DeTOX - Productive whole cell biocatalysis by engineering resistance to toxic products and substrates
Lead Research Organisation:
University of York
Department Name: Biology
Abstract
Product toxicity is a major problem for many IBBE processes involving production of small molecules by living cells. Toxicity causes yield restrictions & cell lysis, & frequently affects the commercial viability of biomanufacturing. Likewise, small molecules in lignocellulosic feedstocks inhibit bacterial fermentations & ultimately depress product yields. In this CBMNet NIBB-led bid, a team of scientists from four Universities apply their fundamental expertise in systems & synthetic biology & membrane function, to engineer increased resistance to small molecules in the industrially relevant bacteria, E. coli & solventogenic Clostridia. Our innovation is to translate BBSRC-funded research in microbial stress responses, membrane structure & membrane transporters, into the development & commercialisation of innovative applications in IBBE by our 5 commercial partners. A key project output will be a commercial chassis strain, DeTox, with generally increased chemical resistance.
Technical Summary
A major challenge in industrial biotechnology & bioenergy is to solve serious problems with yield restrictions due to product or substrate toxicity. Increasing product concentrations by >10-fold would deliver commensurate improvements in revenue from such processes, and is a viable target, given the millimolar product concentrations formed in many proposed bioprocesses at present. This is critical for commercially viable production of bulk & specialty chemicals by living cells, because many of these are toxic & need to be removed rapidly to avoid damage to the intracellular contents & cell membrane. It is also essential for the effective use of lignocellulosic substrates that contain fermentation inhibitors that exert their toxic effects by penetrating the cell. Our objective is to produce host strains with enhanced resistance to a broad range of chemical products & so provide highly-productive chassis for commercial synthetic biology. We will study the mechanisms of chemical toxicity and resistance in E. coli & solventogenic Clostridium spp., both by analyzing cellular responses during bioproduction and by experimental evolution of resistant strains. We will also apply world-leading membrane science (efflux pumps, proteomics, lipidomics & membrane biophysics) to execute novel, rational redesign of cell membranes to enhance resistance. We will combine knowledge of these systems to develop our DeTox strain platform by strain engineering, using synthetic biology standards. The chassis will be tested in small scale replicas of industrial bio-processes, & iteratively redesigned for maximum robustness under process conditions, using models describing cellular responses to toxin exposure. The outcome will be DeTox chassis, to be licensed to our partners, and DeTox gene cassettes, that can be ported to other hosts.
Planned Impact
As described in proposal submitted to IUK
Organisations
- University of York (Lead Research Organisation)
- Engineering and Physical Sciences Research Council (Co-funder)
- YORK ST JOHN UNIVERSITY (Collaboration)
- Lucite International (United Kingdom) (Project Partner)
- Centre for Process Innovation (Project Partner)
- Biocleave Limited (Project Partner)
- ReBio Technologies Limited (UK) (Project Partner)
People |
ORCID iD |
Gavin Thomas (Principal Investigator) |
Publications
Balmer A
(2016)
Five rules of thumb for post-ELSI interdisciplinary collaborations
in Journal of Responsible Innovation
Del Carratore F
(2021)
Multi-omics Study of Planobispora rosea, Producer of the Thiopeptide Antibiotic GE2270A.
in mSystems
Drousiotis K
(2023)
Characterization of the l-arabinofuranose-specific GafABCD ABC transporter essential for l-arabinose-dependent growth of the lignocellulose-degrading bacterium Shewanella sp. ANA-3.
in Microbiology (Reading, England)
Elston R
(2023)
Flipping the switch: dynamic modulation of membrane transporter activity in bacteria.
in Microbiology (Reading, England)
Goode A
(2021)
Interactions of polymyxin B with lipopolysaccharide-containing membranes.
in Faraday discussions
Gyulev IS
(2018)
Part by Part: Synthetic Biology Parts Used in Solventogenic Clostridia.
in ACS synthetic biology
Herman R
(2020)
The Salmonella enterica serovar Typhimurium virulence factor STM3169 is a hexuronic acid binding protein component of a TRAP transporter.
in Microbiology (Reading, England)
Hussain R
(2018)
To Boil an Egg: Substrate Binding Affects Critical Stability in Thermal Unfolding of Proteins.
in The journal of physical chemistry. B
Minde D
(2020)
Biotin proximity tagging favours unfolded proteins and enables the study of intrinsically disordered regions
in Communications Biology
Title | Introduction to MORF for the 2020 Global Bioeconomy Summit |
Description | This 3 minute video shows how MORF can be used to display and interrogate complex multi-omics datasets. The video was hosted on the website for the 2020 Global Bioeconomy Summit. |
Type Of Art | Artistic/Creative Exhibition |
Year Produced | 2020 |
Impact | NA |
URL | https://gbs2020.net/videos/ |
Title | MORF Promotional Advert |
Description | This short video explains how MORF can be used to store, share and analyse omics data. The video is for a general audience such as investors or business development experts. |
Type Of Art | Film/Video/Animation |
Year Produced | 2021 |
Impact | The video is uploaded on the MORF website and is being used to promote MORF to potential clients. |
URL | https://youtu.be/1YrpEuXlgo8 |
Title | MORF Styrene Case Study |
Description | This video describes the work on the DETOX styrene project, focusing on how MORF was used to identify targets that were able to improve styrene production |
Type Of Art | Film/Video/Animation |
Year Produced | 2021 |
Impact | The video will be released to coincide with the styrene paper publication. |
Description | In the DETOX project we aimed to used a multi-omics approach to try and improve bioprocess productivity, by understanding how industrially relevant chemicals produced by bacteria can intoxicate the producing cell and using this knowledge to improve their performance. First using a control system of a cell producing the non-toxic chemical citramalate we were able to test and demonstrate the value of our integrating system, working across 4 Universities. The key test system in the project that we developed was the production of styrene, which is highly toxic to the producing cell. Our multi-omics data let to target selection and the manipulation of these targets resulted in higher levels of production of styrene in the producing system. We also investigated how Clostridia respond to biobutanol production and identified an important cellular response which also appears to confer increased tolerance when overproduced. The software platform, DETOXbase, now MORF, will be developed further through FoF funding and has already been used by other academic and industry users to store and analysis their data using our propriety Web browser based tools. |
Exploitation Route | Specific information about citramalate and styrene could be used by other academic and industry. Our generic MORF platform we hope to continue to develop and perhaps also commercialise. |
Sectors | Chemicals Manufacturing including Industrial Biotechology Pharmaceuticals and Medical Biotechnology |
Description | 17-ERACoBioTech - MEmbrane Modulation for BiopRocess enhANcEment - MeMBrane |
Amount | £632,794 (GBP) |
Funding ID | BB/R02152X/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 04/2018 |
End | 04/2021 |
Description | BBSRC IAA University of York |
Amount | £300,000 (GBP) |
Funding ID | BB/S506795/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 03/2018 |
End | 03/2021 |
Description | Browser-based comparative analysis of industrial fermentations |
Amount | £9,916 (GBP) |
Funding ID | BIV-HVB-2021/05 (THOMAS) |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 01/2022 |
End | 06/2022 |
Description | IAA |
Amount | £15,500 (GBP) |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 09/2021 |
End | 03/2022 |
Description | Inclusion Bodies for Industrial Biotechnology |
Amount | £49,832 (GBP) |
Funding ID | C2019-057 THYME POC Fund |
Organisation | Thyme Project |
Sector | Academic/University |
Country | United Kingdom |
Start | 04/2019 |
End | 03/2020 |
Description | Materials and Manufacturing Round 2 |
Amount | £758,612 (GBP) |
Funding ID | TSB 103564 |
Organisation | Innovate UK |
Sector | Public |
Country | United Kingdom |
Start | 07/2017 |
End | 07/2019 |
Description | Predicting aggregation potential and kinetics of industrially relevant proteins |
Amount | £49,578 (GBP) |
Funding ID | FOF-09 |
Organisation | United Kingdom Research and Innovation |
Department | Research England |
Sector | Public |
Country | United Kingdom |
Start | 09/2021 |
End | 03/2022 |
Title | Biotin labelling |
Description | The Lilley lab have developed a method to use biotin painting as a method to gain unique insights into in vivo condition-dependent subcellular plasticity of proteins by tagging sites of local disorder in proteins with biotin. |
Type Of Material | Technology assay or reagent |
Year Produced | 2019 |
Provided To Others? | Yes |
Impact | Minde, D., Ramakrishna, M. & Lilley, K.S. Biotin proximity tagging favours unfolded proteins and enables the study of intrinsically disordered regions. Commun Biol 3, 38 (2020) doi:10.1038/s42003-020-0758-y |
Title | MOBpsi, Multi-Omics Based Production Strain Improvement |
Description | MOBpsi (Multi-Omics Based Production Strain Improvement) is a strategy for improving the performance of biological cell factories. The three hallmarks of the MOBpsi that represent advances over conventional approaches are: (i) cell factories producing the chemical of interest are analyzed, rather than simply adding the toxic chemical to cultures (i.e. external application); (ii) a systems approach is taken using time-resolved multi-omic data sets and quantitative analytics within an integrated database to facilitate the identification of genetic interventions to enhance cell factory performance; (iii) scalable fermentations, rather than plate or flask cultures, are used to enhance the likelihood of the improved cell factories being used commercially. |
Type Of Material | Technology assay or reagent |
Year Produced | 2021 |
Provided To Others? | No |
Impact | This methodology is submitted for publication in mSystems |
Title | OOPS, A new method to capture the RNA-binding proteome |
Description | The OOPS approach re-purposes acid guanidinium thiocyanate-phenol-chloroform (AGPC) extraction, commonly referred to as the 'TRIzol protocol' after the branded reagent, which has been used for decades to enrich and partition RNA from other cellular components. The Trizol protocol separates RNA and protein in aqueous and organic phases respectively, and instructs the researcher to avoid the interface. However, the Lilley Group and their collaborators reasoned that RNA:protein adducts generated by UV cross-linking will be highly enriched at the interface, including rRNAs, tRNAs, mRNAs, sRNAs and other non-coding RNAs. The OOPS protocol therefore begins from this interface, with repeated phase separations to ensure removal of non-RNA-bound protein from the resulting final interface. Proteins at this final interface can then be recovered by enzymatically digesting the RNA and repeating the Trizol phase separation, taking the resulting protein-rich organic phase. |
Type Of Material | Biological samples |
Year Produced | 2019 |
Provided To Others? | Yes |
Impact | Cited over 100 times in first year. |
URL | https://www.nature.com/articles/s41587-018-0001-2 |
Title | MORFlux FBA tool |
Description | The MORFlux FBA tool enables development of metabolic models through the MORF website. A number of predefined models can be used as a starting point, or custom models can be uploaded. Reactions can be added, constraints edited and the resulting model can be solved and downloaded from the website. No programming language is required to use the tool, allowing bench scientists to use their fermentation data to develop a bespoke metabolic model for their organism, and use in silico analysis to generate testable hypotheses for improving growth or increasing yield. |
Type Of Material | Data handling & control |
Year Produced | 2020 |
Provided To Others? | Yes |
Impact | The tool was initially built for the research described in the paper below. Additional projects using MORFlux are ongoing. Hall RJ, Flanagan LA, Bottery MJ, Springthorpe V, Thorpe S, Darby AC, Wood AJ, Thomas GH. 2019. A tale of three species: adaptation of Sodalis glossinidius to tsetse biology, Wigglesworthia metabolism, and host diet. mBio 10:e02106-18. https://doi.org/10.1128/mBio.02106-18. |
URL | https://morf-db.org/tools/morflux/fba |
Title | Multi-Omics Research Factory (MORF) |
Description | The MORF tool is a software platform that allows scientists to analyse their 'omics data using an online browser, without any coding experience required. The DETOX project uses MORF to securely store, share and interrogate transcriptomic, proteomic, metabolomic and lipidomic data sets. These data are connected to the genome and fermentation data. Significant changes in expression or abundance can quickly be identified through dynamic lists based on user-set thresholds. Genes of interest can be investigated further by clicking through to the gene page, which pulls up all the 'omics data, Go terms and external links associated with that gene. Data can also be exported into journal-quality graphs and published data can be made public on the site for the community to use. Our citramalate data is available at www.morf-db.org. The platform has been developed further through the MeMBrane project, BB/R02152X/1, to incorporate eukaryotic genomes and now stores over 100 bacterial & eukaryotic genomes that are freely available to browse. |
Type Of Material | Data analysis technique |
Year Produced | 2019 |
Provided To Others? | Yes |
Impact | In addition to the DETOX project, in which MORF was first developed, the tool is also instrumental in sharing data across the international ERA CoBiotech MeMBrane team, BB/R02152X/1, and the THYME project with Teesside University and Fujifilm to study Inclusion Bodies in Industrial Biotechnology. Market research is currently ongoing through a Pathfinder Award to develop a business plan for commercialisation of the software. |
URL | http://www.morf-db.org. |
Description | York Visors for Frontline Staff |
Organisation | York St John University |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | During the first COVID-19 lockdown the DETOX project manager Joyce Bennett managed a community project in York to provide free visors for frontline workers in and around the city. A team of around 50 volunteers, also including the DETOX technician Reyme Herman, printed, laser cut, assembled, sterilised, and delivered 10,000 visors between April and June 2020. Fundraising for the project raised an extra £7,400 which was donated to the Cavell Nurses Trust. |
Collaborator Contribution | Joyce Bennett provided overall project management to the effort and, in particular, managed the requests and deliveries. Under her initiative, and with Reyme Herman, a second assembly line was set up at the University of York, using parts directly from the biology workshop and assembling and sterilising them in a biology lab. |
Impact | The team were awarded a #YorkHeros Blue Plaque by the York Civic Trust. |
Start Year | 2020 |
Title | MORF name & logo |
Description | MORF is a browser based tool to store, share and analyse multi-omics datasets. York are commercialising the software and have submitted a trademark application to protect the name and logo. |
IP Reference | |
Protection | Trade Mark |
Year Protection Granted | |
Licensed | No |
Impact | Further funding has been awarded to exploit, develop and commercialise the software, including 2 IAAs, 2 THYME grants, a HVB BIV with Oxford Biotrans and a Pathfinder award. |
Title | MORF: MultiOmics Research Factory |
Description | MORF is a browser based tool to store, share and analyse multi-omics data. The tool is being commercialised for both academics and industry to access bioinformatics insights without the need for programming skills. MORF is available through the University of York and can be written onto grants as a service offered by the Technology Facility. MORF can not only help researchers to analyse their data, but can also be used to disseminate publicly funded data through the website to support a publication. Projects in MORF can be kept secure for the team or made open access for the community to benefit from. |
Type Of Technology | Webtool/Application |
Year Produced | 2019 |
Impact | MORF is a commercial entity within the University of York and the team are preparing to spin out the enterprise if successful. DETOX Project Manager Joyce Bennett has applied for an Enterprise Fellowship to support this work. |
URL | http://www.morf-db.org |
Description | Biology Week event; Synthetic Biology; could we? should we? |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Public/other audiences |
Results and Impact | A public event hosted by the Royal Institution, London as part of Biology Week. A panel of experts - including Susan Molyneux-Hodgson as the only social scientist - debated the topic 'Synthetic Biology: how far could it go: how far should it go?' The audience of a couple of hundred was accessed and the event was filmed by RI for their dedicated YouTube Channel. To date, the video has been viewed 11,900 times. The comments/discussion on the video are extensive and include a very wide range of opinions. |
Year(s) Of Engagement Activity | 2015 |
URL | http://www.rigb.org/whats-on/events-2015/october/public-synthetic-life-how-far-could-it-go-how-far-s... |
Description | IBioIC industry meeting |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Industry/Business |
Results and Impact | leading national industrial biotechnology event with major network opportunities. discussed responsible innovation work with numerous non-academic partners leading to one potential future collaboration, with NPL |
Year(s) Of Engagement Activity | 2019 |
Description | Marvellous Microbes at YorNight |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Public/other audiences |
Results and Impact | The Marvellous Microbes exhibition included family friendly interactive demonstrations to explore how microbes could stop the spread of tropical diseases and reduce our dependence on fossil fuels. Researchers from the Thomas lab explained how they are targeting specific body molecules inside the 'smelly' microbes to prevent body odour and using microbes to produce fuels of the future. Visitors were invited to 'Ask a scientist' their questions about what they wanted to know about life as a scientist, with answer posted online after the event. |
Year(s) Of Engagement Activity | 2018 |
URL | https://www.york.ac.uk/news-and-events/events/yornight/2018/activities/marvellous-microbes/ |
Description | Marvellous Microbes at the Festival of Ideas, York |
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 | 90 school children & teachers visited the teaching labs to experience Marvellous Microbes. They enjoyed the hands-on activities and getting to come into the laboratory at the University. The event was also laid on for members of the public as an after-school event. |
Year(s) Of Engagement Activity | 2019 |
URL | http://yorkfestivalofideas.com/2019/events/marvellous-micros/ |
Description | Panel and talks at Synthetic Biology UK 2018, Bristol |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Other audiences |
Results and Impact | Panel on responsible innovation organised to present social science work to technical audience. Paper and poster also presented |
Year(s) Of Engagement Activity | 2018 |
Description | Presentation at Factories for Advanced Biomanufacturing meeting |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Professional Practitioners |
Results and Impact | presented talk on RRI to technical audiences |
Year(s) Of Engagement Activity | 2017 |
URL | http://cbmnetnibb.group.shef.ac.uk/?s=factories+for+advanced |
Description | Royal Society Panel Debate at Hay Literary Festival |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Public/other audiences |
Results and Impact | Royal Society organised evening session at the Hay Literary Festival (May 2017). Susan Molyneux-Hodgson the representative for the social sciences on the Panel with 3 senior scientist, to discuss 'genetic technologies'. |
Year(s) Of Engagement Activity | 2017 |
URL | https://www.hayfestival.com/wales/index.aspx?skinid=2¤cysetting=GBP&localesetting=en-GB&resetf... |
Description | Social and Political Challenges for the Bioceconomy |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Industry/Business |
Results and Impact | major event to present social science research to technical audiences. Included talks from experts, workshop activities and open discussion and debate. |
Year(s) Of Engagement Activity | 2016,2017 |
URL | http://cbmnetnibb.group.shef.ac.uk/?s=social+and+ethical+challenges |