AT systems with only a single complement of branching enzymes?
The research has proved instrumental in allowing us to establish a range of new collaborations in trans-AT PKS enzymology, specifically with groups in the UK (Birmingham, Cardiff, Newcastle, London) and internationally (Germany, France, Belgium, Indonesia, and USA). It has also allowed the research team to engage with associated consortia and networks, e.g. NPRONET, SynbiCITE, ERASynBio, etc. and to form a number of collaborative links with industrial partners (BioservUK, Centeo, Douglas Instruments). These links have resulted in research publications, presentations at conferences, and the acquisition of additional grant funding.
Exploitation Route Many of the discoveries made during this project are fundamental in nature and will have significant impact on the academic community through the generation of new knowledge. This new knowledge has been, and will continue to be, disseminated through peer-reviewed publication in leading international journals, conference presentations, and involvement in networking activities and collaborative projects. We will also disseminate these discoveries more widely through outreach activities engaging the public fully, e.g. Science Cafes, school outreach programs, schools week, local Science Centres (e.g. @Bristol), Bright Nights Festival, Festival of Nature, etc. Some of the outcomes of this research may be commercially exploitable, for example our work on trans-AT acyltransferase selectivity and beta-methyl branch formation. We have prior experience of establishing collaborative links with industrial sponsors, and plan to forge similar links to realize the commercial impacts of our research. This has been, and will continue to be, achieved through involvement in a range of industrial facing initiatives and networks e.g. NPRONET, BIOCATNET, SynbiCITE, BrisSynBio, etc, exploiting our existing industrial links to highlight the value and impact of our research outcomes, and working with the University of Bristol's Research Enterprise and Development Unit to identify and approach potential industrial sponsors/collaborators.
Sectors Chemicals,Education,Pharmaceuticals and Medical Biotechnology

URL https://royalsociety.org/grants/case-studies/paul-race/
 
Description Many of the discoveries made during this project have been fundamental in nature and have had most significant impact on the academic community. This has been through the adoption of the principles of PKS enzymology established during the course of the study and exploiting this information to inform the research of others. Such discoveries have also impacted upon the industrial natural products community in a manner equivalent to that described above. Although there has not been a direct commercial impact from this grant, work performed during this study did establish preliminary data that has subsequently formed the basis of an industrial collaboration that has attracted further funding from 2 separate sources (£300k) and is seeking to develop a platform technology for the manufacture of high value biologics (see other sections). The outcomes from this grant have also been included in a number of outreach activities (Schools talks, science festivals) that have provided educational and societal impact through knowledge sharing with a range of publics.
First Year Of Impact 2013
Sector Chemicals,Education,Pharmaceuticals and Medical Biotechnology
Impact Types Societal,Economic

 
Description Centre for structural analysis of complex biological systems
Amount £547,203 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2014 
End 10/2015
 
Description IB Catalyst
Amount £247,085 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 04/2015 
End 03/2016
 
Description Newton International Fellowships - 2017
Amount £99,000 (GBP)
Funding ID NF171419 
Organisation The Royal Society 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2017 
End 10/2019
 
Description Next Generation DNA Synthesis
Amount £2,219,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 02/2015 
End 02/2016
 
Description Structural and Mechanistic Investigations of Antibiotic Production in Bacteria
Amount £724,351 (GBP)
Funding ID BB/R007853/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 08/2018 
End 07/2021
 
Description SynbiCITE poof of concept award
Amount £50,000 (GBP)
Funding ID EP/L011573/1 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 12/2014 
End 05/2015
 
Description Synthetic Biology Innovation and Knowledge Centre
Amount £4,990,071 (GBP)
Funding ID EP/L011573/1 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 10/2013 
End 09/2018
 
Description Synthetic Biology Research Centres
Amount £13,528,180 (GBP)
Funding ID BB/L01386X/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 07/2014 
End 07/2019
 
Description Unlocking the potential of engineered C-C bond forming enzymes for biocatalysis
Amount £770,153 (GBP)
Funding ID BB/T001968/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 09/2019 
End 09/2022
 
Description Centeo LTD 
Organisation Centeo
Country United Kingdom 
Sector Private 
PI Contribution Explored the influence of temperature cycling on protein crystallization using Centeo's proprietary TG40 device.
Collaborator Contribution Provided instrumentation, reagents and technical advice.
Impact None to date. Project is a collaboration between biochemists, engineers and physicists.
Start Year 2013
 
Description Douglas Instruments 
Organisation Douglas Instruments
Country United Kingdom 
Sector Private 
PI Contribution PRR and his research group further developed the random matrix microseeding technique for protein crystallization.
Collaborator Contribution Provided technical advice, reagents and instrumentation.
Impact Collaboration has produced one publication. The project was not multidisciplinary.
Start Year 2012
 
Description Peptide Nano-factory project 
Organisation BioServ UK
Country United Kingdom 
Sector Private 
PI Contribution A multilateral collaboration with BioservUK Ltd and Protein Ark Ltd to develop a novel platform technology for the production of high value peptides that are recalcitrant to conventional solid phase synthesis. The technology was devised and developed by PRR in collaboration with Dr Steven Burston, University of Bristol. PRR's team generated preliminary data demonstrating the tractability of the technology.
Collaborator Contribution BioservUK Ltd are performing validation and scale-up studies to further refine the technology. Protein Ark Ltd are liaising with customers to identity specific peptide targets for scale-up production by BioservUK.
Impact Further funding has been awarded from the UK Synthetic Biology IKC (SynbiCITE) and Innovate UK/BBSRC via the IB catalyst, to refine and commercialize the technology (see further funding). This collaboration is multidisciplinary involving biochemists, chemists, microbiologists, fermentation scientists, and process engineers.
Start Year 2013
 
Description Peptide Nano-factory project 
Organisation Protein Ark
Country United Kingdom 
Sector Private 
PI Contribution A multilateral collaboration with BioservUK Ltd and Protein Ark Ltd to develop a novel platform technology for the production of high value peptides that are recalcitrant to conventional solid phase synthesis. The technology was devised and developed by PRR in collaboration with Dr Steven Burston, University of Bristol. PRR's team generated preliminary data demonstrating the tractability of the technology.
Collaborator Contribution BioservUK Ltd are performing validation and scale-up studies to further refine the technology. Protein Ark Ltd are liaising with customers to identity specific peptide targets for scale-up production by BioservUK.
Impact Further funding has been awarded from the UK Synthetic Biology IKC (SynbiCITE) and Innovate UK/BBSRC via the IB catalyst, to refine and commercialize the technology (see further funding). This collaboration is multidisciplinary involving biochemists, chemists, microbiologists, fermentation scientists, and process engineers.
Start Year 2013
 
Description Antibiotic Discovery in the Abyss - presentation at new scientist live 2018 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact PRR gave a 45 min public lecture at New Scientist Live 2018. Efforts in PRR's lab to discover and develop new natural product based antibiotics were described.
Year(s) Of Engagement Activity 2018
URL https://www.newscientist.com/article-topic/new-scientist-live-talks-2018/page/2/
 
Description BBC documentary - The Truth About Antibiotics 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact PRR was interviewed and profiled as part of the BBC documentary "The Truth About Antibiotics". The programme focused on innovate research being undertaken to tackle the emergence of AMR. PRR's work on natural product antibiotic discovery was featured.
Year(s) Of Engagement Activity 2019
URL https://www.bbc.co.uk/programmes/b0c1nl68
 
Description ERASynBio workshop of the European Synthetic Biology Centres 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact The key results of this activity were to inspire and feed further ERASynBio developments and recommendations to the European Commission.

None yet
Year(s) Of Engagement Activity 2014
URL https://www.erasynbio.eu/
 
Description Public engagment activity (Bristol) [Antibiotic Discovery in the Abyss] 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Public engagement activity focused on summarising research efforts at the University of Bristol to discover and exploit natural products as pharmaceutical leads. The specific focus of the activity was antibiotic discovery. >50 members of the public attended and there was considerable discussion and questions. The event lasted > 2 hours.
Year(s) Of Engagement Activity 2017
 
Description RCUK workshop on DNA synthesis 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact Following the first phase of DNA Synthesis investments earlier in 2014, BBSRC, on behalf of the Research Councils, hosted a one day workshop to discuss mechanisms for implementing the second phase of capital investment (approximately £8M) for precompetitive DNA synthesis research/capabilities. The purpose of the capital money is to create a 'national capability' in DNA synthesis which will support the long term growth of UK synthetic biology.
Attendees were required to complete a questionnaire in advance of the workshop identifying the (up to) five biggest research challenges for the next generation of DNA synthesis and the barriers to a coordinated state-of -the-art national capability. At the workshop attendees further considered what capability is already available and therefore what the existing requirements and bottlenecks for a UK capability are.
The key outcome of the activity was the generation of a small number of short strategic cases for capital investment. These are currently being reviewed by BBSRC.
Representatives from the private sector were also present at the workshop and it is anticipated that they will work closely with the academic community in the development of technologies that tackle challenges that underpin the next generation of DNA synthesis, but also in bringing the technologies/ capabilities developed closer to market/ into wider use.

Long term there will be tangible impacts generated from the instruments purchased via this award if successful. The impact is likely to be mainly in the academic and industrial spheres, and in the areas of fine and speciality chemicals, pharmaceuticals, and life science technologies.
Year(s) Of Engagement Activity 2014
 
Description School visit and talk 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Talk and Q and A on careers in scientific research
Year(s) Of Engagement Activity 2015
 
Description School visits and talks 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact I discussed the research undertaken in my laboratory and career opportunities in science to secondary school students at Worle Community School. The talk stimulated discussions about research careers in both academia and industry.

Following this annual event I often host students from the school for 1-2 week laboratory internships in my group in Bristol.
Year(s) Of Engagement Activity 2011,2012,2013,2014
 
Description Synthetic Biology Research Centres Ethical Legal and Social Aspects meeting 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact This was a BBSRC hosted one-day ELSA workshop for the SBRC grant holders to ensure they incorporate appropriate and meaningful ELSA consideration into their Centres. Additionally the workshop networked the SBRCs and the broader synthetic biology community including the EPSRC-BBSRC synthetic biology CDT.
The main results of the activity were to share the ELSA plans across the SBRCs, exploration of strengths and challenges. Joint publications between SynBio researchers and social scientists will be a measurable metric of success in this for the SBRCs.

None yet
Year(s) Of Engagement Activity 2014