Synthetic bacterial vesicles to enhance recombinant protein production, delivery and isolation for Industrial Biotechnology applications.

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

Abstract

The ability to reprogram a cell to direct the packaging of specific molecules into discrete membrane envelopes is a major objective for synthetic biology. This controlled packaging into membrane vesicles will allow biologists to create a plethora of new technologies, which could be applied in both biotechnology and medical industries. These include the generation of novel metabolic factories within a cell for energy production; for rapidly packaging toxic proteins into contained environments before they have a chance to harm any normal metabolic activities, so they can be purified for use in subsequent pharmaceutical applications; the creation of protective packages filled with difficult to isolate biomolecules, which can be kept in stable environment to allow their storage and purification; and also generate simple vehicles for delivery of drugs and vaccines to the patient.

Here, we provide a simple and cost effective solution to the problem. We have discovered a method to program a simple cell to create membrane packages which can be filled with different molecules of interest. We have not only discovered a way to fine-tune the shape of the membrane package (e.g. into long tubular matrices or spherical vesicles), but we have also devised controllable mechanisms that either keep the package within, or secrete the package out of the cell. Thus we have therefore made a landmark breakthrough in synthetic biology research. A major aim of this BBSRC key strategic area is to design from new and improve on natural systems and exploit these for the production of commercially important chemicals and biotherapeutics, which is what we have achieved here.

Our overall aim in this project is to make use of these exciting discoveries to modify cells, making them capable of creating membrane bound packages filled with any protein of interest, which can then either be secreted from the cell and isolated from the culture media using a simple one step filtration technique, or stored within the cell where it can be made to act as a metabolic micro-factories, producing useful and/or valuable molecules without intoxicating the cells. In this way we hope to develop new ways to produce fine and platform chemicals as well as biotherapeutics.

Through the research described in this application we are certain that we will be able to contribute to the development of new sustainable approaches for generating biotherapeutics, which will be assimilated into production techniques by diverse bio-industries.

Technical Summary

The isolation of recombinant biopharmaceuticals from large scale fermentation cultures is a major challenge to industry. The ability to target the secretion of recombinant proteins into culture media is attractive as it improves efficiency and reduces costs. Currently there are no established methods for transporting recombinant proteins across bacterial membranes, or for generating vesicles to facilitate the subsequent isolation and purification of a biopharmaceutical protein from the culture media. We have developed a novel system that utilises the expression of a synaptic vesicle turnover regulating protein, that when expressed in the established recombinant protein production gram-negative bacterium, E. coli, drives the formation of extracellular vesicles, or exosomes. We have also established preliminary methodologies for targeting desired proteins of interest to the interior of the recombinant vesicle. These combined synthetic systems provide an exciting opportunity for delivering high-value recombinant proteins from the bacterial cell, into the culture media, to allow simple protein purification from continuous and perfusion cultures.

Here we will apply molecular cell biology and biochemical methods to develop a detailed understanding of the molecular basis of this inducible membrane reorganisation system by undertaking a biophysical analysis of protein-membrane interactions, subsequent membrane reorganisation, vesicle formation, and methodologies targeting proteins to vesicular lumen. Each iteration of the optomised system will be further validated using industrial culture conditions on site at the industrial partner, FujiFilm-Diosynth. Thus we will identify optimal methods and conditions for the controllable production of recombinant vesicles from E. coli, and establish a series of methodologies for vesicle packaging and increasing yield of recombinant proteins from E. coli in both laboratory and large scale industrial contexts.

Planned Impact

The research described in this application will have a major impact on several areas of science, including synthetic, cell and molecular biology. The research relates to the construction and design of specific membrane-bound vesicles in bacteria and how the process of vesicle formation can be controlled and regulated. The ability to generate membrane bound vesicles within bacteria represents a key development with significant biotechnological applications such as vaccine development, drug delivery, compartmentalisation, recombinant membrane protein production and energy generation.
The understanding gained (along with the potential to apply synthetic biology approaches to re-engineer bacteria cell factories to generate membrane bound compartments) has immediate implication and long-term potential application. The research falls under the remit of synthetic biology as it involves the design and construction of novel biologically based parts, and systems, as well as redesigning existing natural biological systems for useful purposes. The research involves the generation of new functions in cells and organisms and approaches to produce synthetic compartments. The research highlights the potential to engineer improvements in existing biological products and especially improve our understanding of biological systems through researching the role of modularity. The immediate beneficiaries include the academic and industrial community that is actively addressing approaches for more robust recombinant protein production.
The UK is recognized as a leading player (both academically and industrially) across the international sector for research into, and manufacture of, recombinant proteins and biotherapeutics. This research, as a collaboration with Fujifilm-Diosynth Biotechnologies (FDB) will help the UK to maintain its status as an innovator for biotechnology. A major outcome of the project will be to highlight the strong industrial-academic collaborative ethos in the UK and the manner in which BBSRC industrial-academic funding initiatives ensure long-term retention of strong UK-based industrial research focus for multi-national companies. At a training level, the PDRA funded under this LINK project will be embedded into the laboratory environment of FDB in year 2 of the research. They will gain experience of the industrial perspective of direct commercial relevance of the discovery, development and manufacturing focus and will be exposed to the broader FDB environment, participating in project team meetings and attending workshops on technology and enterprise development, and commercialization.
Project partners have discussed and accepted a strategy for knowledge exchange and data dissemination. Partners are members of other existing successful collaborations (with a mixture of industrial and academic partners) and have used a range of knowledge exchange pathways to disseminate data and results of potentially commercial-sensitive information in modes that satisfy the needs of all participants (and research funders). A contract between Kent and FDB will develop agreed ownership of intellectual property (IP) and responsibility for its application, ensuring that the support of BBSRC can be acknowledged and that knowledge generated in the project can be disseminated in an appropriate manner. With the contract in place, the project team (and, wherever possible, specifically the PDRA) will disseminate outputs via peer-reviewed publications and at research conferences. This will include UK-based sector networks e.g. CBMNET (http://cbmnetnibb.group.shef.ac.uk) a Network in Industrial Biotechnology and Bioenergy (NIBB) and major interntional microbiology conferences.

Publications

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Description he ability to reprogram a cell to direct the packaging of molecules into discrete membrane envelopes is a major objective for synthetic biology. This controlled packaging into membrane vesicles will allow biologists to create new technologies for biotechnology and medical industries. These include the generation of novel metabolic factories for energy production; the creation of protective packages filled with difficult to isolate biomolecules; providing a stable environment for isolation and storage of proteins; for delivering nanovesicle cancer therapies, as well as providing vehicles for delivery of drugs and vaccines to the patient. We describe a simple and cost-effective solution to the problem. By adding a small tag onto the protein to be produced a simple bacterial cell will generate membrane packages filled with the tagged molecules of interest. These packages are exported from the cell into the growth broth. Remarkably, protein function is preserved within these packages for months in the fridge. Our approach therefore facilitates simple, efficient and rapid purification of diverse proteins for use in biotechnology and medicinal applications.
Exploitation Route Currently under further development and further funding application.
Patent filed and paper submitted
Sectors Agriculture, Food and Drink,Environment,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description Recombinant vesicle characterisation 
Organisation Anton Paar
Department Anton Paar Ltd
Country United Kingdom 
Sector Private 
PI Contribution Validation of their DLS and fluorescence monitoring technology with analysis the recombinant vesicles.
Collaborator Contribution developing the new system by hyphenating the two technologies allowing the rapid and simultaneous screening of vesicle size and to determine whether they are filled with the target protein via a fluorescent bio-marker.
Impact none
Start Year 2018
 
Description Vesicle packaged recombinant protein production 
Organisation Fujifilm
Department Fujifilm Diosynth Biotechnologies, UK
Country United Kingdom 
Sector Private 
PI Contribution Development of vesicle technology that can be applied to a wide range of biotherapeutics (i.e. development of peptide sequences, culture conditions, media, promoter, etc)
Collaborator Contribution Cash contribution £10,000; Consumables: £30,000; Equipment time: £15,000; Staff costs for research, training and project management: £430000 Test and validate recombinant vesicle packaging technology within large scale fermenter growth reactors.
Impact Patent application.
Start Year 2018
 
Title Sequences and Methods for production of recombinant biological molecules 
Description The invention encompasses a polypeptide sequence and method that provides a simple mechanism for targeted high yield release of specific membrane packaged biological molecules such as proteins into cell media which can be continuously isolated from active cultures. This results in a significant increase in yields of functional soluble molecules, such as proteins, from cultures and facilitates efficient downstream processing for a wide range of biotechnology applications. 
IP Reference  
Protection Patent application published
Year Protection Granted
Licensed Yes
Impact IP is co-held with Fujifilm-Diosynth Biotechnology UK Ltd, with agreed heads of terms. No impact yet as patent filed in December 2021, and associated research paper describing the technology currently under submission