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

Publications

10 25 50
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Balmer A (2016) Five rules of thumb for post-ELSI interdisciplinary collaborations in Journal of Responsible Innovation

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Thomas GH (2017) On the pull: periplasmic trapping of sugars before transport. in Molecular microbiology

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Willson BJ (2019) Evolutionary dynamics of membrane transporters and channels: enhancing function through fusion. in Current opinion in genetics & development

 
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 05/2018 
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 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 05/2019 
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 08/2017 
End 07/2019
 
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 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 ReAlChem 
Organisation Green Biologics
Country United Kingdom 
Sector Private 
PI Contribution Expertise on responsible innovation
Collaborator Contribution Access to people and facilities to conduct research into responsible innovation
Impact Multidisciplinary involving sociology and microbiology. Outputs are in progress.
Start Year 2017
 
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