Development of supramolecular assemblies for enhancing cellular productivity and the synthesis of fine chemicals and biotherapeutics.

Lead Research Organisation: University of Kent
Department Name: Sch of Biosciences

Abstract

The ability to rewire and reorganise the internal metabolic machinery of the cell through the engineering of scaffolds and compartments represents a major aspiration of synthetic biology. Here we address issues to this problem through the design and rational engineering of de novo and natural scaffolds and organelles. Indeed, it is well known that nature uses compartments to efficiently concentrate and/or segregate specific proteins in specific organelles. In this way, active components such as enzymes have better access to their substrates but also toxic substances are prevented from diffusing throughout the cell. In one of the key strategic areas of BBSRC, Synthetic Biology, a major aim is to design from new and / or improve on such existing natural systems and to exploit these for the production of commercially important chemicals and biotherapeutics.

Whereas most compartments inside cells are limited by a lipid membrane, there is increasing interest and potential in compartments limited by a non-lipid / proteinaceous shell. In this proposal we will exploit the use of Bacterial MicroCompartments (BMCs) and Self-Assembling peptide caGEs (SAGEs) as exponents of such non-lipid bounded compartments. One of the possible advantages of proteinaceous compartments over lipid-bounded organelles is transport in and out of the compartment. Our overall aim is to (let the cells) build metabolic micro-factories that will be able to produce useful and or valuable molecules without intoxicating the cells. In this way we may develop new ways to produce fine and platform chemicals as well as biotherapeutics.

The Universities of Kent, Bristol and Queen Mary have been at the forefront of studying both BMCs and SAGEs and have unravelled a large number of the underlying principles. We are therefore in an excellent position to take these studies to the next level; introducing new metabolic processes into compartments, expressing such systems inside cells, and ultimately use them for "large scale" production.

In order to achieve this ambitious goal we have assembled a highly interdisciplinary team of researchers covering such diverse areas as Cell Biology, Chemistry, Bioinformatics, and Engineering. Only via such an integrated approach will it be possible to design the desired functioning bacterial factories. Also, through the exchange of concepts, ideas, and technologies between the 3 sites we will be able to achieve substantially more than each group independently. It is important to highlight the significant interest in this research from the chemical/pharmaceutical sector, who will be able to guide us to valuable targets but may also be a route to further development and translation of specific outcomes of this project.

Through the research described in this application we are confident that we will be able to contribute to the development of new sustainable approaches to the generation of chemicals and biotherapeutics and for their rapid incorporation into manufacturing with leading companies.

Technical Summary

There are many advantages to localising metabolic pathways to or within a scaffold or compartment: the effective concentration of enzymes, substrates and intermediates is greatly increased leading to kinetic enhancement; toxic intermediates are sequestered away from essential cell machinery; and side reactions are avoided and on pathway chemistry promoted leading to thermodynamic enhancement. Many of these advantages extend to protein folding and posttranslational modification potentially transforming the use of bacterial cells for protein production. This project is unique in bringing together two complementary approaches to the synthetic engineering of supramolecular assemblies for catalysis; the first is the redesign and reengineering of the bacterial microcompartment (BMC) and second the exploitation of a synthetic self-assembling peptide cage (or SAGE). There is still much to learn about both systems to inspire their redesign and application in synthetic biology. In BMCs the nature of the targeting peptide shell protein interaction needs to be defined, the assembly process of the shell and organisation of the encapsulated enzymes understood, and methods of control of fluxes across the protein shell need to be elucidated. Importantly, the extent to which new pathways can be introduced needs to be explored. In SAGEs their in vivo construction for synthetic biology applications is essential as is characterising their ability to organize enzymes, retain intermediates and catalyse useful transformations. Importantly, there are considerable benefits to both microcompartment and SAGE research by combining the expertise of the applicants. In addition, there are considerable opportunities for cross-over between the two systems, for example the recruitment of coiled-coil interactions to extend the repertoire of enzyme tags and shell protein interactions in microcompartments and the recruitment of shell proteins to SAGE scaffolds.

Planned Impact

The research described in this application will have a major impact on several areas of science, by employing synthetic biology to devise new strategic approaches to industrial biotechnology. It will permit the generation of bacterial strains with highly organized metabolic pathways to allow for improved efficiency in the production of fine and platform chemicals as well as biotherapeutics. The project involves metabolic engineering, exploiting synthetic scaffolds and catalysts, to generate ergonomically-designed bioreactors. In so doing this project will naturally provide tools and resources to the broader community within the biosciences field. The research falls well within the remit of synthetic biology and is therefore addressing a key priority area. In this respect the project applies the engineering paradigm of systems design to metabolism. The research also has the potential to engineer improvements in existing biological products and especially improve our understanding of biological systems through researching the role of modularity.

The beneficiaries of this research will be researchers in academia and industry who are interested in synthetic biology and its applications. The research will not only furnish essential information about how pathways and enzymes can be investigated and modified, but it will also provide greater insight into the provision and procurement of pathway substrates. The research will be published in high impact journals and oral communications given at international conferences. It is important to note the complementarity of research skills and the critical mass achieved between the collaborating groups in this project. The academic research outputs will be influential in the UK and in the USA where the major competitors on microcompartments and nanocages are based. We currently have a world lead on the development of microcompartments for efficient production of chemicals and biotherapeutics and want to maintain and extend this lead to give industrial advantage to the UK. The intellectual property resulting from this project will be protected and used via the Innovation and Enterprise Office. Using the infrastructure of the new Centre for Molecular Processing within the University of Kent, the research will be brought to the attention of many leading industrial companies.

The research addresses an industrial demand for more efficient and greener production of platform chemicals as well as proteins with disulfides and post-translational modifications. Many companies have expressed an interest in this project including HealthKTN, Fujifilm, Isogenica and Pfizer. This programme of work will ensure the UK continues to lead the exploration and exploitation of microcompartment and nanocage research with benefits for the academics involved, for the Universities involved, for the UK reputation in biotechnology and biotherapeutics, but importantly feeding into UK Industry. This research will provide an important edge for UK biotechnology companies, existing and new, via providing greater productivity and new molecules, peptides and proteins for various purposes including fine chemical and therapeutic use.
The Kent, Bristol and Queen Mary groups are heavily involved in outreach programmes, through interactions with local schools and community groups. All are members of the Authentic Biology Project, which is funded by a Wellcome Trust society award to bring real research into schools. Regular talks and demonstrations are given through organized events during science week and at other times by invitation via the biology4all website, ensuring there is good dissemination with the general public on a range of important issues.

Publications

10 25 50
 
Description We have discovered that it is possible to develop molecular scaffolds that can be used to enhance metabolic organisation within the cell. By developing such frameworks have shown that it is possible to localise, compartmentalise and concentrate specific biochemical pathways and molecular processes within the cellular cytoplasm. The confinement of such activities to particular regions within the cell has not only result in higher subcellular concentrations of enzymes but, through the provision of compartments, also acts to protect the cell from toxic metabolites and proteins. We have been developing the use of artificial Self-Assembling caAGs (SAGEs) and Bacterial MicroCompartments (BMCs) for these purposes. We have achieved the following:
1. We have characterised a range of other bacterial organelles associated with metabolic functions such as choline, fucose and ethanol utilisation.
2. We have developed further SAGE parts from the design and assembly of new building blocks, and looked at their ability to self assemble both in vivo and in vitro.
3. We have analysed these macromolecular assemblies in vivo by employing both live cell and cryo EM techniques.
4. We have enhanced protein targeting to these molecular scaffolds through the development of targeting strategies to ensure a high level of cargo delivery.
5. We have provided some molecular detail of the macromolecular assemblies from protein crystallography and AFM studies, as well as from high resolution structures of the individual components.
6. We have engineered more complex functions into these artificial systems to allow the synthesis of relevant high value products, including chemicals and protein-based reagents.
7. We are developing novel protein fibres that can be used as metabolic scaffolds across the full length of the cell, and which can also be used to change cell morphology.
Exploitation Route We are working with a number of companies to develop the use of intracellular scaffolds as a way to enhance metabolic flux through biochemical pathways.
Sectors Chemicals,Education,Manufacturing, including Industrial Biotechology

 
Description Our understanding of compartmentalisation has allowed us to re-engineer bacterial micro compartments to allow for the redesign of organelles with new biochemical properties. We have thus, for instance, generated microcompartments for the synthesis of polyphosphate and have submitted a patent application based on how this technology can be sue to help reduce phosphate levels in patients with elevated phosphate. Similarly, we have shown how the use of adding BMC-targeting peptides to enzymes allows for their aggregation into large shell-less aggregates, which remain metabolically active. We again have patented this technology and are trying to use this technology for improved commodity chemical production.
First Year Of Impact 2015
Sector Chemicals,Education,Healthcare,Manufacturing, including Industrial Biotechology
Impact Types Societal,Economic

 
Description Development of supramolecular assemblies for enhancing cellular productivity and the synthesis of fine chemicals and biotherapeutics.
Amount £3,484,653 (GBP)
Funding ID BB/M002969/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2014 
End 10/2019
 
Description Research collaboration 
Organisation University of Bristol
Department School of Biochemistry Bristol
Country United Kingdom 
Sector Academic/University 
PI Contribution Our research into Bacterial MicroCompartments (BMCs) led us to interact with Prof Woolfson's research group at the University of Bristol, where they were working of synthetic peptide cages. By comparing our research approaches and techniques we realised that our combined effort could help in developing a new area of in cell protein design. We developed these ideas further and used these ideas to apply for further funding.
Collaborator Contribution Prof Woolfson's group brought peptide chemistry technology to the project and helped develop powerful ideas concerning the development of cellular compartments that could be targeted through peptide-peptide interactions. Moreover, the Bristol group also had excellent imaging facilities that could be sued to visualise the structures that were being generated within bacterial cells. The ideas generated by this interaction resulted in a successful application for a sLoLa grant to BBSRC.
Impact The outcomes from this partnership can be seen from the sLoLa outputs, which have resulted in a number of high profile papers to journals such as Nature Chemical Biology and Nature Communications. It has also attracted interest from a number of Biotechnology companies.
Start Year 2013
 
Title Genetically Modified Microorganisms 
Description The discovery relates to the use of N-terminal peptide sequences that allow enzymes to aggregate together to form an active aggresome. The invention relates to genetically modified microorganisms and their use in the production of desired products of metabolic pathways, and particularly improving the levels of production of said products. In particular the microorganisms of the invention are modified to comprise enzymes of a metabolic pathway for a product of interest, wherein the enzymes are each tagged with a bacterial microcompartment (BMC)-targeting signal peptide, and wherein the microorganism lacks bacterial microcompartments. The present invention also relates to cell-free systems comprising said (BMC)-targeting signal peptide tagged enzymes in the absence of BMCs. 
IP Reference WO2017077320 
Protection Patent application published
Year Protection Granted 2015
Licensed No
Impact The process is currently being developed for specific processes with ZuvaSyntha
 
Description BBSRC PR: Designer barrel proteins 
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 Other academic audiences (collaborators, peers etc.)
Results and Impact Science publication, Computational design of water-soluble a-helical barrels [Science 24 October 2014: Vol. 346 no. 6208 pp. 485-488 DOI: 10.1126/science.1257452] was publicised on the BBSRC news page.

Unknown.
Year(s) Of Engagement Activity 2014
URL http://www.bbsrc.ac.uk/news/industrial-biotechnology/2014/141024-pr-bristol-team-creates-barrel-prot...
 
Description Parliamentary Science Committee presentation 2014 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Policymakers/politicians
Results and Impact Approximately 200 people attended the Parliamentary and Scientific Committee meeting on the 17th June 2014. The audience included Parliamentarians, members of scientific bodies, science-based industry and academics.
http://www.scienceinparliament.org.uk/sample-page/programme/
This meeting has subsequently been written up and included in the Autumn 2014 Science in Parliament (Vol 71 No 4: pgs 20 - 26) publication.

Unknown
Year(s) Of Engagement Activity 2014
URL http://www.scienceinparliament.org.uk/wp-content/uploads/2013/09/Autumn-Contents-page.pdf
 
Description Pint of Science Festival: Dark side of protein science 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact As part of the Pint of Science Festival, researchers from BrisSynBio participated in the 'Dark Matters' event. The event was held in Friska café, Bristol, and involved scientific crafts and discussions between researchers and the public.
Director of BrisSynBio, Professor Dek Woolfson, along with Gail Bartlett, Jack Heal, Drew Thomson and Chris Wood organised the event 'Dark Matters'. Analogous to the idea of dark matter, protein science focuses on the protein structures that could theoretically exist but are not present in natural biology.
Year(s) Of Engagement Activity 2015
URL http://www.bristol.ac.uk/publicengagementstories/stories/2015/dark-side-protein-science.html
 
Description Pint of Science, Bristol, UK, May 2015, "From galaxies of stars to a new universe of proteins" 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Part of the Pint of Science 2015 Programme in Bristol. About 60 people attended.
Year(s) Of Engagement Activity 2015
URL https://pintofscience.co.uk/event/dark-matters/
 
Description RSC: Synthetic Biology: The Free Edinburgh Festival Fringe Show (Heal) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Supported by an award from the Royal Society of Chemistry,the Edinburgh Fringe Festival hosted its first science stand-up on the subject of synthetic biology. Jack Heal's 'Do Scientists Dream of Synthetic Sheep?' show took a comedic approach to genome engineering, de-extinction and more - with the crowd helping to shape its direction with questions and discussion. The show considered questions from artificial life to Jurassic Park, and ran for 21 days.
Purpose: To interest the public in science.
Outcome: The comic felt freshly enthused about doing [synthetic biology] research.
Reflection: Free shows encourage people to take risks in their choices of which shows to see. This spirit is perfect for science outreach events which have to try hard to avoid becoming 'by scientists, for scientists'.

None yet.
Year(s) Of Engagement Activity 2014,2015
URL http://www.bristol.ac.uk/publicengagementstories/stories/2016/jack-heal.html?platform=hootsuite
 
Description Synthetic proteins for a synthetic biology: faster, fitter, stronger 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Better Humans Science Café, Bristol, UK, October 5 2016, "Synthetic proteins for a synthetic biology: faster, fitter, stronger"
Year(s) Of Engagement Activity 2016
 
Description The rise and rise of synthetic biology in the UK: science, policy and public perception 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Policymakers/politicians
Results and Impact Invited to Houses of Parliament, London, UK, June 2014, to speak to the Parliamentary and Scientific Committee.
Year(s) Of Engagement Activity 2014
URL http://www.scienceinparliament.org.uk/wp-content/uploads/2014/05/17-June-AGM-agenda.pdf