Biochemical characterisation of the Glycyl Radical Enzyme Containing Microcompartment (GRM2) from Proteus mirabilis

Lead Research Organisation: University College London
Department Name: Biochemical Engineering

Abstract

The production of bio-therapeutic proteins, small molecule drugs and chemical intermediates of industrial interest in recombinant hosts is becoming an increasingly popular alternative to production in mammalian cell culture, natural hosts (e.g. plants, fungi) or chemical synthesis, which can be slow and expensive. Achieving high product yields in recombinant synthetic pathways requires that the fluxes of each enzymatic reaction of the pathway of interest be balanced to limit the accumulation of intermediates, particularly those toxic to the host. Conventional strategies include the modulation of expression levels of individual enzymes or directed evolution of rate-limiting enzymes and have focused on balancing pathway flux, but don't consider other crucial factors such as diffusion, transport, localisation of intermediates and the cost-effective purification and recovery of products. Nature has developed strategies to deal with many of these issues by confining certain biochemical pathways to various organelles. In prokaryotes, bacterial microcompartments (BMCs) encapsulate enzymes associated with certain metabolic processes within a large protein shell, bringing sequentially acting enzymes into spatial proximity thus increasing local enzyme concentration, protecting the cell from potentially reactive intermediates and facilitating co-factor recycling. In the past decade significant progress has been made to engineer BMCs to permit the construction of BMC bioreactors with novel functions within the cell.

The overall aim of this project is to develop BMCs as synthetic biology platforms for spatial organization of enzymes for enhanced production of valuable compounds in E. coli. The project will expand our understanding of the fundamentals of engineering synthetic compartmentalisation, the benefits and limitations of pathway localisation and inform the rational design of future microcompartment-derived technologies.

The project is aligned with the EPSRC centre for Doctoral Training in Bioprocess Engineering Leadership. It provides new synthetic biology approaches to the redesign of cellular chemistry and will impact in the development of cost-effective and sustainable bio-based manufacture.

BMC tools and technologies will be developed with industrial applications in mind. The work is supported by in-kind contribution from the industrial biotechnology company Ingenza, a world leader in the application of synthetic biology for the provision of efficient bioprocesses for the manufacture of chemicals, biologics, pharmaceuticals and biofuels. Compartmentalisation of enzymes/pathways may provide a valuable strategy for the improvement of industrial strains of interest. The industrial partner will offer enzymes/pathways of interest and advice/consultancy on how industry would like to see the BMC technology applied.

Publications

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Studentship Projects

Project Reference Relationship Related To Start End Student Name
EP/R512400/1 30/09/2017 31/12/2022
1930945 Studentship EP/R512400/1 24/09/2017 24/02/2022 Gawain Moody
 
Description Bacteria contain a variety of complex organelles, each with a unique function which optimizes bacterial survival. My project looks at a proteinaceous organelle called the bacterial microcompartment. This organelle has a variety of functions, from carbon fixation to aldehyde oxidation. Broadly speaking, they encapsulate key enzymes of a particular pathway with a selectively permeable proteinaceous shell. Recently, a new class of microcompartments, encapsulating a glycyl radical enzyme, has been discovered - my project focusses on a subclass of this microcompartment, which has been found associated to a choline utilization pathway, the GRM2. So far within my project, key insights into the fundamental biology of the GRM2 microcompartment found within Proteus mirabilis have been gained. First and foremost, most of the individual shell proteins of this microcompartment do not seem to be able to self assemble into higher order structures, and instead seem to form large aggregates at the pole of the cell. However, the last shell protein in the GRM2 operon is capable of forming long, filamentous like structures. Upon closer inspection through tomography, these are revealed to be several sheets, arranged in parallel to each other. Similar types of higher order, self assembling structures like nanotubes, rosettes and honeycombs have been observed in other kinds of microcompartments. (Young et al., 2017)
When all the GRM2 shell proteins are arranged in the same vector, in their natural order, with the native ribosome binding sites, only few of these sheets are observed. This is an unusual result, as other types of microcompartment, such as the Propanediol-utilizing (pdu) microcompartment, can form self assembling closed, empty microcompartments when expressing all the shell proteins. As some literature has suggested, the presence of an enzymatic core around which the shell proteins form a closed microcompartment may be necessary. We know that if the core enzyme of pathway associated to the GRM2 microcompartment, a choline TMA lyase (cutC), is knocked out from the operon encoding the microcompartment, the structure of the microcompartment is disrupted, and aberrant "rosette" structures form. If this enzyme is reintroduced back on a plasmid, the structure is recovered, and a closed microcompartment is formed (Jameson et al 2015). Therefore, it was of interest to this project to further investigate this enzyme's role in microcompartment structure, stability and assembly. An investigation is underway to test the role of cutC in the formation of higher order structures, possibly even closed microcompartments, when expressed with all the GRM2 shell proteins.

Another important facet of microcompartments are natural sequences called targeting peptides, found within the enzymes encapsulated by the microcompartment. These act as molecular anchors which allow the enzymes to be attached to the inside of the closed shell. They have been used in the past to target non native enzymes to the inside of an empty microcompartment, to enhance the output of a biosynthetic pathway converting pyruvate to ethanol (Lawrence et al 2014). Two targeting peptides have been found within the GRM2 microcompartment encoding locus, within the enzymes Aldehyde dehydrogenase (CutF) and Phosphotransacetylase (CutH). There were amplified by gene synthesis, and cloned into a vector so that they fused with a fluorescent protein in a position that imitated their natural position within the protein. Control plasmids without targeting peptides were also synthesized. These were then expressed under different conditions, and samples were collected and processed into soluble and insoluble fractions for viewing on an SDS gel. Peculiarly, these targeting peptide fused fluorescent proteins remained in the soluble fraction - in other microcompartments which harness targeting peptides, the targeted protein is rendered extremely insoluble, and can be viewed at the puncta of the cell when using fluorescence microscopy. When these targeting peptide fused proteins are coexpressed with the GRM2 shell proteins, similar results are observed - the fluorescent protein seems to form an aggregate at the poles or periphery of the cell, even with the sheet structures from the last shell protein present. Perhaps an additional component from the GRM2 is necessary for the targeting peptides to interact with the shell proteins (Kalnins et al. 2020). This should be investigated in future work.
Modularity between microcompartment components of different types was also tested. Here, GRM2 targeting peptides were targeted to an empty Pdu compartment - it seems that there could be an interaction between the targeting peptides and the empty Pdu compartment. The deposition of the fluorescent protein in these samples is no longer just uniformly aggregated at the poles of the cell - it looks slightly uneven and speckled, which is not observed in other coexpressions.

From these results, there could be potential to scaffold insoluble proteins such as enzymes associated to a biosynthetic pathway, and observing if these proteins are solubilized. Furthermore, it would be of interest to see if enzymes scaffolded with these targeting peptides exhibit an increased output of the desired product.
Exploitation Route Through this project, we aim to gain a deeper biological understanding of the GRM2 microcompartment in a number of areas, like shell assembly, enzyme association and microcompartment biotechnology. This has implications for areas like biological compartmentalization, protein self assembly, protein scaffold assembly, targeting of heterologous proteins, proteinaceous microbial organelles, and synthetic biology for the purposes of pathway enhancement.
With some more data, we plan to publish what we've found, and the results from that could inform future work and research initiatives in areas like those mentioned above.
Furthermore, since my project is based in a biochemical engineering department, there is potential for scale-up studies, once a better understanding of this microcompartment is gained, potentially leading to industrial interest and collaborations.
Sectors Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology