Development and Application of Next Generation Synthetic Biology Tools

Lead Research Organisation: University of Manchester
Department Name: School of Medical Sciences

Abstract

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.

Technical Summary

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.

Planned Impact

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.
 
Description We have developed new engineered bacterial strains for the expression of recombinant proteins, this is important for the expression of high-value proteins such as enzymes and protein-based pharmaceuticals. The more efficient protein production afforded by these engineered strains, could for example find great promise to reduce the cost of producing protein-based therapeutics ultimately aiding access to therapy for patients and reducing the cost-burden on national healthcare provider such as the NHS. Secondly we have discovered a novel excretion pathway in bacterial cells. Protein translocation is an essential feature of cellular organisms. Bacteria, like all single-cell organisms, interact with their environment by translocation of proteins across their cell membranes via dedicated secretion pathways. Proteins destined for secretion are directed toward the secretion pathways by the presence of specific signal peptides. This study demonstrates that under conditions of both osmotic stress and translation stress, E. coli cells undergo an excretion phenomenon
whereby signal peptide-less proteins are translocated across both the inner and outer cell membranes into the extracellular environment. Confirming the presence of alternative translocation/excretion pathways and understanding their function and regulation are thus important for fundamental microbiology and biotechnology.
Exploitation Route In the following research areas and industrial sectors: Applied Biotechnology, protein production, industrial biotechnology
Sectors Chemicals,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

URL https://dixonlab.org
 
Description Has led to the adoption of new working practices within the industrial collaborators UK-based R&D facility, which has led to new business opportunities.
First Year Of Impact 2014
Sector Manufacturing, including Industrial Biotechology
Impact Types Economic

 
Description BBSRC, DTP
Amount £80,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2015 
End 09/2020
 
Description BBSRC-FAPESP joint call in advanced biofuels
Amount £2,082,439 (GBP)
Funding ID BB/P01738X/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 03/2017 
End 02/2022
 
Description BBSRC-FAPESP pump-priming award
Amount £35,000 (GBP)
Funding ID BB/L026244/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 08/2014 
End 07/2016
 
Description BioCatNet Proof of Concept
Amount £35,032 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2015 
End 02/2016
 
Description EPSRC CDT UCL
Amount £80,000 (GBP)
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Academic/University
Country United Kingdom
Start 10/2015 
End 08/2020
 
Description Engineering Novel Allosteric Transcription Factor Recognition: Applications in Bio-catalysis and Synthetic Biology
Amount £80,000 (GBP)
Funding ID 2110033 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 09/2018 
End 09/2022
 
Description Enhanced Production and Process Analysis of Biotherapeutics - Antibody Fragments and Alternative Scaffolds
Amount £60,000 (GBP)
Funding ID 1621074 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Academic/University
Country United Kingdom
Start 10/2015 
End 09/2019
 
Description IB Catalyst - Early Stage Feasibility
Amount £121,512 (GBP)
Funding ID BB/M011259/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 04/2015 
End 03/2016
 
Description Indonesian Government Home funded PhD Srudenship
Amount £100,000 (GBP)
Organisation Government of Indonesia 
Sector Public
Country Indonesia
Start 09/2016 
End 08/2020
 
Description Proof of Concept - CMBNet
Amount £35,032 (GBP)
Funding ID BIOCATNET PoC-012 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Department Networks in Industrial Biotechnology and Bioenergy (NIBB)
Sector Academic/University
Country United Kingdom
Start 07/2015 
End 10/2015
 
Description Rutherford Fund Strategic Partner Grants
Amount £149,500 (GBP)
Funding ID RF-2018-41 
Organisation Department for Business, Energy & Industrial Strategy 
Sector Public
Country United Kingdom
Start 04/2018 
End 03/2019
 
Description Science Without Boarders
Amount £120,000 (GBP)
Organisation National Council for Scientific and Technological Development (CNPq) 
Sector Public
Country Brazil
Start 10/2015 
End 08/2020
 
Description Synthetic Biology Centre Bid 2
Amount £10,100,000 (GBP)
Funding ID BB/M017702/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start  
 
Description Synthetic Biology: Development and Application Cellular Computation Devices
Amount £80,000 (GBP)
Funding ID 1618786 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2015 
End 09/2019
 
Description UK Catalysis Hub
Amount £172,000 (GBP)
Funding ID EP/M013219/1 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Academic/University
Country United Kingdom
Start 05/2018 
End 04/2020
 
Description Welcome Trust ISSF Research Consortia Award
Amount £44,000 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2015 
End 09/2016
 
Title Proteome analsyis of Translocation phenomenon 
Description Proteome analsyis of Translocation phenomenon 
Type Of Material Database/Collection of data 
Year Produced 2018 
Provided To Others? Yes  
Impact Manuscript published, Translation Stress Positively Regulates MscL-Dependent Excretion of Cytoplasmic Proteins MBio 2018, doi: 10.1128/mBio.02118-17. 
URL http://proteomecentral.proteomexchange.org/cgi/GetDataset?ID=PXD006474
 
Description AS project 
Organisation Biocatalysts Ltd
Country United Kingdom 
Sector Private 
PI Contribution The research project.
Collaborator Contribution Supply of materials to enable the research to proceed and desk analysis of commercial viability of the processes to be developed as part of the research.
Impact Too early
Start Year 2015
 
Description AS project 
Organisation Chirotech Technology
Country United Kingdom 
Sector Private 
PI Contribution The research project.
Collaborator Contribution Supply of materials to enable the research to proceed and desk analysis of commercial viability of the processes to be developed as part of the research.
Impact Too early
Start Year 2015
 
Description Collaboration with CMO for biologics production 
Organisation Cobra Biologics
Country United Kingdom 
Sector Private 
PI Contribution We have contributed IP, know-how, and research expertise to this collaboration
Collaborator Contribution The partners have contributed industry R&D and know-how
Impact Too earlier to say, the collaboration has led to successful joint grant funding.
Start Year 2013
 
Description Collaboration with CTBE, Brazil 
Organisation Bioethanol Science and Technology Centre (CTBE)
Country Brazil 
Sector Private 
PI Contribution During a Faculty-led Research Development visit to Brazil, we visited the Brazilian National Laboratory for Bioethanol (CTBE). Following this visit, myself and Fabio Squina (CTBE) have been in communication about applying ours interests in synthetic biology to facilitate enzyme discovery associated with lignin-biomass valorisation. We hosted visitors from the CTBE and other research institutes in Brazil at Industrial Biotechnology Bioenergy Meeting that I co-organised. This led to a successful funding application to the BBSRC/FAPESP (FAPPA) pump-priming call, to allow us to explore these preliminary ideas and facilitate further networking and research consortia building activities. Most recently we re-visited the CTBE to further develop collaborators links and build a research proposal. This has led to exchange of materials, a proposed exchange of personnel between my laboratory and the CTBE, and most recently to a large funding application to the BBSRC/FAPESP (>£2.5 UK).
Collaborator Contribution During a Faculty-led Research Development visit to Brazil, we visited the Brazilian National Laboratory for Bioethanol (CTBE). Following this visit, myself and Fabio Squina (CTBE) have been in communication about applying ours interests in synthetic biology to facilitate enzyme discovery associated with lignin-biomass valorisation. We hosted visitors from the CTBE and other research institutes in Brazil at Industrial Biotechnology Bioenergy Meeting that I co-organised. This led to a successful funding application to the BBSRC/FAPESP (FAPPA) pump-priming call, to allow us to explore these preliminary ideas and facilitate further networking and research consortia building activities. Most recently we re-visited the CTBE to further develop collaborators links and build a research proposal. This has led to exchange of materials, a proposed exchange of personnel between my laboratory and the CTBE, and most recently to a large funding application to the BBSRC/FAPESP (>£2.5 UK).
Impact Outcomes: A proposed funding application to the BBSRC/FAPESP (>£2.5 UK). Multi-discplianry collaboration: Synthetic Biology, Meta-transcriptomics, Enzyme discovery, biocatalysis, bioprocess development, life-cycle analysis.
Start Year 2013
 
Description Collaboration with International biopharma company (2017- on going) 
Organisation Novo Nordisk
Country Denmark 
Sector Public 
PI Contribution Following on from research data present and US conference, this international biopharma companies made contacted and expressed and expressed an interested in getting access to ort technology and establishing a collaboration
Collaborator Contribution We have discussed and applied a work program together over several months, and executed the appropriate legal agreements. The technical validation work is due to be initiated within Q1 2018
Impact Outputs are still pending.
Start Year 2017
 
Description BBSRC Enterprise Fellowships Networking Event 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact BBSRC Enterprise Fellowships Networking Event
Year(s) Of Engagement Activity 2017
 
Description British Science Week Schools' Fair 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Members of Dixon Lab participated in the British Science Week Schools' Fair (13-16th March 2018), 100 attendees
Year(s) Of Engagement Activity 2018
 
Description Croda Open Innovation Day 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact New project ideas discussed.
Year(s) Of Engagement Activity 2017
 
Description EuroScience Open Forum (ESOF) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact EuroScience Open Forum (ESOF):

+provide an open forum for debate on science and technology and research policies in Europe
+strengthen the links between science and society
+contribute to the creation of an integrated space for science and technology in Europe, linking research organisations and policies at national and EU levels; strive for a greater role of the EU in research and
+influence science and technology policies.
Year(s) Of Engagement Activity 2016
URL http://manchester2016.esof.eu/en/
 
Description FLS Community Open Day 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact An outreach stand about biotechnology for the production of fragrance compounds and public opinion of synthetic biology
Year(s) Of Engagement Activity 2016
 
Description Institute Open Day (Outreach) 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Prepare promotional material in advance of the meeting, and hosted the interactive stand 1,5 days

tbc
Year(s) Of Engagement Activity 2014
 
Description Institute Open Day (Outreach) 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Synthetic Biology stand at MIB open-day attended by >200 A levels students
Year(s) Of Engagement Activity 2014,2015,2016
 
Description Invited Speaker at Centre of Excellence in Biopharmaceuticals, UK 4th Annual Scientific Symposium 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Participants in your research and patient groups
Results and Impact Q&A

Contact with new potential industrial collaborators
Year(s) Of Engagement Activity 2015
 
Description Invited Speaker at European Congress on Biotechnology 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Participants in your research and patient groups
Results and Impact Q&A

Lead to new contacts
Year(s) Of Engagement Activity 2014
 
Description Invited Speaker at the UK-South Korea Synthetic Biology Workshop. Heathrow UK 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Participants in your research and patient groups
Results and Impact Q&A

New potential collaborators in Korean
Year(s) Of Engagement Activity 2015
 
Description Invited speaker at CTBE and UNICAMP 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Participants in your research and patient groups
Results and Impact Talks sparked Q&A, and supported subsequent discussions around research proposal

Further funding applications
Year(s) Of Engagement Activity 2014
 
Description Invited speaker at ELRIG Advances in Protein Technology 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact Sparked Q&A immediately afterwards. Led to follow up discussions with three different researchers, two of which have led to ongoing collaborations

We have transferred the technology discussed in the talk into a new collaborators institute, who is a leading national service provider in the relevant field.
Year(s) Of Engagement Activity 2013
URL http://elrig.org/portfolio/advances-in-recombinant-protein-technology-2013/
 
Description Invited speaker at Society of General Microbiology AGM, 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Participants in your research and patient groups
Results and Impact Q&A

Follow up contact with members of the research community
Year(s) Of Engagement Activity 2014
 
Description Rainbow Seed Fund Catapult Investor Insight Event, Court of Justice, London 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact Presentation on emerging areas from the academic research base to investors and press
Year(s) Of Engagement Activity 2017
 
Description SEB Synthetic Biology Symposium 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Talk sparked questions and discussions. A number of new potential collaboration contacts were made.

Was asked to come for a Faculty visit at another institute
Year(s) Of Engagement Activity 2014
URL http://www.sebiology.org/meetings/Past_Meetings/synthetic_biology/overview.html
 
Description SMART-Map Industrial Dialogue on Synthetic Biology 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Study participants or study members
Results and Impact Workshop on the responsible development of Synthetic Biology which forms part of the EU H2020 SMART-map project.
Year(s) Of Engagement Activity 2017
URL http://projectsmartmap.eu/
 
Description SynBioChem Workshop on Responsible Research and Innovation 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Study participants or study members
Results and Impact Roundtable discussant at SynBioChem Workshop on Responsible Research and Innovation
Year(s) Of Engagement Activity 2015
 
Description iGEM Giant Jamboree 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Synthetic Biology iGEM Giant Jamboree
Year(s) Of Engagement Activity 2016
URL http://2016.igem.org/Giant_Jamboree