University of Manchester UKRI Innovation Fellowships: BBSRC Flexible Talent Mobility Accounts

Lead Research Organisation: University of Manchester
Department Name: Alliance Manchester Business School

Abstract

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Publications

10 25 50
 
Description See details of Individual Awards in Partnerships & Collaborations
Exploitation Route See details of individual Awards in Partnerships & Collaborations and in other sections e.g. Awards / Further Funding
Sectors Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description SYNBIOCHEM Centre awarded a £ 5K BBSRC Flexible Talent Mobility Account Fellowship to Dr. Yun Wang
Amount £5,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start  
 
Title Collaborative effort to develop synthetic genomics technologies for industrial biotech - 
Description The project established a work flow to solve the genomes of 12 SCRaMbLEd yeasts strains in parallel, starting from individual colonies within two weeks. A manuscript for the pipeline is in preparation. The project established a new approach to solve SCRaMbLEd yeast genomes with complex structural variations which previously could not be solved by conventional short read NGS. A manuscript for the tool NANOPIPE is in preparation. 
Type Of Material Biological samples 
Year Produced 2018 
Provided To Others? No  
Impact The project established a work flow to solve the genomes of 12 SCRaMbLEd yeasts strains in parallel, starting from individual colonies within two weeks. A manuscript for the pipeline is in preparation. The project established a new approach to solve SCRaMbLEd yeast genomes with complex structural variations which previously could not be solved by conventional short read NGS. A manuscript for the tool NANOPIPE is in preparation. 
 
Title An academic and industrial partnership to exploit methyl transfer enzymes 
Description Protein Data Bank: Entry 6I3C: S-COMT:SAM:DNC complex Entry 6I3D: S-COMT:sinefungin:DNC complex BioMagRes-Bank: Entry 26848: S-COMT:SAM:DNC complex Entry 26851: S-COMT:sinefungin:DNC complex 
Type Of Material Database/Collection of data 
Year Produced 2018 
Provided To Others? Yes  
Impact Deposition of two X-ray crystal structures of catechol O-methyltransferase in complex with inhibitors bound (S-COMT:SAM:DNC and S-COMT:sinefungin:DNC with the accession codes 6I3C and 6I3D, respectively) in the protein data bank. Deposition of two backbone 1H, 13C and 15N NMR chemical shift assignments in the BioMagRes-Bank (under the BMRB accession codes 26848 and 26851, respectively). 
 
Description An academic and industrial partnership to exploit methyl transfer enzymes 
Organisation C4X Discovery Ltd
Country United Kingdom 
Sector Academic/University 
PI Contribution MIB researchers and C4X Discovery have considerable interest in methyltransferases. In the MIB they are studied to extend our understanding of fundamental processes underlying enzyme catalysis. C4X Discovery is interested in their inhibition as a model for developing drug discovery protocols against enzymes. The MIB team, primarily through the work of Sylwia Czarnota, had made a step-change in the expression, purification, X-ray crystallography and NMR spectroscopy of catechol O-methyltransferase, which made this partnership especially appealing to C4X Discovery. The project involved developing robust protocols for the expression, purification, high-resolution X-ray and NMR investigation of protein-ligand complexes that are pertinent to examining the breadth of strategies through which methyltransferases may be targeted successfully. The primary achievements are the publication of all of this work in a high impact journal (Czarnota et al., ACS Catal. 2019 9 4394-4401) and the deposition of two X-ray crystal structures of catechol O-methyltransferase in complex with inhibitors bound (S-COMT:SAM:DNC and S-COMT:sinefungin:DNC with the accession codes 6I3C and 6I3D, respectively) in the protein data bank and the corresponding backbone 1H, 13C and 15N NMR chemical shift assignments in the BioMagRes-Bank (under the BMRB accession codes 26848 and 26851, respectively). The original objectives, listed below, focussed on knowledge transfer between MIB researchers and C4X Discovery and were achieved in full. Specifically, they were: 1. Knowledge transfer of state-of-the-art approaches in structural biology, particularly in X-ray crystallography and NMR spectroscopy, and in enzymology, particularly in novel approaches to measuring the turnover of methyltransferases with high reproducibility. 2. Expertise in COMT sample handling. 3. Access and exposure to MIB facilities, include the specialist capabilities in NMR spectroscopy, X-ray crystallography, enzyme kinetics, protein purification and expression, with a possible expansion to EPR spectroscopy and computational approaches. 4. Development of a longer-term partnership with researchers at the MIB.
Collaborator Contribution as above
Impact The primary benefits and outputs are: Publication of results in Czarnota et al., ACS Catal. 2019 9 4394-4401 Deposition of two X-ray crystal structures of catechol O-methyltransferase in complex with inhibitors bound (S-COMT:SAM:DNC and S-COMT:sinefungin:DNC with the accession codes 6I3C and 6I3D, respectively) in the protein data bank. IAA Award: Developing Bud23 as a feasible therapeutic target. BBSRC IAA106 KTP Award: 2019-22 Protein:Ligand Complex Dynamics by Solution NMR: Application to Rational Drug Design for Challenging Protein Targets. InnovateUK 1025484 Deposition of two backbone 1H, 13C and 15N NMR chemical shift assignments in the BioMagRes-Bank (under the BMRB accession codes 26848 and 26851, respectively). The key benefit accrued by the company partner through this project has been knowledge transfer from the expert academic group. Approaches to the biochemical characterisation and structural biology of methytransferases as a target class will result in improved success in prosecution of such targets for drug discovery. This constitutes an important area for C4X Discovery as a business and for the wider industry. Secondment of C4X staff to MIB has provided access to MIB facilities. In [particular experitise has been gained in protein expression and purification which capability will now be embedded in the company and add value across the discovery portfolio. The project established a relationship between C4X and the group at the University of Manchester. This relationship has blossomed and will proceed having secured additional funding in the form of an InnovateUK KTP award.
Start Year 2018
 
Description Collaborative effort to develop synthetic genomics technologies for industrial biotech 
Organisation Beijing Genomics Institute
Country China 
Sector Academic/University 
PI Contribution The synthetic biology market is driven by the need for affordable, sustainable and high-value products such as pharmaceuticals, fuels and other consumer products for the growing global population. Most of these products were originally derived from natural sources, or produced through synthetic organic chemistry approaches, but the latter are limited by scalability, profitability and accessibility. Cai lab has been pioneering the design and synthesis of synthetic genomes (the Cai lab has 7 papers in Science around synthetic chromosomes in 2017), and we are now looking to deploy a genome-scale inducible rearrangement technology (termed SCRaMbLE) to producing high-value compounds, in collaboration with a world leading genomics company BGI and China National GeneBank. The base of this collaboration was initially funded by a BBSRC grant (BB/M005690/1) which supports the construction of synthetic chromosomes, and now it is taken further by another BBSRC IPA grant (BB/P02114X/1) to scale up the construction of synthetic chromosomes with lab automation. This BBSRC IFF grant will enable us to translate our academic excellence to tangible industrial usages of synthetic genomes and open up new exciting opportunities for the future. There will be extensive knowledge and skill exchange during Yun's visit to Manchester. UoM offers the expertise in lab automation, synthetic genomics and nanopore sequencing, while BGI will provide expertise in next-gen sequencing and trans-omics analysis. Both are world leading and complementary to each other. To take this joint effort further, BGI has committed an in-kind contribution of ?100K and will support future applications such as a BBSRC IPA or a LINK award led by Prof. Cai. Marco Monti and Dr. Daniel Schindler previously established nanopore sequencing and data analysis using existing tools in Manchester. Until now 7 sequencing experiments have been conducted which solved in total 72 genomes. Dr. Yun Wang visited the laboratory at the Manchester Institute of Biotechnology for three months. During this time he worked close with Marco Monti and Dr. Daniel Schindler, established a sequencing analysis pipeline (NANOPIPE) to combine long and short read sequencing data to solve SCRaMbLEd genomes. The established pipeline was challenged using low coverage nanopore sequencing data of highly complex SCRaMbLEd strains which were unable to be solved with conventional short read sequencing techniques. The pipeline managed to reduce the number of solutions significantly in one case to only a single solution: 1,732,332 to 8,311 solutions in strain MMy005 (coverage 12.7-fold) 48 to 12 solutions in strain MMy006 (coverage 18.3-fold) 160 to 1 solution in strain MMy007 (coverage 31.4-fold) Dr. Daniel Schindler travelled to the 7th International Yeast 2.0 and Synthetic Genomes Conference in Sydney, Australia and to the GASBII Meeting in Berlin, Germany to present and discuss data. In addition, he visited BGI-Shenzhen to coordinate the project and strengthen the collaboration. Marco Monti graduated his master's, which was integrated into this project, with honours.
Collaborator Contribution As above
Impact Nanopore sequencing coupled to the NANOPIPE software allows now to solve complex rearranged genomes in a fast and high throughput manner. 12 yeast strains can be analysed at the same time which drops the price for a single genome based on long reads to less than £ 100 incl. all necessary consumables. Notably, starting from 12 individual yeast strains to solving their respective genomes takes only 2 weeks. Genomic Engineering and large-scale DNA assembly are the expertise of the Cai Lab and highly benefit from this project in terms of an easy to handle and cheap established in-house genome sequencing pipeline. In addition, a highly motivated and outstanding PhD student, Marco Monti, could be acquired by the Cai Lab through this project. BGI-Shenzhen has gained knowledge in application and dealing with long read sequencing data based on the nanopore sequencing technology. The combination of long and short read sequencing data is highly interesting in an application based manner, many diagnostic relevant sequences, especially in highly repetitive genome regions, can not be analysed by standard short read sequencing techniques. BGIShenzhen has a strong commercial interest in diagnostics this is one of the reasons of their commitment in long read sequencing applications. Many synergies between BGI-Shenzhen and the laboratory of Prof. Patrick Yizhi Cai were encountered which strengthen current collaborations and will lead to further collaborations in future to bundle forces within big questions in Synthetic Genomics
Start Year 2018
 
Description Developing a commercial pipeline for high value recombinant proteins (Anil Day) 
Organisation Protein Technologies Ltd
Country United Kingdom 
Sector Private 
PI Contribution The University has developed a novel high-yield and low-cost production platform to manufacture recombinant proteins for industrial biotechnology. The platform utilizes plant cells rather than traditional animal cells, eliminating the risk of contamination with human pathogens and is driven by photosynthesis making it sustainable. High-value proteins with applications ranging from tissue engineering, stem cell and inflammation research to combatting antibiotic-resistant bacteria will be expressed in plant cells. The harvested raw material will be converted into commercial products using the specialist purification and processing knowledge available at Protein Technologies Ltd. The project supports the two-way exchange of early career postdoctoral researchers (ECRs) between the University and Protein Technologies Ltd. This allows the academic researcher to learn the downstream processing steps in an industrial lab and the industrial researcher to work on the plant system in academia. The business knowledge of the company will identify the best route to commercialization.  The project falls in the BBSRC priority areas of synthetic biology and industrial biotechnology. The innovation fellowship enables knowledge exchange between the University and Protein Technologies Ltd (PTL) to translate world leading recombinant protein expression technologies into commercial products. The project focuses on manufacturing in-demand recombinant proteins used in R&D that are extremely expensive (£4,000 to £10,000 per mg) and a phage bacteriolytic protein to combat antibiotic-resistant bacteria, that would be too expensive to manufacture by traditional fermentation technologies. Through past BBSRC support, we have demonstrated that we can attain commercial yields: over 2g of recombinant TGFß3 protein per kg of fresh material (the highest achieved in plants) at a low cost (£100 per g of recombinant protein before purification). In the first phase, we will manufacture five TGFß superfamily members: bone morphogenetic protein 2 (BMP2), BMP14, transforming growth factor ß1 (TGFß1), TGFß2, TGFß3 and the bacteriolytic protein (modified cpl lysin). A rate-limiting step is the conversion of our raw plant material into commercial products. This requires the downstream processing expertise and the business knowledge provided by PTL. Specifically, the University will provide the company with raw material, knowledge and training in isolating and handling user-designed plants expressing recombinant proteins and the company will provide training and knowledge of the downstream purification/formulation steps and business knowledge needed to develop commercial products. This collaborative work programme moves the project from technology readiness level 4 (TRL4) to TRL5/6 ready for operation at the industrial scale.  
Collaborator Contribution As above
Impact The main events and achievements of the IFF project relate to the four work packages: (WP1) TGFß3-expressing plants were grown in a controlled-environment lab scale chamber (total area ~ 2 m2) at a lower temperature (23°C) density 20 plants per m2 and 16-hr light and 8-hr dark cycles. Under these conditions, plants develop bigger leaves and hence the leaf biomass that could be harvested from our chamber is higher. The average annual leaf biomass is estimated to be 15 kg per m2. We have previously reported that TGFß3-expressing plants can produce around 2 g of the recombinant protein from 1 kg of leaf material (Gisby et al., 2011). In the published article, however, only 43.75 mg of the 2 g of TGFß3 could be recovered from a crude preparation of plant chloroplasts (i.e. <3% recovery). We have optimised the recombinant protein isolation protocol and we can now recover around 500 mg of the denatured and reduced recombinant protein from 1 kg of processed leaf material (i.e. 25% recovery) at 80% purity as determined by Coomassie-stained SDS-PAGE. The retail price of denatured human TGFß3 produced in Escherichia coli is $1440 per 1 mg of the protein (ATGen, USA). In comparison, it costs us less than £500 to make a gram of the denatured protein. Also related to this WP, we have designed codon-optimised genes using our proprietary method for four new members of the TGFß3 superfamily (TGFß1, TGFß2, BMP2 and BMP14) and a phage lysin. Chloroplast transformation vectors with the synthetic genes were successfully assembled and delivered into the chloroplast of our proprietary ?rbcL tobacco plants using a biolistic bombardment device with 1,100 and 650 PSI rupture disks. A total of 20 resistant shoots emerged from spectinomycin-bleached leaf explants and propagated at least once on fresh selective regeneration media before moving shoots to a Magenta™ jar for root formation. Plants expressing TGFß1 to >2g/kg leaf biomass were isolated. The other resistant shoots were escapes and the transformation experiments were repeated. (WP2) A total of sixty combinations of [salt], [buffer], oxidising agent: reducing agent ratio and [protein] were tested for refolding and dimerisation of the (1) urea-denatured and (2) Guanidine hydrochloride denatured and solubilised TGFß3. Rapid dilution of the denatured protein in the refolding buffer (final volume 2 mL) helped identify the best combination to enable refolding. Using this method, the correctly folded TGFß3 dimer accounted, at day 15, Developing a commercial pipeline for high-value recombinant proteins 07/05/2018 25/06/2019 30/06/2019 Protein Technologies Ltd Anil Day to 30% of total proteins in sample which include the TGFß3 oligomeric aggregates, denatured TGFß3 monomer and the renatured TGFß3 monomer (two conformations). When upscaling production of TGFß3 to mg quantities, rapid dilution failed to refold and dimerise the protein. A faster and more efficient protocol was devised whereby the concentration of the denaturant and reducing agent in the protein sample is gradually decreased by diafiltration with five volumes of the refolding buffer using a labscale tangential flow filtration system (TFF; company equipment). Refolding using this method could be achieved within hours but leaving the solution mixing at room temperature (21°C) for another three days helps make more TGFß3 dimers. Successful refolding using the TFF system was also achieved using cheaper brands of reagents which is essential to reduce costs of manufacturing. Purification of TGFß3 was by ion exchange chromatography using the AKTA start system (company equipment). Strong cation (SP FF and SP HP), weak cation (CM FF), strong anion (Q FF) and weak anion (DEAE FF) columns were tested for optimal purification of the refolded TGFß3 dimer. Highest purity (>95%) and best recovery (~60%) was achieved on a strong cation column (SP HP). The purified TGFß3 was stored in 20% ethanol (formulation buffer optimisation using the UNCLE machine owned by the Biomolecular Analysis Facility at FBMH) and tested for bioactivity in the lab of Mark Travis (Catherine Smedley) using a luciferase-based cell assay. The plant-made TGFß3 dimer was functional and followed the same pattern as a commercial TGFß3. Stepby-step instructions for protein extraction and purification have been compiled in a standard operating procedure (SOP). (WP3) The IP landscape was evaluated and involved searching databases for prior art and relevant patents. We found many granted patents that had lapsed which would provide us with the freedom to operate. Additionally, we have found that our method is novel. The cost of the prototype process was estimated to be £100 per 1 mg of purified TGFß3. We have also identified within FBMH, with the help of Dr Bruce Humphrey, the Strategic Funding Manager at FBMH, several research groups interested in a sustainable and cheap source of TGFß proteins. The low-cost production pipeline and the need/demand for these proteins make this research project commercially feasible which led to invention disclosure to UMIP and routes to commercialisation were discussed including protecting IP by patent filing or as a trade secret, patent ownership and forming a company spin-out (WP4) We were awarded the BBSRC Impact Acceleration Account - Push-to-IP and drafted an application for the BBSRC Follow-on-Funding Pathfinder call. These are required to increase the portfolio of TGFß-related proteins which was advice from UMIP to help us build a strong business case for a BBSRC Followon-Funding application and Innovate UK grant application. By completing this work, MEH has engaged in almost all the upstream and downstream processes required to produce recombinant proteins, a day-to-day task in biotechnology companies. This hands-on research experience should place MEH in a strong position to compete for strong follow-on employment opportunities. This includes employment in follow-on projects in the industrial expansion phase of this project. The secondment to PTL and the negotiations/ meetings with UMIP, allowed MEH to gain insight into the commercial sector and become aware of the milestones and problems when translating academic research and intellectual property to start-up companies. MEH has also built a network of business contacts from attending the Catapult Ventures event at UMIP and the BioProNet meeting. This network will help us locate potential UK and overseas investors that would provide funding for the industrial phase. The PI gained the track record of industrial research with PTL. This will strengthen collaborative industry led grant application to Innovate UK. Establishing a low-cost manufacturing pipeline for recombinant TGFß proteins will support research at the UoM in areas of tissue engineering, stem cell and regenerative medicine. This will support Manchester research at the forefront of the latest innovations in regenerative and healing therapies. As mentioned above, two collaborations emerged from meeting with FBMH members who will use TGFß proteins to make high-value biotherapeutics. Protein Technologies Ltd (PTL), as a CRO, provides services to their clients namely in the expression and purification field (upstream and downstream processing). The secondment of LR to our lab has provided PTL with very insightful information relating to tobacco expression, a system not currently in their portfolio of services. Consequently, through this collaboration we have new opportunities to work with PTL to provide our expression methods as a production platform. Moreover, and importantly PTL has provided invaluable downstream processing expertise and knowledge that has resulted in producing a functional TGFß3. PTL and MEH have drafted a SOP for the production of TGFß3. This SOP will be tested for scaling up to assess commercial feasibility.
Start Year 2018