A novel, fast and efficient resource recycling system for improving the performance of engineered bacteria
Lead Research Organisation:
Imperial College London
Department Name: Bioengineering
Abstract
The proposed project aims to design, mathematically model and experimentally implement in E. coli a novel synthetic resource recycling system that:
(a) Automatically releases ribosomes wastefully sequestered by mRNA-ribosomes "non-stop" complexes, which often result from the overexpression of synthetic biology proteins
(b) Accelerates the degradation of mistranslated proteins resulting from non-stop complexes and thereby the recycling of the amino acids sequestered by these mistranslated proteins
(c) Improves host cell fitness, thereby improving both growth and protein production rates, by automatically increasing in response to burden the degradation rate of proteins overexpressed from synthetic DNA.
Our synthetic resource recycling system relies on the modular re-engineering of the ribosome rescue mechanism naturally used by bacterial cells to detect and alleviate the wasteful sequestration of two of their most important cellular resources, i.e. ribosomes and amino acids. To do this we will re-engineer the primary ribosome rescue mechanism, i.e. the tmRNA mediated trans-translational system, to create a modular system that automatically detects mistranslated proteins and adds a variety of tags to them (e.g. mf-Lon, his, HIV-tat tags) or fuses mistranslated proteins with other proteins (e.g. GFP, BFP, mCherry). Through this system, mistranslated proteins can be easily quantified or quickly degraded to efficiently recycle their constituent amino acids.
Our novel synthetic ribosome rescue system will allow us to easily quantify the occurrence and impact of non-stop complex formation and amino acid consumption imposed by synthetic biology constructs on their host cells. This will provide the research community with a deeper understanding of the core feedback interactions between synthetic biology circuits and their host cells and of the main mechanisms responsible for these interactions. In particular, this system will allow to test whether the main cause for the stalling of ribosomes on mRNAs and the formation of non-stop complexes is the lack of charged tRNAs (due a severe depletion of free amino acids) and whether this can be alleviated by increasing the recycling rate of amino acids sequestered in overexpressed or misfolded proteins, thereby reducing the formation of "non-stop" mRNA-ribosome complexes.
We will demonstrate the gain in fitness and production capacity of E. coli cells equipped with our synthetic resource recycling system and the associated increase in production yields of antibody fragments, which account for nearly 40% of the global biopharmaceutical market.
Another exciting direction of this research is that an understanding of how to control ribosome rescue and the re-use of amino acids will also allow us to purposefully design systems that have a controllable fitness disadvantage. This could be used as a novel biosafety mechanism for synthetic biology, as cells could be designed to predictably perform below the level of their natural counterparts, thereby offering a direct means of controlling bacterial colonisation capability. The construction of a controllable resource recycling system could therefore also be beneficial to those seeking to have greater control over genetically modified technologies.
In summary, through this project, we will make a crucial step forward in the creation of engineered cells that are "more fit for purpose" by equipping them with a controllable resource recycling system which will (a) increase host cell fitness while maintaining synthetic biology functionality and protein production yield, (b) improve biosafety through the control of the ribosome rescue mechanism, which is essential for host cell viability, (c) improve reliability and portability of synthetic biology constructs across different strains of the same or even different bacterial hosts, and (d) provide a deeper understanding of resource allocation and how it impacts host fitness and productivity.
(a) Automatically releases ribosomes wastefully sequestered by mRNA-ribosomes "non-stop" complexes, which often result from the overexpression of synthetic biology proteins
(b) Accelerates the degradation of mistranslated proteins resulting from non-stop complexes and thereby the recycling of the amino acids sequestered by these mistranslated proteins
(c) Improves host cell fitness, thereby improving both growth and protein production rates, by automatically increasing in response to burden the degradation rate of proteins overexpressed from synthetic DNA.
Our synthetic resource recycling system relies on the modular re-engineering of the ribosome rescue mechanism naturally used by bacterial cells to detect and alleviate the wasteful sequestration of two of their most important cellular resources, i.e. ribosomes and amino acids. To do this we will re-engineer the primary ribosome rescue mechanism, i.e. the tmRNA mediated trans-translational system, to create a modular system that automatically detects mistranslated proteins and adds a variety of tags to them (e.g. mf-Lon, his, HIV-tat tags) or fuses mistranslated proteins with other proteins (e.g. GFP, BFP, mCherry). Through this system, mistranslated proteins can be easily quantified or quickly degraded to efficiently recycle their constituent amino acids.
Our novel synthetic ribosome rescue system will allow us to easily quantify the occurrence and impact of non-stop complex formation and amino acid consumption imposed by synthetic biology constructs on their host cells. This will provide the research community with a deeper understanding of the core feedback interactions between synthetic biology circuits and their host cells and of the main mechanisms responsible for these interactions. In particular, this system will allow to test whether the main cause for the stalling of ribosomes on mRNAs and the formation of non-stop complexes is the lack of charged tRNAs (due a severe depletion of free amino acids) and whether this can be alleviated by increasing the recycling rate of amino acids sequestered in overexpressed or misfolded proteins, thereby reducing the formation of "non-stop" mRNA-ribosome complexes.
We will demonstrate the gain in fitness and production capacity of E. coli cells equipped with our synthetic resource recycling system and the associated increase in production yields of antibody fragments, which account for nearly 40% of the global biopharmaceutical market.
Another exciting direction of this research is that an understanding of how to control ribosome rescue and the re-use of amino acids will also allow us to purposefully design systems that have a controllable fitness disadvantage. This could be used as a novel biosafety mechanism for synthetic biology, as cells could be designed to predictably perform below the level of their natural counterparts, thereby offering a direct means of controlling bacterial colonisation capability. The construction of a controllable resource recycling system could therefore also be beneficial to those seeking to have greater control over genetically modified technologies.
In summary, through this project, we will make a crucial step forward in the creation of engineered cells that are "more fit for purpose" by equipping them with a controllable resource recycling system which will (a) increase host cell fitness while maintaining synthetic biology functionality and protein production yield, (b) improve biosafety through the control of the ribosome rescue mechanism, which is essential for host cell viability, (c) improve reliability and portability of synthetic biology constructs across different strains of the same or even different bacterial hosts, and (d) provide a deeper understanding of resource allocation and how it impacts host fitness and productivity.
Planned Impact
This project, which is aligned with the EPSRC strategic priority in Synthetic Biology and the UK Synthetic Biology Strategic Plan (Biodesign for the Bioeconomy), will directly contribute to the growing excellence of synthetic biology in the UK and facilitate its industrialisation. Biotechnology companies use single cells (bacteria, yeast, or mammalian) as 'cell factories' to produce molecules of use in many different sectors, such as pharmaceuticals, enzymes, biofuels, cosmetics or fragrances. To date, innovation in biotechnological processes has focused on maximising output, but now the challenge is to use cell factories more efficiently by reducing the required input of energy and nutrients and maximising efficient (re-)use of internal cellular resources.
Impact on Society. Synthetic biology's considerable anticipated impact in biotechnology, biomedicine, energy, food technology, and society in general has been acknowledged worldwide by the OECD and UK, EU and US policy makers. The availability of controllable cellular mechanisms that automatically detect and alleviate the main causes of wasteful usage of precious cellular resources constitutes a crucial, open problem in the field. Solutions to this problem are key to increase the predictability, reproducibility, performance, and portability of synthetic biology constructs. This project will offer such solutions by re-engineering mechanisms that control the efficient (re-)use of the costliest gene expression resources, i.e. ribosomes, and the recycling of essential protein building blocks, i.e. amino acids. An important deliverable of this work will be engineered cells with improved fitness and tolerance for functional synthetic biology constructs. This will open up avenues for new research and world-leading developments in the UK lasting well beyond the project duration.
Impact on Economy and Industry. The biotechnology and nascent synthetic biology industries constitute the main target for economic and industrial impact. With large amounts of money spent on the development of high-performance cells capable of robustly and efficiently producing biofuels, commodity chemicals, pharmaceuticals, etc., the economic impact of resource-efficient whole-cell biosynthesis can be very large (see also letters of support from identified industrial collaborators). The results of this project will have an important impact on (a) the creation of host cells with improved performance in terms of their gene expression capacity through automatic recycling of their translational resources, (b) the implementation of new biosafety mechanisms based on the control of our engineered resource recycling system, and (c) the increased portability of synthetic biology constructs across different host strains. As a first example of improved performance, we will use engineered E. coli strains to demonstrate increase in the production yields of antibody fragments. Antibodies represent 40% of the global bio-production industry with numerous applications in research and medicine, including novel immune-system-based cancer treatments.
Impact on Knowledge and Training. Synthetic biology is an increasingly popular subject with students, specifically because of its multidisciplinary nature and its anticipated significant impact on the future. I play an important role in the training and education of the next generation through synthetic biology teaching at Imperial and intend to incorporate the findings, models and genetic designs of our novel recycling system into examples for courses such as the "MRes in Systems and Synthetic Biology" and "Modelling in Biology" that I teach at Imperial. The UK has had remarkable success in teaching parts-based synthetic biology, producing many world-class undergraduate projects, so investment in further research in the UK is critical to retaining the best students in the country and building a successful UK-based bioeconomy.
Impact on Society. Synthetic biology's considerable anticipated impact in biotechnology, biomedicine, energy, food technology, and society in general has been acknowledged worldwide by the OECD and UK, EU and US policy makers. The availability of controllable cellular mechanisms that automatically detect and alleviate the main causes of wasteful usage of precious cellular resources constitutes a crucial, open problem in the field. Solutions to this problem are key to increase the predictability, reproducibility, performance, and portability of synthetic biology constructs. This project will offer such solutions by re-engineering mechanisms that control the efficient (re-)use of the costliest gene expression resources, i.e. ribosomes, and the recycling of essential protein building blocks, i.e. amino acids. An important deliverable of this work will be engineered cells with improved fitness and tolerance for functional synthetic biology constructs. This will open up avenues for new research and world-leading developments in the UK lasting well beyond the project duration.
Impact on Economy and Industry. The biotechnology and nascent synthetic biology industries constitute the main target for economic and industrial impact. With large amounts of money spent on the development of high-performance cells capable of robustly and efficiently producing biofuels, commodity chemicals, pharmaceuticals, etc., the economic impact of resource-efficient whole-cell biosynthesis can be very large (see also letters of support from identified industrial collaborators). The results of this project will have an important impact on (a) the creation of host cells with improved performance in terms of their gene expression capacity through automatic recycling of their translational resources, (b) the implementation of new biosafety mechanisms based on the control of our engineered resource recycling system, and (c) the increased portability of synthetic biology constructs across different host strains. As a first example of improved performance, we will use engineered E. coli strains to demonstrate increase in the production yields of antibody fragments. Antibodies represent 40% of the global bio-production industry with numerous applications in research and medicine, including novel immune-system-based cancer treatments.
Impact on Knowledge and Training. Synthetic biology is an increasingly popular subject with students, specifically because of its multidisciplinary nature and its anticipated significant impact on the future. I play an important role in the training and education of the next generation through synthetic biology teaching at Imperial and intend to incorporate the findings, models and genetic designs of our novel recycling system into examples for courses such as the "MRes in Systems and Synthetic Biology" and "Modelling in Biology" that I teach at Imperial. The UK has had remarkable success in teaching parts-based synthetic biology, producing many world-class undergraduate projects, so investment in further research in the UK is critical to retaining the best students in the country and building a successful UK-based bioeconomy.
People |
ORCID iD |
| Guy-Bart Stan (Principal Investigator) |
Publications
Borkowski O
(2018)
Cell-free prediction of protein expression costs for growing cells
in Nature Communications
Ceroni F
(2018)
Burden-driven feedback control of gene expression
in Nature Methods
Kylilis N
(2019)
Whole-Cell Biosensor with Tunable Limit of Detection Enables Low-Cost Agglutination Assays for Medical Diagnostic Applications.
in ACS sensors
Kylilis N
(2018)
Tools for engineering coordinated system behaviour in synthetic microbial consortia.
in Nature communications
Pan W
(2018)
Identification of Nonlinear State-Space Systems From Heterogeneous Datasets
in IEEE Transactions on Control of Network Systems
Tomazou M
(2018)
Computational Re-design of Synthetic Genetic Oscillators for Independent Amplitude and Frequency Modulation.
in Cell systems
| Description | Key findings include: Key Finding #1: The introduction of synthetic genetic circuits in bacterial cell strains causes an "unnatural burden" to the host cell as there is intense competition between the synthetic components and the bacterium native processes for intracellular resources. Such valuable intracellular resources could include energy, metabolites, enzymes and co-factors amongst others. The project has identified three key intracellular resources that are limited upon the introduction of synthetic genetic circuits: ribosomes, RNA polymerases and folding chaperone proteins. To quantitatively monitor the utilisation of these key resources in engineered bacteria, this project has designed and constructed genetically encoded monitor devices. These monitor devices are composed of the sensor component that senses the intracellular available levels of each resource and produces a signal in the form of a fluorescence reporter gene to allow non-invasive monitoring of resource utilisation and thus quantitative measurement of the molecular competition. The initial tests of these monitor devices by use of chemical reagents that artificially manipulate the availability of the three aforementioned intracellular resources demonstrated good correlation between resource depletion and signal from the monitor devices. Key Finding #2: Bacterial cells are commonly used for the bioproduction of chemicals and valuable proteins such as for use in medicine or biotechnology. To maximise yields of product accumulation, scientists need to balance cell resources devoted to the production of the desired product against the resources needed by the cell host to reproduce. As both of these factors are important but increase in one of them causes reduction to the other, there is a point of optimality for high yield product accumulation. Current methods for determining this point of optimality include laborious and capital-intensive design-build- test-learn cycles of genetic constructs via the use of libraries of genetic parts that control the rate of expression of the heterologous genes. To offer a more direct route to the point of optimality for recombinant protein expression, we have designed a biomolecular controller that allows automatic control of cellular burden imposed by the introduction of synthetic genetic circuits. The design achieves this objective by limiting the number of sequestered ribosomes associated with heterologous gene expression, which we demonstrated by mathematical modelling and robustness analysis. In addition, we have experimentally validated the sensor and actuator components of the designed controller and integrated these components into a genetic construct encoding the designed biomolecular feedback controller. Further experiments are being carried out to demonstrate its functionality in vivo as it will be used to enhance the heterologous production of therapeutic antibodies. |
| Exploitation Route | The outcomes and key finding of this study could have important economic impact for the industrial biotechnology sector. The tools, designs and methods developed here for the quantitative assessment of intracellular resources utilisation and the implementation of novel biomolecular mechanisms that optimise resource allocations can be very large, as these open avenues for the development of high performance engineered cells capable of producing pharmaceuticals, commodity chemicals, biofuels, etc., in a more cost-effective manner. To demonstrate how the finding can be used by others to improve robustness and performance of engineered cells, we will produce synthetic strains that heterologously produce antibody fragments, which account for nearly 40% of the global pharmaceutical market. |
| Sectors | Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology |
| Description | EPSRC Centre for Doctoral Training in BioDesign Engineering |
| Amount | £7,001,622 (GBP) |
| Funding ID | EP/S022856/1 |
| Organisation | Engineering and Physical Sciences Research Council (EPSRC) |
| Sector | Public |
| Country | United Kingdom |
| Start | 03/2019 |
| End | 09/2027 |
| Description | Genetically Encoded Nucleic Acid Control Architectures |
| Amount | £642,353 (GBP) |
| Funding ID | EP/P02596X/1 |
| Organisation | Engineering and Physical Sciences Research Council (EPSRC) |
| Sector | Public |
| Country | United Kingdom |
| Start | 08/2017 |
| End | 02/2021 |