Development and Application of Next Generation Synthetic Biology Tools

A variety of micro-organisms including E. coli and S. cerevisiae (baker's yeast), are used in commercial bio-production processes to manufacture a number of products, ranging from high-value low-volume products, such bio-therapeutics (e.g. recombinant insulin), to mid-value products, (e.g. biocatalysts and specialised chemicals), to low-value bulk commodity products (e.g. succinic acid and biofuels). In addition, the same biotechnologies used in these production processes are commonly used in basic life science research. As such, the development of new gene control systems would be of great benefit in a number of applied and fundamental areas of biological and biomedical R&D. In particular, the research here seeks to develop novel protein production and metabolic engineering tools, and to demonstrate the applications of these novel synthetic biology tools in the context of the bioprocessing industry.

Within the pharmaceutical sector, biopharmaceuticals constitute 7 out of the 10 bestselling products. The global protein-based biopharmaceutical market had sales worth $70b in 2010, and this figure is set to increase to $110b by 2015. The reasons for the increase in projected global sales are thus: i) many of the targets of these therapeutic proteins are deemed 'undruggable' by traditional small molecule approaches, ii) therapeutic proteins often have superior safety and efficacy profiles, iii) the development times on average are shorter for therapeutic proteins than traditional therapies, iv) there is often limited or absent competition. However, although biopharmaceuticals offer many health benefits along with substantial commercial opportunities, their production remains a significant technical challenge.

Through this current study, I shall develop and demonstrate four important flavours of a novel gene co-expression technology, to allow multimeric protein products to be produced more effectively, along with the potential to provide a simpler and more efficient manufacturing process. Additionally, these co-expression technologies will be used to optimise a number of multivariate co-expression challenges, helping to guide metabolic engineering efforts. The outputs from this fellowship will lead to improved bioprocess efficiencies, with the potential to reduce both drug development times and manufacturing costs, and therefore the financial burden upon national healthcare providers. This research project will also generate technology and knowledge that will help maintain the UK's competitive edge, and will produce highly trained and skilled research personnel.

This project will involve the development and application of a series of novel gene expression control biotechnologies in E. coli, to provide feedback control and multiple gene co-expression control for the controlled enhancement of the expression of multiple and/or difficult proteins. This work will include examples of practical application of these tools and systems for use in academia, the bioprocessing sector and other areas of the knowledge based bio-economy. Results from this study will also enable fundamental questions regarding bacterial physiology, stress response mechanisms and pathway flux engineering to be addressed, resulting in the establishment of a strong independent research career.

Firstly, a modular co-expression platform will be developed, which utilise the unique translational control of previously-developed orthogonal riboswitches, to regulate the expression of multiple viral RNA polymerases. This platform will be demonstrated by controlling the co-expression stoichiometry of the heavy and light chains of a Fab' antibody fragment. Secondly, a stress response sensing feedback system will be developed, utilizing a trans-acting antisense ncRNA input signal to permit recombinant gene expression to be matched to cellular synthetic capacity, enabling auto-regulatory optimisation of high-density fermentation cultures. Thirdly, multivariate pathway flux engineering will be used, in parallel with transcriptome profiling, to optimise glycosylation of proteins of bio-therapeutic importance within E. coli. Finally, a proof-of-principle study will seek to demonstrate the potential of using the bacterial c-di-GMP allosteric self-splicing ribozyme from C. difficile as a gene expression tool within a eukaryotic background. The systems developed here will be demonstrated with a number of practical examples of interest, and under fermentation conditions of relevance to the bioprocessing sector.

WHO WILL BENEFIT: Pharmaceutical, biotech and contract manufacturing organisations charged with producing human therapeutic proteins on the hundred gram to kilogram scale could also use the co-expression systems developed here to produce biopharmaceuticals in E. coli. Interest from industry members has indicated how these technologies could find application in protein-based biopharmaceutical production processes. Drug discovery companies would benefit from novel co-expression technologies that permit the supply of toxic and/or insoluble drug targets e.g. Domainex. Additionally, many chemical companies that employ biocatalysts, such as Novozymes, DSM, Lonza, BASF and Dr. Reddy's, could also use these new co-expression tools for the production of multi-subunit enzymes. Similarly, oil companies such as Shell and BP have invested heavily in synthetic biology programmes to engineer microorganisms to produce new biofuels, where new co-expression tools would be equally important. Finally, there are many companies, such as Invitrogen, Qiagen, Promega, Stratagene, EMD Bioscience, Thermo Fisher, Bio-Rad, GE healthcare and Sigma-Aldrich, who sell commercial expression systems for use in bacteria. Any number of these companies could benefit through licensing agreements to use new systems based on the co-expression technologies developed in this project.

HOW WILL THEY BENEFIT: We will actively seek to communicate our findings to the wider community, through scientific meetings and scholarly publications (by continuing to publish in top journals such as: JACS, PNAS, Nat Biotechnol. & Nature Chem. Biol.). However, in order for the technology we develop to become widely adopted, particularly in industry, it will be important to first secure any intellectual property rights for all new inventions we discover. As the research progresses and our relationships with interested commercial partners develop, we will seek to commercially exploit these new technologies and license the IP for use in industrial-scale protein production processes. Improvements in bioprocess efficiency, generated from this fellowship, could lead to a potential reduction in both drug manufacturing costs and the financial burden upon national healthcare providers. This research project will also generate technology and knowledge that will help maintain the UK's competitive edge and will produce highly trained and skilled research personnel.