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

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.

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.

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.