DETOX Publications
Robust systematic approaches for the metabolic engineering of cell factories remain elusive. The available models for predicting phenotypical responses and mechanisms are incomplete, particularly within the context of compound toxicity that can be a significant impediment to achieving high yields of a target product. This study describes a Multi-Omic Based Production Strain Improvement (MOBpsi) strategy that is distinguished by integrated time-resolved systems analyses of fed-batch fermentations.
The tolerance of E. coli towards the feedstock-derived inhibitor furfural can be improved by the expression of NADH-dependent enzymes with furfural reductase activity. The York team were able to identify this activity in enzymes from the benzyl alcohol dehydrogenase family and showed that these enzymes can provide substantial improvements in furfural tolerance by increasing the rate of furfural reduction in vivo, thus demonstrating that the benzyl alcohol dehydrogenases could be a valuable source of furfural-detoxifying enzymes for industrial bioprocesses.
Membrane Stability in the Presence of Methacrylate Esters: Langmuir, August 2020, 36, 33 pages 9649-9657
Vivien Yeh, Alice Goode, Graham Eastham, Robert P. Rambo, Katsuaki Inoue, James Doutch, and Boyan B. Bonev
The Bonev lab investigated the effect of methacrylate esters on the structure and stability of model membranes using solid-state nuclear magnetic resonance (NMR) and small-angle X-ray scattering (SAXS).
In a poster prepared for the cancelled Microbiology Society Annual Conference in 2020, the authors present MORF, the online tool created by the DETOX project for analysing and interrogating multi-omics datasets.
Building on their analysis of MFS transporter fusions, the authors evaluate how horizontal gene transfer, recombination and fusions have brought new functionality to membrane transporters. They describe a range of bacterial fission and fusion examples and find fusions of regulatory domains to be prevalent, while noting that complete enzyme fusions are much rarer.
Systems Analyses Reveal the Resilience of Escherichia coli Physiology during Accumulation and Export of the Nonnative Organic Acid Citramalate: mSystems, 11 June 2019, 4, 4, e00187-19
Joseph Webb, Vicki Springthorpe, Luca Rossoni, David-Paul Minde, Swen Langer, Heather Walker, Amias Alstrom-Moore, Tony Larson, Kathryn Lilley, Graham Eastham, Gill Stephens, Gavin H. Thomas, David J. Kelly, Jeffrey Green
The DETOX team performed multi-omic analyses of E.coli fermentations producing the unnatural metabolite citramalate. This paper presents the metabolite, transcript, protein, and lipid profiles of high-cell-density, fed-batch fermentations of E. coli expressing either citramalate synthase or an inactivated enzyme. The results show that, even at 25 g liter−1, the non-toxic citramalate has very little effect on the cell. Other than the citramalate precursor pyruvate, the metabolic profile was not perturbed. Gene expression changes were limited and none of the general stress response regulons were activated. Changes in membrane lipid composition relating to growth phase were observed during the fermentation but these were also unaffected by citramalate production. This systems analysis of the citramalate fermentation shows that E. coli has capacity to readily adjust to the redirection of resources toward recombinant protein and citramalate production, suggesting that it is an excellent chassis choice for manufacturing organic acids. For the DETOX project, this data provides an essential control dataset against which we can compare the effect of producing more toxic compounds. The data is all available to browse on MORF.
Calum A. Pattrick, Joseph P. Webb, Jeffrey Green, Roy R. Chaudhuri, Mark O. Collins, David J. Kelly
Our colleagues at Sheffield have investigated the effects of vanillin on E.coli using proteomic profiling, transcription factor modeling and adaptive lab evolution. They find that vanillin causes activation of regulatory networks associated with preventing damage by reactive oxygen species. At the same time, the presence of vanillin caused the upregulation of the copA gene and the reduction of copper ions. Copper homeostasis, which is linked to FeS clusters and iron and sulphur acquisition in bacteria, was found to be essential for growth in the presence of vanillin. The authors identify numerous candidates for engineering vanillin tolerance, including the potential vanillin efflux pumps AcrD and AaeAB. This paper was the editor’s pick for this issue of mSystems.
Benjamin J. Willson, Lindsey Dalzell, Liam N. M. Chapman, Gavin H. Thomas
Our York team reviewed the bacterial major facilitator superfamily transporters arising from fusion events using the InterPro and pFam databases. Eight groups of fusions were identified, including two previously unidentified fusions.
Rayner M. L. Queiroz, Tom Smith, Eneko Villanueva, Maria Marti-Solano, Mie Monti, Mariavittoria Pizzinga, Dan-Mircea Mirea, Manasa Ramakrishna, Robert F. Harvey, Veronica Dezi, Gavin H. Thomas, Anne E. Willis, Kathryn S. Lilley
The Lilley lab present orthogonal organic phase separation (OOPS) method to recover unbiased cross-linked protein–RNA and free protein, or protein-bound RNA and free RNA from any organism. Gavin reviewed the bacterial RNA-interactome for E.coli, which included 277 previously unidentified ribosome binding proteins (RBP) and suggest novel RBP functions in prokaryotes.
Rebecca J. Hall, Lindsey A. Flanagan, Michael J. Bottery, Vicki Springthorpe, Stephen Thorpe, Alistair C. Darby, A. Jamie Wood, Gavin H. Thomas
DETOX bioinformatician Vicki Springthorpe created an online FBA tool (MORFlux) to help the authors develop a model to describe the metabolic relationship between symbionts Sodalis glossinidius and
Massive over-representation of solute-binding proteins (SBPs) from the tripartite tricarboxylate transporter (TTT) family in the genome of the α-proteobacterium Rhodoplanes sp. Z2-YC6860: Microb Genom. 2018 May; 4(5): e000176.
Leonardo T. Rosa, Vicki Springthorpe, Matheus E. Bianconi, Gavin H. Thomas, and David J. Kelly
Researchers from Sheffield and York present the massive over expansion of the tripartite tricarboxylate transporter (TTT) family. Vicki developed the circular genome browser, now used on MORF, to depict the location of the TTT genes within the genome. In the 8.2 Mb genome of the α-proteobacterium Rhodoplanes, 434 genes encoding solute-binding proteins (SBPs) from the tripartite tricarboxylate transporter (TTT) family were found. The SBP genes were located close to genes encoding membrane components of transport systems from different families, as well as regulatory proteins such as histidine-kinases and transcription factors, indicating a broad range of functions around the sensing, response and transport of organic compounds.
Rohanah Hussain, Charlotte S. Hughes, Tamás Jávorfi, Giuliano Siligardi, Paul Williams, Boyan B. Bonev
The Bonev lab use synchrotron radiation circular dichroism to monitor the thermal evolution of secondary structure in proteins as they approach the melting point and the impact of substrate on their thermal behaviour.
Ivan S. Gyulev, Benjamin J. Willson, Rosanna C. Hennessy, Preben Krabben, Elizabeth R. Jenkinson, Gavin H. Thomas
This review of synthetic biology elements in solventogenic Clostridia identifies promoters, transcriptional terminators and ribosome binding sites in use and in need of further development. This comprehensive analysis will be essential for the engineering Clostridia strains on the DETOX project.
Joseph P. Webb, S. Alison Arnold, Scott Baxter, Stephen J. Hall, Graham Eastham, Gill Stephens
This paper from Joe’s PhD work describes the development of the citramalate-producing E.coli strain and fermentation that we used for our DETOX control dataset, published in mSystems, 2019.
Time, space, and disorder in the expanding proteome universe: Proteomics 2017, 17, 7, 1600399
David‐Paul Minde, A. Keith Dunker, and Kathryn S. Lilley
David et al. explore the dynamic world of proteins revealing multiple protein structures caused by alternative sequences, alternative conformations, alternative interactions with a range of biomolecules, cellular localizations and alternative behaviours in different cell types. The authors present computational tools that are able to expand on multiple orthogonal data types to provide insights into the understanding of the dynamics of cellular mechanisms.