Synthetic biology approaches to compartmentalisation in bacteria and the construction of novel bioreactors

Synthetic biology has emerged from the development of techniques that allow for significant alterations in the metabolism/physiology of the cell to produce desirable products, including drugs, chemicals, vitamins, biofuels etc. Ultimately synthetic biology could lead to the construction of new metabolic pathways and even new life forms. A major advance in the area of synthetic biology would be the ability to make compartments in the cell to house specific metabolic processes. The development of such bespoke organelles would allow for greater control and regulation of the process, detaching the encased process from any negative influences within cellular metabolism. We have demonstrated that it is possible to synthesise such organelles in E. coli by the metabolic engineering of the 1,2-propanediol utilizing (pdu) operon, a complex that contains around 20,000 polypeptide subunits and has a molecular mass of approximately 300 mDa. More recently, we have shown that it is possible to make empty bacterial microcompartments (BMCs) by the coordinated overproduction of 5 gene products that compose the shell (outer casing) of the organelle. We have also demonstrated that proteins normally encased within the BMC are targeted to this empty vesicle and that other non-vesicle proteins can be incorporated to the BMC by fusing them onto target proteins. By labelling the proteins with GFP it has been possible to undertake live cell imaging of the organelles and evidence for movement between organelles via filaments has been observed. We now wish to extend this study by directing specific enzymes to the BMC in order to make bespoke bioreactors. Such an approach will provide insights into the metabolic advantage of compartmentalisation. Engineering of the shell proteins will be undertaken to help in the rational design of a semi permeable shell with a broader substrate specificity. The intramolecular orientation of the shell proteins within the organelle will be investigated by a combination of antibodies and proteolytic processing. Detail on the intermolecular arrangement of shell proteins within the BMC will be obtained from studies using a range of different GFP-shell protein fusions and the analysis of the BMC by FRET. In this way we will be able to generate a model of the organelle and this, in combination with a number of other experimental approaches, will permit an investigation into how proteins are targeted and incorporated into the BMC. The rate of protein exchange in the organelle and the order of incorporation into the organelle will be defined not only using live cell imaging by also by employing FRAP. Detail on how the organelle is held within the cell will be investigated using a range of directed cell cytoskeleton mutants. The role of a Ras-type GTPase, PduV, in organelle dynamics will also be probed. The research outlined in this application combines a mixture of basic science with exciting applied opportunities. The project falls squarely in the remit of the BBSRC by advancing the fundamental understanding of complex biological processes and directly addresses the synthetic biology priority area.

We have now shown that it is possible to generate empty bacterial microcompartments (BMCs) through the coordinated expression of the genes encoding the outer shell proteins of these proteinaceous macromolecular assemblies. Using a synthetic biology approach we plan to build on this ability in order to generate bioreactors with specialised functions. We plan therefore to determine whether the BMC can be used as an area for the post-translational modification of proteins via proteolytic processing. The catabolism of methylglyoxal will also be targeted to the organelle to determine if this two-step process can be contained effectively within the organelle and determine if compartmentalisation can have a metabolic advantage. A range of different experimental approaches will also be employed in order to determine some basic biochemical principles and properties of these empty BMCs. By tagging the shell proteins with a range of GFP analogues we will be able to follow the synthesis of the individual components of the BCM and follow their incorporation into the organelle. Following on from this, we will be able to use Fluorescence Resonance Energy Transfer (FRET) to determine the relative spatial organization of the shell proteins within the complex. This will permit the construction of an accurate model for this bacterial organelle and make a major contribution towards our understanding of how this assembly can act as a semi-permeable metabolite barrier. We will also tackle the question of how these large bodies are held within the cell. Initial studies with PduV, a Ras-like GTPase, have shown that the protein becomes associated with filaments within the cell. Moreover, in the presence of PduV, movement is observed between the BMCs along these filaments. We have outlined a number of experiments to investigate the possibility that the BMCs are associated with the bacterial cytoskeleton.

The beneficiaries of this research will be researchers in academia who are interested in cell biology and its applications. The research will lead to a more detailed structure of one of the largest proteinaceous bodies observed within the cell - blurring one of the distinguishing features between eukaryotes and prokaryotes, compartmentalization. These bacterial microcompartments have been studied in only a few instances in the past, but genome analysis suggests that around a quarter of all bacteria have the ability to make these organelles. There appear to be a wide range of activities associated with these novel bacterial microcompartments, so there is still a great deal to learn about them. Thus from a general biological standpoint, the research will be of interest to teachers and lecturers (A-level and above) and those involved in educating students on the various organelles found within cells. The research outlined in this application will make a significant contribution towards our understanding of the basic physical and structural properties of these bacterial organelles. Moreover, it will explore the potential that these structures have for application in a range of biotechnological processes. The research will also help define an important area of synthetic biology, permitting the exploitation of compartmentalization and addressing the basic advantages of compartmentalization in biology. In this respect, the research will of general interest to those in applied areas of the biotech industry, especially those interested in metabolic engineering. The research outlined in this application deals with the synthesis and biology of bacterial microcompartments. These are commonly found in a range of pathogenic bacteria and early reports suggest that these structures are important during infection. Those interested in enteric bacteria will thus benefit from an improved understanding of the roles that these organelles play in the disease process. The bacterial microcompartments also offer the opportunity for significant translational application. For instance, the microcompartments can be used to introduce novel pathways into cells, especially pathways that contain toxic intermediates as the bodies will protect the cell from potential harm. Similarly, the organelles can be used to protect processes from the damaging effects of oxygen within the cell and thus can be used to cage sensitive enzyme complexes such as the hydrogenase or nitrogenase. As a large proteinaceous body with a significant surface area, the organelle could be used to expose proteins on its exterior, thereby allowing the complex to be used as an adjuvant in antibody production. There are thus many important applications for which these microcompartments can be used and thus there is likely to be some considerable interest from industry. The realization of the impact of this research in terms of translational applications will take at least 5 years. The skills acquired by those involved in this project include not only a wide range of important biological techniques ranging from imaging and structural biology through to microbiology and recombinant DNA technology but also the chance to contribute towards a basic understanding of bacterial physiology. The knowledge and techniques will provide those employed with skills that can be used across education and industry. The intellectual property resulting from this project will be protected and used via the Research and Enterprise Office. The research will be published in high impact journals and oral communications given at international conferences. Using the infrastructure of the new Centre for Molecular Processing within the University of Kent, the research will be spun out through a new start up company.