Metals in Biology: The elements of Biotechnology and Bioenergy

Lead Research Organisation: Durham University
Department Name: Biosciences

Abstract

The purpose of this network is to make the UK the premier destination for research and development that demands Metals in Biology expertise. Metals catalyse the reactions of life and these demands are expected to grow.

Some of the earliest discoveries, uncovering metals as vital components of proteins and not mere contaminants, were made in the UK. Today we have world leaders uncovering how cells handle metals and the mechanisms of metal-dependent enzymes. Now is an opportune moment to exploit this knowledge to the benefit of industry and society. UK businesses which will innovate with this knowledge include established world leaders in their sectors and new entrants.

Metallo-proteins contribute to bio-energy production, bioremediation, biomedicine, synthesis of high value industrial feed-stocks and more. The metal-handling systems of cells are vital to sustain adequate metal-protein speciation in vivo. As nearly a half of enzymes, perhaps one third of all gene products, are estimated to require metals, the impact of this network will be pervasive.

Metal-handling circuits can also be subverted to inhibit microbes. The exploitation of metals and metal-chelators as antimicrobials is a form of bio-mimicry since we are now starting to discover that immune systems manipulate metal-supply to inhibit microbial growth. Indeed, many common antibiotics tightly bind to metals and some antibiotics are closely related to iron-scavenging molecules (siderophores).

Metal-supply pathways can be engineered for the manufacture of supplements such as cobalt-requiring vitamin B12 and enzymes with metallo-prosthetic groups. Talented individuals are currently scattered in different UK research communities. The efforts of biologists, chemists, mathematicians, engineers and others will be drawn together by this network to allow effective knowledge and skills transfer with industry and with the relevant regulatory authorities.

The network will run for three years with structured workshops, a large community building meeting and additional ad hoc workshops throughout the tenure of the network.

Technical Summary

The prevalence of metallo-enzymes means that success in synthetic biology may pivot upon an ability to engineer metal-supply inside microorganisms, plants and animal cells. For example, the sustainable manufacture of isobutanol has required the engineering of cellular iron-circuits: Aft1 and Aft2 levels were adjusted to activate genes for iron uptake to provide sufficient metal-cofactor for an introduced dihydroxyacid dehydratase (DHAD) enzyme.

The abundance of each metal is controlled inside cells by sensors (such as Aft1 and Aft2) that regulate metal import, metal export, metal trafficking and metal storage systems, they also switch metabolism to take advantage of more available metals and to minimise demand for those in deficiency. Metals which form tight complexes with proteins are held at low abundance while weaker binding metals are maintained at concentrations many millions of times higher. Under these controlled conditions the metallo-enzymes acquire the correct metals.

Network members will work with the bio-processing sector to optimise metal availability inside cells to give consistent biologic quality and yield. They will develop antimicrobial-chelants, ionophores and metal nano-particles that subvert the metal-handling systems of micro-organisms, they will explore the impact of microbes on the mobility of metal contaminants in the environment and engineer metal bio-accumulation and bio-detection. Members will collaborate with multiple companies to engineer synthetic metallo-enzymes and will optimise metal uptake and assimilation into biomolecules required for bio-energy production, bioremediation, biomedicine and synthesis of high value industrial feed-stocks.

This network will consolidate the activities of communities working on Metals in Biology and speed the exploitation of those aspects of their research relevant to industrial biotechnology and bioenergy.

Planned Impact

The network will have diverse impacts due to the pervasive nature of metals in biology (refer to case for support). Contributions to Industrial Biotechnology and Bioenergy can be categorised under the following headings:

Metals in bio-processing
Metal-related antimicrobials
Metal circuits for synthetic biology, bio-energy and industrial biotechnology
Metals in the environment
Metal-related nutrition and supplements
Metallo-enzyme engineering for bio-energy and industrial biotechnology
Tools and technologies for metals in biology

A purpose of this network is to promote interactions between the Metals in Biology research community and non-academic beneficiaries.

We already have over 100 members and yet consider that a major role for the network will be to disseminate awareness of the significance of the sub-discipline and the opportunities that it presents, most especially outside the confines of academia. We intend to grow these numbers especially from industry. The names on the members' lists provide an early indication of some of the many beneficiaries, in academia and also within industry, regulatory and advisory bodies. Most of these individuals approached the network to request membership and all have been re-contacted in the closing week before submision of this proposal to ensure that they wish to be on these lists (this efficiency has avoided innumerable letters of support).

Throughout the term of the award, and in response to enquiries for expertise (from BBSRC, industry, government, outreach organisations and others) the network manager will liaise with the membership to source the best individuals to provide advice. The PI and CoI are actively involved in outreach activities including the generation of press releases, and the CoI has written popular science books and contributed to BBC TV series. Throughout the network such best practice will be disseminated within this community.

The network manager and clerical assistant will deliver communications and promote the network, support members, maintain databases, gather information, assemble reports and brochures throughout the term of the award (see work-plan). The network manager is already part of a business engagement team so will bring contacts and sector knowledge into this role.

The research emanating from funded projects will be published in open access high impact journals and oral communications given at international conferences (after adequate protection of IP where needed). The track records of the PI, CoI and management team, attest to their commitment to such forms of impact activities.

Knowledge and skills acquired as a result of network activities (by PI's and also by RAs linked to funded projects) will be applicable to a broad swathe of academia and industry.

Crucially, the intellectual property (IP) resulting from the projects associated with the network will be protected via company/institutional innovation and enterprise offices. The network will foster rapid routes to exploitation of such IP.

Publications

10 25 50
 
Description Refer to BBSRC extranet for details.

Network statistics:
£11.6 million of additional linked funding has been won by BBSRC NIBB members
43 collaborative projects have been initiated/funded with Industry and Academic partners (79% new collaborations and >93% keen to advance to higher TRL's)
Case studies have been generated for all projects and are available on the web linked to each collaboration.
Proof of concept awards (and seeding catalyst awards):
5 rounds (28% success rate)
Other Network Activities:
Overall tally 136 activities: 79 events, 53 industry engagement, 2 public engagement, 2 media interaction (exemplars have been added to Researchfish and more details can be obtained from the BBSRC Extranet). 840 participants have attended these events. An estimated 86 jobs have been created or safeguarded.
Refunding of phase II BBSRC NIBB for 5 years.
Exploitation Route Refer to BBSRC extranet plus the collaborations/partnerships and industry engagement items associated with this award and listed on Researchfish.
Sectors Agriculture, Food and Drink,Chemicals,Energy,Environment,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

URL http://community.dur.ac.uk/MiB_NIBB/outcomes-of-completed-biv-projects-poc-projects-case-studies-and-articles/
 
Description This award funds a National Network in Industrial Biotechnology and Bioenergy which is working to promote the exploitation of Metals in Biology research. The (many) impacts are currently being cataloged on a BBSRC extranet database. By the end of 2017 the network had supported 136 activities with academic and industrial participants, including events, other types of specific engagements with industry, public lectures, media interactions and has had 539 members (with about one third from business/non-academic organizations). The network has funded 43 projects in the form of proof of concept funds, seeding catalyst awards and business interaction vouchers, all of which involve industry and academic partnerships. Several of the above projects have already come to fruition providing underpinning data enabling the launch of products on the open market and large scale follow-up funding in support of end users. The total additional funding awarded to network members exceeds £11 million and there has been both IP protected and products launched (not itemised on Reseachfish but listed on the BBSRC Extranet).
First Year Of Impact 2015
Sector Agriculture, Food and Drink,Chemicals,Energy,Environment,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
Impact Types Economic

 
Description Advice to BBSRC regarding biomanufacturing of higher value chemicals such as speciality, performance/effect, or fine chemicals
Geographic Reach National 
Policy Influence Type Implementation circular/rapid advice/letter to e.g. Ministry of Health
 
Description Advice to BBSRC regarding the removal of metals from e-wastes and other sources
Geographic Reach National 
Policy Influence Type Implementation circular/rapid advice/letter to e.g. Ministry of Health
 
Description Advice to the BBSRC regarding bio-based approaches to precious metal recovery from waste for sustainable manufacturing.
Geographic Reach Multiple continents/international 
Policy Influence Type Implementation circular/rapid advice/letter to e.g. Ministry of Health
 
Description BBSRC NIBB phase II
Amount £1,360,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 03/2019 
End 02/2024
 
Description JN Funding
Amount £44,000 (GBP)
Organisation Procter & Gamble 
Sector Private
Country United States
Start 10/2017 
End 09/2021
 
Title Calculating in vivo metalation from the sensitivities of metal-sensors. 
Description Equations, software and spreadsheets to calculate the sensitivities of metal sensors and in turn to determine metal availability inside a cell. This enables the calculation of metalation inside living cells with implications for engineering a half of the reactions of life. Includes: 1. Excel Spreadsheet (with instructions) to enable calculation of fractional DNA occupancy. 2. MATLAB codes (with instructions), to determine the buffered metal concentration from given value(s) of ?D or ?DM. 3. Supplementary equations and unique Supplementary Note 2 references in support of the above. 
Type Of Material Technology assay or reagent 
Year Produced 2019 
Provided To Others? Yes  
Impact Too early 
URL https://www.nature.com/articles/s41589-018-0211-4.pdf
 
Title Rhodococcus molecular toolkit 
Description We organised an event (in response to a community request) which led to the development of a Rhodococcus molecular toolkit: http://prospect.rsc.org/MiB_NIBB/wp-content/uploads/2017/01/Workshops-kickstart-Rhodococcus-molecular-toolkit.pdf 
Type Of Material Improvements to research infrastructure 
Year Produced 2015 
Provided To Others? Yes  
Impact As a result of the workshops, the community-­building that researchers hoped to achieve has indeed worked and they are now starting to construct a shared resource for molecular biology tools in Rhodococcus. "This has been a brilliant result". 
URL http://prospect.rsc.org/MiB_NIBB/wp-content/uploads/2017/01/Workshops-kickstart-Rhodococcus-molecula...
 
Description A pilot study to characterise plant-derived compounds that promote the synthesis of copper nanoparticles from contaminating copper ions in waste water 
Organisation Northumbrian Water
Country United Kingdom 
Sector Private 
PI Contribution Contamination of land and waterways by toxic metals is a serious environmental problem particularly in areas of the UK where mineral mining was once widespread. However, if the polluting metal can be sequestered into bioactive metal nanoparticles (NPs) then these NPs have important commercial values e.g. Copper NPs have diverse uses ranging from industrial catalysts to antimicrobials in food packaging. Previous work has established that crude plant extracts when mixed with solutions of metal ions extracts stimulate the synthesis of metal nanoparticles. The aim of this project is to determine the identity of the bio-active molecules within the plant extracts that are required for Copper NPs synthesis and their mode of action. The results from this pilot study will inform future use of plants, plant cell cultures or specific plant-produced compounds to remove contaminating copper, and other trace metal ions from waste water in order to synthesize commercially valuable metal nanoparticles for further exploitation in a specific collaboration with our industrial partners.
Collaborator Contribution We funded a Business Interaction Voucher in collaboration with Keith Lindsey of Durham University (BIVMiB040)
Impact Project on-going
Start Year 2017
 
Description Adding Value to Biocatalytic Hydroxylation Products for Synthesis and Drug Discovery 
Organisation Oxford Biotrans Ltd.
Country United Kingdom 
Sector Private 
PI Contribution We funded and administered a Seeding Catalyst Proof of Concept Award in collaboration with Jeremy Robertson of University of Oxford (ISCFPOCMiB035)
Collaborator Contribution This project exploits the special chemical properties of a set of complex biological catalysts (enzymes) that contain iron. The enzymes will be produced in growing engineered E. coli cells, and the iron centre, when bound to oxygen from the air, is uniquely able to introduce a polar 'handle' onto organic molecules. We intend to use these iron-containing enzymes to produce compounds of value to the pharmaceutical industry whose drug discovery campaigns usually begin by screening compound collections for 'hits' or, in fragment-based approaches, the features of weakly-active 'fragment' compounds are combined to identify promising 'leads'. This depends crucially on access to small organic molecules, whose nature dictates the trajectory of the whole drug discovery campaign. The highest chance of identifying leads that develop successfully into successful drugs arises when the initial compound/fragment collection is structurally diverse; therefore, we aim to diversify compound collections in a two-stage process that mimics the biosynthesis of known medicines such as the anti-cancer drug taxol. Stage one is the above-mentioned enzymatic introduction of the 'handle'; in stage two, the chemical properties of this handle will be exploited to introduce features that promote favourable interactions with drug targets.
Impact Project on-going
Start Year 2017
 
Description Adding Value to Galactomannan Polysaccharides with Cu Enzymes 
Organisation Schlumberger Limited
Department Schlumberger Cambridge Research
Country United Kingdom 
Sector Academic/University 
PI Contribution We funded and administered a Business Interaction Voucher in a collaboration with Julea Butt of University of East Anglia (BIVMiB009)
Collaborator Contribution Concerns over fuel security are frequent news headlines and the rising costs of fuel are a daily reminder of the challenges faced by a global society with ever increasing energy demands. Medium- to long-term solutions to these challenges will require effective access to renewables alongside the development of infra-structures that enable such energy to be delivered to the point of need with the ease that is presently enjoyed with our use of fossil fuels. Improved access to the oil and gas reserves that are found in shale present an attractive option for the short- to medium-term. Here we aim to investigate opportunities to improve the recoveries of shale oil/gas through the use of copper-containing enzymes that can modify the properties of a natural biopolymer, guar gum, that plays a key role in the shale extraction process.
Impact Galactomannans are naturally occurring polymers with high solubility in water. The resulting solutions have high viscosity at relatively low polymer concentration such that they are widely used as industrial colloids. Galactomannans find application as thickening agents in pharmaceutics, cosmetics, food products and water-based fracturing fluids. However, the properties of galactomannans can be augmented by chemical modification. The traditional routes to modification require protecting groups and the use of non-aqueous solvents. However, enzymatic modification offers prospects of more sustainable routes to modified galactomannans. The galactomannan polymer is comprised of covalently linked sugar molecules, namely, mannose and galactose. Polymer hydrolysis resulting in chain shortening was demonstrated using a commercially available enzyme cellulase. Oxidation of the galactose sidechains within the polymer in aqueous solution was demonstrated using a commercially available enzyme, galactose oxidase. In addition, electrochemical oxidation of the galactose and mannose sugars was demonstrated by oxidation of aqueous guar suspensions at a graphite electrode. The results demonstrate that commercially available enzymes offer routes to controlled modification of guar in aqueous solution and this offers prospects for the development of more sustainable routes to industrial scale galactomannan modification.
Start Year 2015
 
Description Analysis of ferritin iron in pea flour 
Organisation Agritopics Ltd
Country United Kingdom 
Sector Private 
PI Contribution We funded and administered a Business Interaction Voucher in a collaboration with Janneke Balk, John Innes Centre (BIVMiB015)
Collaborator Contribution Peas provide a rich source of proteins and nutrients for human diets. They also contain relatively high levels of iron in a special form which is very easy for the body to absorb. To extract this high value component or use pea flour directly in food products, the first step is to mill the dry peas. Together with the industrial partner AgriTopics, we will evaluate different milling procedures, milling fractions and particle sizes to optimize this first step for producing nutritional flour and iron extraction. The project will provide the basis for the development of new iron supplements, which could also be used clinically to treat iron deficiency anaemia, and specialist flours for the food industry. Our application fits within the remit of the Metals in Biology BBSRC NIBB as it investigates the industrial processes for refining peas to produce a bio-available iron nanoparticle for the health (and food) industries.
Impact Peas are rich in nutrients and protein, but highly undervalued as a healthy part of our diet. Janneke Balk's laboratory has developed a method to extract ferritin, an iron-rich protein nanoparticle, from dried peas. It has further shown in collaboration with Prof Susan Fairweather-Tait (UEA Med School) that purified pea ferritin has excellent iron bio-availability in cultured intestinal cells. AgriTopics is a specialist agribusiness developing high value products from processed pea flours for the "free-from" (gluten-free) and health food markets. In this project we tested the effect of different milling techniques on the extraction of ferritin. We also investigated the nutritional profile of pea flour before and after ferritin extraction. We found that a specialised milling process, developed and optimised by the Industry partner, is as good, or even better than a range of alternative milling processes for the extraction of ferritin. The extraction procedure removes sugars from the pea flour, but these are only 3 g / 100 g to start with. Fatty acids, protein and micronutrients remain in sufficient quantities for the flour to be used for food products.
Start Year 2015
 
Description Arginine terminated LPMOs-a new face in biomass breakdown? Follow-on funding 
Organisation Novozymes
Country Denmark 
Sector Public 
PI Contribution We funded and administered a Seeding Catalyst Proof of Concept Award with Paul Walton of University of York (ISCFPOCMiB038)
Collaborator Contribution The generation of fuels/commodity chemicals from sustainable biomass hinges on a single key issue. This issue is that biomass (e.g. wood, plant matter) is very hard indeed to break down in a controlled manner, severely hindering its sustainable conversion into useful materials such as bioethanol and other chemicals. It is an issue which has bedevilled the bio-based industry. However, in a major breakthrough in 2010/11 we discovered copper-containing lytic polysaccharide monooxygenases (LPMOs) which are natural enzymes that can break down cellulose (a plant based polysaccharide) in a highly efficient manner. LPMOs are now used in biorefineries to generate bioethanol and have transformed the industry. In this project, we seek to maximise the ability of a new type of LPMO to break down woody biomass. If the ability of this enzyme to convert lignin could be harnessed then it would be a significant addition to the biomass industry.
Impact Project on-going
Start Year 2017
 
Description Arginine-terminated LPMOs: a new face in biomass breakdown? 
Organisation Novozymes
Country Denmark 
Sector Public 
PI Contribution We funded and administered a Proof of Concept Award in collaboration with Paul Walton of University of York (PoCMiB028)
Collaborator Contribution The efficient conversion of abundant biomass into liquid biofuel is of vital importance in meeting the world's energy demands. Until recently, however, and despite its unrivalled calorific potential it has not been possible to take abundant biomass, which is composed mostly of lignocellulose, and industrially convert it through to bioethanol. The reason for this is the chemical recalcitrance of the cellulosic biomass. Of the available methods, the use of enzymes to perform the breakdown looks promising, especially enzymes called, lytic polysaccharide monooxygenases (LPMOs). LPMOs have overturned our understanding of biomass conversion as they boost significantly the conversion of biomass to ethanol. This particular project aims to study a whole new exciting class of metal- containing LPMOs which do not contain the usual active site amino acids (histidine), thereby offering new insight into how biology performs the conversion of biomass, and-consequently- humankind's ability to use biomass as a sustainable fuel source.
Impact The efficient conversion of abundant biomass into liquid biofuel is of vital importance in meeting the world's energy demands. Until recently, however, and despite its unrivalled calorific potential it has not been possible to take abundant biomass, which is composed mostly of lignocellulose, and industrially convert it through to bioethanol. The reason for this is the chemical recalcitrance of the cellulosic biomass. Of the available methods, the use of enzymes to perform the breakdown looks promising, especially enzymes called, lytic polysaccharide monooxygenases (LPMOs). LPMOs have overturned our understanding of biomass conversion as they boost significantly the conversion of biomass to ethanol. This particular project aims to study a whole new exciting class of metal- containing LPMOs which do not contain the usual active site amino acids (histidine), thereby offering new insight into how biology performs the conversion of biomass, and-consequently- humankind's ability to use biomass as a sustainable fuel source.
Start Year 2017
 
Description Assessing the bioavailability of metal ions accumulated by DRAM® filters 
Organisation Epona Technologies Ltd
Country United Kingdom 
Sector Private 
PI Contribution We funded and administered a Business Interaction Voucher in a collaboration with Louise Horsfall of University of Edinburgh (BIVMiB011)
Collaborator Contribution The environmental quality standard for copper in groundwater in the UK is set at 1 - 28 µg/l, due to its toxic nature, but copper compounds are one of the few pesticides allowed for organic food production. They are also used to control fungal diseases in vineyards and coffee plantations; as molluscicides and insecticides; as a disinfectant in the farming and fishery industries and to prevent algal blooms. Whilst the spirits industry produce whisky, vodka and gin in a process which involves a universal step of distillation in copper pot stills, producing waste contaminated with solvated copper ions. Consequently this has led to increasing concern over environmental copper levels, their toxicity and the adverse effects on humans and wildlife. Epona Technologies Ltd has developed DRAM® filters, which are able to accumulate polluting copper ions from industry and agriculture but, without steps to allow its reuse, the problem is just being transferred elsewhere. With the finite supply of copper and its ever increasing cost, employing bacteria for the recycling of such contaminants to allow their reuse may provide a long-term sustainable solution. The Horsfall Lab is currently examining the process of copper nanoparticle production by bacteria. We have already determined that Morganella sp. can transform copper ions from whisky waste into nanoparticles of solid, zero-valent copper in addition to the reported biotransformation of model solutions and we would like to determine whether the copper ions accumulated by DRAM® filters could be transformed into copper nanoparticles too. This would be dependent upon the bioavailability of the copper ions accumulated and whether the conditions of bioavailability coincide with the conditions under which transformation occurs. It would also be of significant importance and impact if the nanoparticles were retained by the DRAM® filters, allowing for a separate elution step.
Impact This project assessed the potential to recover copper(II) from DRAM® media filters and its bioconversion to metallic nanoparticles harnessing the ability of M.psychrotolerans to reduce Cu(II) to Cu(0). Using a CuSO4 solution, copper(II) was accumulated on these filters through filtration or stirred incubation. Both methods resulted in 1g of DRAM® capturing about 2.4 mg Cu(II). Approximately 20% of this bound Cu(II) can be desorbed into the surrounding medium and made available for biotransformation into copper(0) nanoparticles. These particles were visualised and positively identified by electron microscopy. Thus M.psychrotolerans can form nanoparticles from copper(II) accumulated on DRAM® filters. As DRAM is a biological material non-uniform in size its presence was a significant obstacle in the positive identification of metal nanoparticles. To facilitate electron microscopy visualisation of nanoparticles in this study DRAM® media was isolated from the culture medium containing M.psychrotolerans cells using a copper-permeable membrane. It can however be reasonably assumed that reduction of copper(II) can take place when bacterial cells are incubated together with DRAM® in the absence of a membrane. Moreover, a culture of M.psychrotolerans cells with a DRAM® media suspension might increase bioconversion through improved availability of copper(II). In this study, approximately 12% of Cu(II) ions are reduced by M.psychrotolerans to metallic Cu(0) nanoparticles after overnight incubation in LB medium containing 5 mM CuSO4. It is expected that the gradual release of copper from the DRAM® media may alleviate copper toxicity to live cells and thereby increase productivity. However, while the proof of principle study was successful with model solutions and controlled copper solutions, the industrially used DRAM® media with its unknown contaminants is currently beyond our methods of nanoparticle isolation and analysis, and therefore no nanoparticles could be identified from this source.
Start Year 2015
 
Description Bioaccumulation of platinum from waste 
Organisation Teegene Biotech Limited
Country United Kingdom 
Sector Private 
PI Contribution We funded and administered a Business Interaction Voucher in a collaboration with Helen Carney of Teesside University (BIVMiB031)
Collaborator Contribution Platinum is a scarce metal, being one of the least abundant elements in the earth's crust and as such has a high material value. This research will focus on the recovery of platinum from wastewaters where it is in its soluble, ionic form. Platinum is present in wastewater from a diverse range of sources such as metal refining, chemical industries and hospital waste where it can be found as a component of chemotherapy drugs. Bacteria have been found to take up and accumulate platinum using both active and passive methods, often referred to as biosorption and bioaccumulation. The proposed research, a collaboration between TeeGene, Teesside University, and University of York, will investigate the potential of bacteria to recover platinum from industrial wastewater with the aim of re-using the recovered metal. The project aims to identify a suitable bacterium to be used in a waste refining process and identify any physicochemical factors that influence platinum recovery.
Impact Project on-going
Start Year 2016
 
Description Biosynthesis of bimetallic nanoparticles for fine and speciality chemical production 
Organisation Johnson Matthey
Country United Kingdom 
Sector Private 
PI Contribution We funded a Seeding Catalyst Proof of Concept Award in a collaboration with Jon Lloyd of University of Manchester (ISCFPOCMiB041)
Collaborator Contribution This project combines biotechnologists from the University of Manchester and industrial catalysis experts at Johnson Matthey, a multinational speciality chemicals and sustainable technologies company. This "Proof of Concept" award will exploit the ability of microorganisms to produce bimetallic nanocatalysts for fine and speciality chemical production. Metal-reducing bacteria are able to recover a wide range of metals from process environments as catalytically active nanoparticles. By taking advantage of the unique biochemistry of metal-reducing bacteria, we will produce highly reactive and tunable metallic nanoparticles for speciality chemical production. These will include bimetallic nanoparticles that offer advantages over monometallic catalysts due to the combination of properties from the presence of two metals (rather than one) and from additional properties due to the synergy of the two metals, offering increased efficiency and specificity for speciality chemical production. This novel biotechnological process also offers a simple, cost-effective, environmentally friendly synthesis route for bimetallic catalyst production.
Impact Project on-going
Start Year 2017
 
Description Chelation Therapy in the Washing Machine 
Organisation Procter & Gamble
Country United States 
Sector Private 
PI Contribution We funded and administered a Business Interaction Voucher in a collaboration with Nick Jakubovics, Newcastle University (BIVMiB012)
Collaborator Contribution Biofilms are a major problem in all sorts of industrial settings, including bioprocessing facilities. Mechanical biofilm removal is not always possible due to the chemical and physical properties of the contaminated surface and the use of chemical agents is the most appropriate approach for biofilm control. This proposal involves working with an industrial partner (P&G) to develop novel chemical technologies for biofilm removal at low temperatures on a complex surface (laundry). We envisage that successful outcomes can be translated to biofilm control in many different settings including bioprocessing plants.
Impact Project on-going
Start Year 2016
 
Description Cloning and metal analysis of recombinant aldehyde ferredoxin 
Organisation ZuvaSyntha Ltd
Country United Kingdom 
Sector Academic/University 
PI Contribution We funded and administered a Business Interaction Voucher in a collaboration with Martin Warren, University of Kent (BIVMiB022)
Collaborator Contribution The aim of the project is to enhance the recombinant production of a key enzyme of biotechnological importance with a view to the production of metalcofactor complete protein. Specifically, the aim is to find optimal conditions that allow for overproduction of recombinant aldehyde ferredoxin oxidoreductase (AOR), an enzyme that allows the transformation of carboxylic acids into aldehydes. AOR has an unusual metal requirement in that it contains an oxotungsten centre bound to a pair of molybdopterin cofactors and a 4Fe4S cluster. We will clone two mesophilic forms of AOR from Clostridium ljungdahlii and another thermophilic form of the enzyme from Pyrococcus furiosus. The enzyme will be produced with a Histag to allow for easy purification. Moreover, the protein will also be produced with a tag to allow for the protein to be targeted to a bacterial microcompartment (BMC). BMCs are utilised by nature to help accommodate enzymes that produce aldehydes as pathway intermediates.
Impact Project on-going
Start Year 2016
 
Description Creating new starch active copper LPMOs through the generation of loop libraries 
Organisation WeissBioTech GmbH
PI Contribution The efficient deconstruction of plant biomass into biofuels and other chemicals is a key challenge to secure a low carbon economy. In nature, many microorganisms secrete enzymes that can break down recalcitrant biomass that is composed mostly of lignocellulose into soluble substrates. Harnessing the catalytic power of these enzymes to treat biomass outside of their natural habitats is challenging and a major goal of industrial biotechnology. Recently, a new class of enzyme that drastically increases the efficiency of biomass conversion has been identified. These enzymes contain a copper ion and are called lytic polysaccharide monooxygenases (LPMOs). The aim of this project is to assess whether second generation LPMOs with enhanced substrate activities can be created. As a proof of principle, we will use a starch degrading LPMO as a template to design and synthesize DNA libraries that will then be screened for substrate activity.
Collaborator Contribution We funded and administered a Business Interaction Voucher in collaboration with Jonathan Worrall, University of Essex (BIVMiB042)
Impact Copper containing lytic polysaccharide monooxygenases (LPMOs) greatly aid the deconstruction of plant biomass to value added products and the production of "renewable" bioenergy. The Business partner, WeissBioTech has a vested interest in using enzyme cocktails for the liquefaction and saccharification of starches. To improve the economic outputs of such processes the creation of second generation enzymes with better substrate interaction and more efficient substrate turnover is desirable. The long-term aim of this project is to create second generation starch active LPMOs that are more efficient through better substrate interaction and higher substrate turnover. To create second generation starch active LPMOs, loops that form the active surface surrounding the catalytic copper ion were targeted. In total, there are five active surface loops that may be considered important for interaction and specificity with starch. Active surface loop libraries have been designed in silico with saturating mutations in selected amino acid positions in each loop based on sequence variability within the starch active LPMO family. This Business Interaction Voucher has allowed for the synthesis, through combining Site Evaluation Library and Combinatorial Library technology, of two out of the five loop libraries in a starch active LPMO. By using an in vivo assay these two loop libraries will be screened for enhanced activity relative to the wild type LPMO.
Start Year 2017
 
Description Developing an ultra-compact integrated hyperspectral monolithic fluorescence biosensing system 
Organisation Zinir Ltd
Country United Kingdom 
Sector Private 
PI Contribution We funded and administered a Business Interaction Voucher in a collaboration with Po-Wah So of King's College London (BIVMiB007)
Collaborator Contribution We are using magnetic resonance imaging (MRI) to look into the distribution of iron in the brain without surgery or biopsy, to learn whether iron accelerates/promotes ageing or disease. However, changes in the MRI signal can also be due to changes in cell structure and/or biological molecules in the brain. We aim to determine the specifications for a new ultra-compact, easily transportable device for simultaneously visualising a number of cells/biological molecules, individually labelled to emanate a characteristic type of light. We will assess the feasibility of incorporating such a device within a MRI system such that we can see cells/molecules using the new device but in a 3D anatomical/structural context provided by MRI. This readily transportable and unique device will monitor specific biological processes in living systems used in industrial biotechnology, enhancing efficiency (production) and improving manufacturing methods, and able to perform in environments that other devices cannot operate.
Impact KCL and ZiNIR collaborated on a project to identify the technical specifications of an ultra- compact, easily transportable hyperspectral imaging system for detecting a range of biomolecules in vivo. The system would employ arrays of novel miniature solid-state spectrometer chips developed by ZiNIR, and could potentially be incorporated into a MRI system for multimodal imaging. A literature review and patent search were conducted to assess the current status of hyperspectral fluorescence imaging and multimodal MRI-optical imaging, both in biomedical applications and in industrial biotechnology. A number of parallels were found between the requirements for fluorescence imaging in preclinical studies and chlorophyll fluorescence imaging, a technique used in a wide variety of plant biology and biotechnology applications. Accordingly, a set of technical specifications for the imaging system was defined to satisfy the requirements of both application areas.
Start Year 2014
 
Description Development of new refolding methodologies for expression of heme protein targets 
Organisation F. Hoffmann-La Roche AG
Country Global 
Sector Private 
PI Contribution We funded and administered a Business Interaction Voucher in a collaboration with Emma Raven of University of Leicester (BIVMiB013)
Collaborator Contribution Heme (iron)-containing enzymes are a mainstay of industrial biotechnology, and the industry depends on fundamental improvements in methodology emerging from academic groups around the world to harness the potential of their investments in biopharmaceuticals, bioenergy, biocatalysis and drug design. For a number of complex reasons, the interactions between industry/biotechnology and academic laboratories are often less facile and less extensive than they could be, so that new (often Specialist and/or unpublished) information is not transferred fluently to industrial partners. Our overall objective is to use this project to develop new refolding methodologies for expression of difficult (insoluble) heme protein targets, and to set up an on-going dialogue between industrial and academic partners with mutual cognate interests in specific heme enzymes targets. The methodologies that we develop will open up new avenues for industry partners in cases where they have intractable (insoluble) protein targets.
Impact In this project, protocols for expression methods were developed for a range of different heme enzymes that are not readily expressed using conventional methodologies in E. coli. Early stage targets from the Raven laboratory were used as a "test bed" for other heme systems.
Start Year 2016
 
Description Embedding technical expertise in the optimisation of trace metal supplementation strategies for successful biomethane production 
Organisation Tropical Power Ltd
Country United Kingdom 
Sector Private 
PI Contribution We funded and administered a Business Interaction Voucher in a collaboration with Yue Zhang of University of Southampton (BIVMiB032)
Collaborator Contribution Transformation of waste biomass into bioenergy and useful resources is a key component in 21st century industrial biotechnology. It is increasingly clear that successful biomethanisation of mixed biomass requires complex enzyme systems that are produced by both natural and engineered synthetic microbial communities. Trace quantities of metals play a role in certain essential metallo-enzymes, and thus in ensuring that microbial communities function in the most effective and productive way. There is a growing commercial market in trace metal supplements, but these are often generic rather than based on specific requirements. The current project involves transfer of knowledge and expertise in determining trace metal requirements to a UK company using novel waste feedstocks in Africa for renewable biomethane production. This will enable the company to formulate specific trace metal mixtures for optimum plant performance, while the scientific knowledge gained will contribute to creating future markets for UK suppliers of tailored supplements.
Impact Project on-going
Start Year 2017
 
Description Engineering metal dependent biotin synthase for the biotechnological production of biotin 
Organisation VideraBio Ltd
PI Contribution We funded and administered a Proof of concept Award in collaboration with Christof Jäger of University of Nottingham (PoCMiB029)
Collaborator Contribution Biotin (vitamin B7) is an essential cofactor in bacteria, fungi and plants. It is used in vitamin food supplements and primarily for the enhancement of animal feed. Its production is challenging and particularly expensive with sales prices of ~$1600/kg. In this project we aim to demonstrate a potential way towards a sustainable and cost effective biotechnological production of biotin. We will initiate engineering the key bottleneck enzyme biotin synthase which is responsible for an iron sulfur cluster-induced sulfur insertion reaction. We are going to investigate the first steps into rational computational enzyme design in alignment with the development of automated high throughput, multidimensional, in vivo assays for this enzyme for use in our on-site robotics suite. Our approach will act as starting point for not only rational informed directed evolution strategies, but also will integrate regulatory elements for the repair mechanisms of the host cells.
Impact The essential cofactor Biotin (Vitamin B7) has industrial use in the enhancement of animal feed, and in the pharmaceutical and biotechnology industries. Its production is particularly challenging and costly, favouring development of a sustainable and cost-effective biotechnological approach towards producing biotin. The enzyme biotin synthase, containing a delicate iron-sulfur cluster, represents one key bottleneck in the biosynthetic pathway producing biotin. However, engineering this radical SAM enzyme, which catalyses the complex and chemically difficult transformation of dethiobiotin via sulfur insertion into a non-activated carbon chain, remains highly challenging. During this BBSRC NIBB PoC project, fundamental development of high throughput, multidimensional biotin detection assays, making use of our on-site robotics suite at the Synthetic Biology Research Centre (SBRC), was achieved, in alignment with proof of concept computational investigations to support a rational enzyme design approach. We tested and successfully developed two biotin assays; one based on a previously described Corynebacterium glutamicum indicator strain, alongside a second assay making use of a biotin analogue, 4-Hydroxyazobenzene-2-carboxylic acid (HABA), bound to Avidin (HABA/Avidin). We have found that both the C. glutamicum-based biosensor assay (detection range between 0-1 µg.L-1) and the HABA/AVIDIN (detection range between 1-50 µg.mL-1) have the potential to be adapted for automation of large library screens in multi-titre plate format. The C. glutamicum-based bioassay measured cell growth (Abs600), both with biotin prepared from stock (0 to 5 µg.L-1) and biotin produced in E. coli cells, by lysing the E. coli cells and serially diluting the cytoplasmic fractions. A standard curve was obtained using biotin concentrations between 0 and 1µg.L-1, in two-fold dilution series. Manual loading of the microtiter plates, as well as preparing the dilutions and plates using the liquid handling unit of the robotic suite, demonstrated the biotin concentration was directly proportional to the ability to support C. glutamicum growth in the range from 0 and 1 µg.L-1. In the second affinity-based assay, the amount of free HABA reflects the amount of biotin that is present, once it is displaced from the Avidin. This quantity is measured by the change in absorbance at 500 nm. Sigma/Aldrich-supplied HABA/Avidin reagent (Product No. H2153) was utilised with the provided standard protocol. The assay was optimised for 96-well microtiter plate format with biotin standards added to HABA/Avidin reagent in eight replicates for each concentration, alongside HABA/Avidin-only and HABA/Avidin with media as blanks and background, respectively. The computational approaches focused on targeting potential mutation sites of BioB that influence the redox reactivity of one or both iron sulfur clusters and thus potentially influence sulfur insertion and cluster repair kinetics. For that reason scripts have been developed to analyse the electrostatic effect of the protein environment (due to directional polarity) on the cluster. This type of analysis now makes it possible to spot individual amino acids that influence the field significantly, without being directly in contact with the active site. These amino acids will be taken forward to mutation studies.
Start Year 2017
 
Description Enhancing E. coli for optimal cofactor insertion into heme and iron-sulfur cluster proteins 
Organisation Biocatalysts Ltd
Country United Kingdom 
Sector Private 
PI Contribution We funded and administered a Proof of Concept Award in a collaboration with Nick Le Brun of University of East Anglia (POCMiB002)
Collaborator Contribution Metals as cofactors of proteins and enzymes are essential for all of life. Many metalloproteins have properties that are useful outside of the cell, for example in the synthesis of useful materials or medicines. However, to exploit this, the metalloprotein must be purified with the metallo-cofactor fully incorporated; incomplete incorporation results in major inefficiencies in the production process. A strategy to improve cofactor insertion is to more carefully match protein synthesis with cofactor synthesis/insertion. While in some cases this can be achieved by manipulation of the growth conditions, for example by slowing down protein synthesis, for commercial processes where protein yields are key, important economic and production benefits should be achievable through the engineering of cell factories to increase their capacity to incorporate the cofactor. In this project, the academic partners, who are experts in iron metabolism and iron-protein chemistry, propose to generate strains of E. coli that have significantly increased capacity to incorporate iron sulfur clusters and heme into a range of protein targets. These strains, together with growth protocols developed to use them to best effect, will be tested for the production of examples of commercially relevant iron-sulfur and heme proteins by the commercial partner.
Impact Enzymes and proteins are widely used in commercial processes, for example in the food industry, for the production of fine chemicals, in the detergent industry, as medicines, in diagnostic kits and in emerging fields such as bioenergy. Metals as cofactors of proteins and enzymes are essential for all of life, and metallo-proteins/enzymes represent a significant subgroup of commercially important enzymes/proteins. A potential problem for the use of metallo-enzymes is that their activity is wholly dependent on the cofactor (which is usually the active site at which the catalytic process occurs) and so the efficiency of production is dependent on the efficiency of cofactor insertion. Expression systems are capable of producing a target peptide up to 50% of total cell protein; in many cases, the metallo-cofactor and insertion apparatus of the cell cannot keep up with protein synthesis. This results in major inefficiencies in the production process. An obvious answer is to carefully match protein and cofactor synthesis/insertion. This can be achieved, in some cases at least, by slowing down the rate/reducing the extent of, protein synthesis such that the cofactor pathways can better keep up. However, this is unlikely to be suitable for a commercial context where achieving maximum yield is key. In 2015 we applied for and were awarded a PoC grant from the BBSRC Metals in Biology.
Start Year 2015
 
Description Evaluation of the potential of the molybdenum-containing enzyme DMSO reductase as an oxygenation catalyst 
Organisation Piramal Healthcare UK Ltd
PI Contribution Over the past 30 years science has made huge advances in understanding how biological systems work and this understanding is now being translated into valuable tools for the manufacture of products. This new technology, referred to as biotechnology, can bring many advantages over more traditional methodology. For example, pharmaceuticals are usually very complex molecules that have traditionally been manufactured using conventional chemistry techniques that rely on reactive and sometimes difficult to handle materials; in contrast, biotechnological approaches make use of Nature's own catalysts (called enzymes) to carry out reactions under very benign conditions and unlike conventional catalysts enzymes are biodegradable and non-toxic. This project is exploring the potential of enzymes to carry out complex oxidation chemistry which can be applied in the manufacture of pharmaceuticals and other important products. Emphasis will be placed on getting the enzymes to work effectively in the non-natural environment of a chemical reactor.
Collaborator Contribution We funded a Business Interaction Voucher in collaboration with Gary Black of Northumbria University (BIVMiB044)
Impact Over the past 30 years science has made huge advances in understanding how biological systems work and this understanding is now being translated into valuable tools for the manufacture of products. This new technology, referred to as biotechnology, can bring many advantages over more traditional methodology. For example, pharmaceuticals are usually very complex molecules that have traditionally been manufactured using conventional chemistry techniques that rely on reactive and sometimes difficult to handle materials; in contrast, biotechnological approaches make use of Nature's own catalysts (called enzymes) to carry out reactions under very benign conditions and unlike conventional catalysts enzymes are biodegradable and non-toxic. This project explored the potential of enzymes to carry out complex oxidation chemistry which can be applied in the manufacture of pharmaceuticals and other important products. In total six DMSO reductase enzyme preparations were produced and their capacity to perform complex oxidation chemistry was determined.
Start Year 2017
 
Description Exploiting a copper-dependent chaperone system to improve bioprocessing of therapeutic antibodies 
Organisation Fujifilm
Department Fujifilm Diosynth Biotechnologies
Country United States 
Sector Private 
PI Contribution The production of biotherapeutics has a total market value of around £100 Billion per year. This project expands the repertoire of a novel system that has been shown to improve the production of antibody fragments in E. coli, a product with wide ranging applications in diagnostics (e.g. pregnancy tests), human therapeutics and as fundamental research tools. In addition, future consumer applications might include the use of antibodies in shampoos to prevent the formation of dandruff or in toothpaste to protect against tooth decay. This project focusses upon copper-dependent protein folding catalysts (Scs proteins), with the overall aim of improving the yield and quality of antibody fragments produced in E. coli. Previous antibody targets include the cancer therapeutic heceptin, whereas the current work has a particular focus on Lucentis, a therapeutic antibody used to treat macular degeneration.
Collaborator Contribution We funded and administered a Business Interaction Voucher in a collaboration with Mark Shepherd of the University of Kent (BIVMiB041)
Impact Project on-going
Start Year 2017
 
Description Exploiting the commercial potential of novel biometallic catalysts 
Organisation Johnson Matthey
Country United Kingdom 
Sector Private 
PI Contribution We funded and administered a Business Interaction Voucher in collaboration with Jon Lloyd of the University of Manchester (BIVMiB037)
Collaborator Contribution This project brings together biotechnologists from the University of Manchester and experts in industrial catalysis at Johnson Matthey, a leading multinational specialty chemicals and sustainable technologies company headquartered in the United Kingdom. This Business Interaction project will facilitate collaborative discussions required to underpin the development and exploitation of a new generation of "biometallic" industrial catalysts. They are based on naturally occurring metal- reducing bacteria that are able to accumulate metals from process environments, as catalytically active nanoparticles, while also expressing enzymes that are able to extend the range and complexity of industrial reactions that can be produced from these novel microorganisms. This novel extension of "synthetic biology" has the potential to transform several sectors of UK industry including those of industrial biotechnology and makers and users of catalysts, simplifying current processes, underpinning novel reactions and extending the range of available products.
Impact Project on-going
Start Year 2017
 
Description Extracting mercury from industrial waste using microalgae 
Organisation Reym B.V
Country Netherlands 
Sector Private 
PI Contribution We funded and administered a Business Interaction Voucher in a collaboration with Mark van der Giezen of University of Exeter (BIVMiB017)
Collaborator Contribution Affordable and sustainable energy is an important global challenge. Biofuels are seen as possible solutions; however, currently they have limited environmental benefits and put pressure on land and water. Algal biofuels could overcome many drawbacks of terrestrial plant-based biofuels but are currently more energy-intensive and expensive. Water pollution is another global problem and causes over 14,000 deaths each day. By 2025, 1.7 billion people are faced with absolute water scarcity and two-thirds will have drinking water shortages. Unfortunately, many essential industrial activities contribute greatly to water pollution and more sustainable production methods or waste management practices are required to support the global demand for products but protect water sources. With relevance to both challenges, we will grow algae on metal contaminated industrial waste streams followed by hydrothermal liquefaction. This process will separate waste into four fractions: water, gas, oil and solids, the latter containing the metal waste, thereby valorising waste.
Impact Project on-going
Start Year 2016
 
Description Improving biocatalytic processes by enzyme stability enhancement 
Organisation Oxford Biotrans Ltd.
Country United Kingdom 
Sector Private 
PI Contribution We funded and administered a Business Interaction Voucher in a collaboration with Stuart Ferguson of University of Oxford (BIVMiB036)
Collaborator Contribution The industry partner has licensed biocatalytic oxidation technology from the University of Oxford for the production of fine chemicals such as flavours, fragrances, agrochemicals and active pharmaceutical ingredients. The first product is nootkatone, the high-value grapefruit flavour compound. This industrial biotechnology approach not only replaces classical, more energy- demanding and polluting chemical processes, it also enables non-fossil fuel, sustainable feedstocks to be used. A key improvement to the process is enhancement of the stability of the enzyme catalyst that lies at the heart of the technology, making it applicable to a wider range of products.
Impact Project on-going
Start Year 2017
 
Description Interaction with industrial sponsor 
Organisation Procter & Gamble
Country United States 
Sector Private 
PI Contribution Regular teleconference meetings (in excess of 50 over 24 months including all forms of interaction) with industrial collaborator Reciprocal exchange of materials and biologics with industrial collaborator Reciprocal visits with industrial collaborator (associated PhD students and academic staff etc) Analytical services provided for industrial partner and others, and vice versa
Collaborator Contribution See above
Impact Ongoing and confidential
Start Year 2012
 
Description Investigating platinum group metals in wastes from roadside verges 
Organisation Yorwaste Ltd
Country United Kingdom 
Sector Private 
PI Contribution We funded and administered a Business Interaction Voucher in a collaboration with Neil Bruce of University of York (BIVMiB018)
Collaborator Contribution Platinum group metals (PGMs) are rare elements that are essential components in a widening number of industrial applications and particularly used in catalytic converters on road traffic vehicles. Over time, these metals are lost from the converters via exhaust fumes, with significant levels being deposited onto roads and verges. This increasing dispersal and dilution into the environment is of growing concern. Plants can take up PGMs, and our work at the University of York has demonstrated that the subsequently harvested, PGM-rich plant biomass has catalytic activity. This project aims to measure the levels of palladium and platinum in roadside verge wastes from rural, inner city and highway road stretches. Plants will be grown on the wastes to see if they can take up PGMs. This study will enable us to understand if significant levels of PGMs exist in road sweeping waste and whether a phytoextraction process is a viable technology to recover it.
Impact We have achieved our objectives to investigate NP formation using synthetic peptides and analyse catalytic activity in the subsequently pyrolysed NP-containing plant biomass. Our promising ICP-OES, TEM and catalysis results demonstrate that the expression of synthetic peptides in plants can be used to alter gold NP size and subsequent catalytic activity in planta. As part of our third objective, to determine if plants could be used to selectively take up PGMs from sweeper wastes, we have shown that sweeper wastes contain detectable levels of valuable metals. However, our studies show that further work is needed to understand the phytotoxicity behind these wastes so that they can be optimised to allow plant growth. Our wider studies indicate that synthetic biology could be used to develop plants that can selectively take up PGMs from sweeper wastes.
Start Year 2016
 
Description Investigating the antimicrobial properties of copper infused fabrics 
Organisation Copper Clothing Ltd
Country United Kingdom 
Sector Private 
PI Contribution We funded and administered a Business Interaction Voucher in a collaboration with Bill Keevil of University of Southampton (BIVMiB030)
Collaborator Contribution The University of Southampton have been testing the antimicrobial effects of copper for many years, and have shown that copper can kill bacterial cells in several ways, including rupturing the cell, and destroying the DNA. Copper Clothing Ltd has begun investigating new processes for incorporating copper into their antimicrobial fabrics. Initial work will investigate the antimicrobial properties of fabrics infused with copper using novel technologies. The new fabrics developed by Copper Clothing Ltd. will be tested for levels of antimicrobial kill in a range of bacteria, both by culture and by using fluorescence microscopy to determine the presence of living but non culturable organisms - bacterial that are not killed by the copper, but survive in a stressed form. Future work plans to use bio-produced nano-copper for incorporation into the fabrics and determine their improved efficacy against superbugs compared to the current novel fabric chemistries.
Impact Project on-going
Start Year 2016
 
Description Investigating the link between metal homeostasis, sporulation, and solvent production in the Clostridial ABE fermentation process 
Organisation Green Biologics
Country United Kingdom 
Sector Private 
PI Contribution Clostridia are exemplars of fermentative microbes that convert biomass to renewable chemicals. Green Biologics Limited use this process commercially to produce the platform solvent chemicals butanol (with diverse uses including consumer fuels, paints and coatings and food additives) and acetone (used in cosmetics, plastics and numerous other markets). Solvent yield is limited by the physiology of the microbes under production conditions, including the onset of sporulation. Endospore formation diverts energy to generate biomass, which does not contribute to solvent production. A detailed understanding of the link between metal ions and Clostridium metabolism and physiology during solvent production will provide the means to improve strains for greater solvent yields, an important factor in the economic viability of the process.
Collaborator Contribution We funded and administered a Business Interaction Voucher in collaboration Peter Chivers of Durham University (BIVMiB043)
Impact Clostridia are exemplars of fermentative microbes that convert biomass to renewable chemicals. Solvent yield is limited by the physiology of the microbes under production conditions, including the onset of sporulation. Endospore formation diverts energy to generate biomass, which does not contribute to solvent production. A detailed understanding of the link between metal ion homeostasis and Clostridium metabolism and physiology during solvent production can be applied by GBL to commercial processes. This project focused on obtained detailed information on metal content and changes in gene expression within a 12-h window of growth identified in previous study (BIVMiB035 - Metal utilisation in Clostridium microbial biocatalysts). Biomass samples were collected by GBL at 3-h intervals for metal content analysis and RNA isolation. The metal content analysis (Durham) demonstrated that the previously identified increase in metal content occurred over the 12 h window, rather than more rapidly within a shorter time window. An aporogenic mutant did not show the changes in metal content and composition. Replicate RNA samples at the same 3 h intervals were isolated (Durham) from a wild-type strain and an asporogenic strain for RNASeq analysis. Because of the short time frame for the project, the RNASeq results and their analysis are not complete. The project was beneficial for knowledge transfer - the metal content and RNA sample analysis were carried out at Durham by a GBL scientist. The informal interactions that occurred during the 3-week visit will enhance future work at both sites.
Start Year 2017
 
Description Investigating uptake and catalytic potential of miscanthus grown on palladium mine wastes 
Organisation AgriKinetics Ltd
Country United Kingdom 
Sector Private 
PI Contribution We funded and administered a Business Interaction Voucher in a collaboration with Neil Bruce of University of York (BIVMiB024)
Collaborator Contribution We have shown that Arabidopsis plants grown in liquid culture can take up palladium, depositing it as nanoparticles. Following a processing step, the nanoparticle-containing biomass can be used directly as a catalyst for industrially important reactions. Palladium mining wastes contain significant levels of palladium which are uneconomical to recover using conventional methods; mining areas also need to be re-vegetated. Phytoextraction using field-relevant species could be a viable and environmentally sustainable method to re-green these sites while generating a catalytic product of higher value than the bulk metal. Our preliminary studies at the Centre for Novel Agricultural Products (CNAP) have demonstrated that the grass, miscanthus, can take up palladium from synthetic mine wastes, but not yet at levels that confer catalytic activity. A major limitation to uptake is the insolubility of palladium in the wastes. This project will enhance palladium availability, measure accumulation in miscanthus and provide biomass for catalysis testing.
Impact Project on-going
Start Year 2016
 
Description Light-activated caged-iron chelator for skin photoprotection based on the natural product pulcherrimic acid 
Organisation Croda Europe Ltd
Country United Kingdom 
Sector Private 
PI Contribution We funded and administered a Proof of Concept Award in a collaboration with Charareh Pourzand of University of Bath (POCMiB019)
Collaborator Contribution At present, there is a significant need to counteract the cellular mechanisms that cause skin damage upon prolonged exposure to the UV component of sunlight. Exposure of skin cells to UVA, the oxidizing component of sunlight promotes the generation of harmful reactive oxygen species and leads to an immediate release of labile iron and susceptibility to both oxidative membrane damage and necrotic cell death. Research at Bath since 2006 has resulted in the synthesis and biological validation of light-activated protective compounds (i.e. light-activated caged-iron chelators, CICs) that respond to the UVA-component of sunlight. Upon activation by sunlight these 'intelligent' compounds release an active iron trapping agent (iron chelator) to remove the potentially harmful free labile iron released in skin cell and thereby to protect against iron-catalysed oxidative damage and cell death. A critical requirement for CIC technology is readily available, chemically tractable iron chelators, in which the iron-binding motif can be reversibly modified (caged). In this context, we plan to isolate and modify (cage) the pulcherrimic acid, a natural product from the yeast M. pulcherrima with iron chelating activity and subsequently evaluate its photoprotective activity against UVA-induced iron damage in cultured skin cells.
Impact This project aimed to demonstrate that the natural iron chelator pulcherrimic acid (PA) derived from the yeast Metschnikowia pulcherrima (Mp) may be used as the basis for the development of novel light-activated skin photoprotectants. For this purpose, three overlapping work packages were designed to first evaluate the photoprotective potential of either isolated PA from yeast culture or the chemically synthesized PA and closely related analogues (PAA) against UVA damage in skin monolayer culture. The next step was to synthetically modify either the isolated PA from the yeast or the synthetic PA and its analogues in order to obtain UVA-activated caged derivatives in which the iron-binding motif of the chelator was temporally blocked by a photolabile caging group. The final step was to biologically evaluate the photoprotective potential of the caged PA/PAA compounds against UVA damage in skin cells. The project started by producing pulcherrimin and purifying PA from the culture grown in non-sterile conditions. Studies on lab scale showed that carbon sources like glucose and saccharose are able to trigger the production of pulcherrimin by the Mp yeast. The production of around 150mg/L of pulcherrimin was achieved on a 10L scale culture of an over-producing strain of Mp (DH5) in non-sterile conditions using an air-lift reactor. An optimization of the purification protocol for pulcherrimin was necessary for an effective recovery. Also, the purification of PA proved to be challenging due to the unstable nature of the compound that requires stringent conditions of cold temperature and protection from sunlight. In addition to isolated PA from the yeast, synthetic authentic samples of PA were successfully prepared in the chemistry laboratory, thus permitting the initial evaluation of this natural product as a photoprotective agent. The synthetic approach devised was robust and scalable, and should be suitable for the preparation of a range of amino-acid derived PA analogues and caged compounds. The project also demonstrated promising chemical routes for the synthesis of various light-activated caged PA derivatives. Nevertheless further chemical development work is required to build on the promising results obtained so far in order to obtain light-activatable caged compounds either from a versatile compound generated from the Mp-derived PA, or directly from the latter, both as renewable feedstocks. Although, the natural PA extracted from the Mp yeast was found indistinguishable from the authentic synthetic material, due to lack of optimum purity of PA isolated from the yeast, the biology work concentrated only on the highly pure synthetic PA. The results showed that PA is not cytotoxic per se when exposed to cultured primary skin fibroblasts overnight up to the concentration of 50uM. PA at concentrations in the range of 20-30uM provided a significant photoprotection against UVA-induced damage and cell death. Photoprotection by PA was much superior to that offered by the clinically used bidentate iron chelator Deferiprone at equimolar concentration of 20uM. These promising results provide the proof of concept for the potential development of photolabile caged PA as topical sunscreen ingredients against the damaging effects of solar UVA radiation.
Start Year 2015
 
Description Mag-Tag: magnetite nanoparticle affinity tags for industrial biotechnology protein purification 
Organisation Biocatalysts Ltd
Country United Kingdom 
Sector Private 
PI Contribution We funded and administered a Proof of Concept Award in a collaboration with Sarah Staniland of University of Sheffield (POCMiB011)
Collaborator Contribution Enzymes are protein catalysts which can perform highly specific biotransformations to convert starting materials into desired (often) complex products using mild, aqueous reaction conditions. These key capabilities are difficult to achieve with conventional catalysts, making enzymes ideally suited to the industrial manufacture of foodstuffs, biofuels, pharmaceuticals and a range of other industrial biotechnology. The current challenge to the widening the use of enzymes is the expense of producing them. Large scale industrial purification of enzymes is often prohibitively costly due to the need for expensive, highly functionalised purification resins. This cost barrier limits the use of enzymes to industrial applications where unpurified, crude enzymes are suitable, or where the end product is sufficiently prized to enable the cost of purification to be recouped. We propose a revolutionary, cheap, universally applicable, enzyme purification method to widen the use of purified enzymes in industry. We will use protein fusion-tag technology to purify enzymes directly from crude preparations using cheap, unfunctionalised magnetic iron-oxide nanoparticles, which can then be bulk purified through magnetic separation. By substantially reducing the costs of purification we seek to make enzymes an affordable, green and sustainable method of producing a wide range of products.
Impact In this study, we used a protein that had high binding affinity and selectivity for certain magnetic materials that we previously identified. We used our affinity protein as a fusion tag (MagTag) to a test protein, GFP (green fluorescent protein), as this allowed us to track the binding and release of the target (the GFP fusion protein) via simple fluorescence measurements. During the course of the project we made the GFP-MagTag fusion construct and showed that the presence of the magnetic material binding tag had no detrimental impact on production of the GFP. We optimised a simple synthetic route to the fabrication of cheap magnetic nanoparticles and demonstrated that the fusion protein could bind these under industrially relevant conditions, namely using crude cell lysate with a high optical density. Fluorescence measurements showed that we could successfully capture the GFP fusion protein from the lysate, out-competing other proteins within the sample. Importantly, we were able to show that it was possible to recover the GFP from the nanoparticles after binding and clean-up.
Start Year 2015
 
Description Maximising Biomarker Detection Sensitivity through Metal Enhanced Fluorescence 
Organisation Aeirtec
Country United Kingdom 
Sector Private 
PI Contribution We funded and administered a Business Interaction Voucher in a collaboration with Lu Shin Wong of University of Manchester (BIVMiB023)
Collaborator Contribution Fluorescence-based immunosorbent assays have become a key technology for detection and quantification of microbial contamination (in water, chemical, food and drug production), as well as for molecular biomarkers in agriculture, drug discovery, industrial biotechnology, medical diagnostics and cellular imaging. Here, the selectivity of antibody binding combined with fluorescence spectroscopy, has led to huge advances in sensitivity, selectivity and speed. Typically, these assays employ Fluorescence-based immunosorbent assays have become a key technology for detection and quantification of microbial contamination (in water, chemical, food and drug production), as well as for molecular biomarkers in agriculture, drug discovery, industrial biotechnology, medical diagnostics and cellular imaging. Here, the selectivity of antibody binding combined with fluorescence spectroscopy, has led to huge advances in sensitivity, selectivity and speed. Typically, these assays employ an immobilised antibody to capture the target molecule from the test sample, followed by the immobilisation of a second antibody bearing a fluorescent label. To maximise the fluorescence output from the label, many researchers have started to harness metal enhanced fluorescence (MEF) as a means to improve diagnostic sensitivity. Here, the co-location of the antibody in the vicinity of a metallic nanoparticle results in a large enhancement of fluorescence output (in the order of 100-fold). This project aims at improving the sensitivity of these MEF-based assay systems by the application of state-of-the-art bioconjugate methods to control the orientation of the immobilised antibodies with respect to the nanoparticle.
Impact This interaction voucher was used to develop a collaborative relationship on the production of protein-metallic nanoparticle conjugate materials, for use in Aeirtec's diagnostics platform. It particular, it concentrated on the bioconjugate chemistries needed for the immobilisation of these materials. Specifically, comparative analyses were conducted with various diamine linker molecules using a variety of protecting groups. Robust and quantifiable methods for linking fluorescence marker materials were developed and delivered to Aeirtec. From an academic perspective, the voucher was used to part-fund the research undertaken by students in Dr. Wong's laboratory. In particular, it provided the basis for a mini-project for one of our MSc students. It also part-funded two undergraduate summer internships and provided them with further research experience. Aeirtec has benefited from this interaction through access to chemistry in relation to our capacity to generate a metallic-protein microparticle surface. The interaction has now set a direction for incorporation of metals alongside proteins and microparticle identifying dyes.
Start Year 2016
 
Description Metal demands during protein overexpression in bacteria 
Organisation Biocatalysts Ltd
Country United Kingdom 
Sector Private 
PI Contribution We funded and administered a Busisness Interaction voucher in a collaboration with Peter Chivers of Durham University (BIVMiB001).
Collaborator Contribution Protein and enzyme overexpression is a major facet of industrial biotechnology. The capacity of host organisms for protein overexpression is not naturally optimized. Transition metals are key components of the cellular machinery required for protein synthesis. The effects of protein overexpression on metal allocation within the cell have not been examined. This project will explore the effects of protein overexpression on metal utilization in Escherichia coli, a widely used platform for biologics production. The objective is to gain insight into the cellular response to the metal demands of protein overexpression. Metal allocation and utilization will be studied using RNASeq to monitor changes in gene expression that are metal-regulated or encode metal-requiring enzymes and proteins related to protein synthesis.
Impact Transcript levels and metal content of E. coli cells were measured at different time points post-induction during a representative fermenter run. Increased transcript levels were observed in genes important for Mg, Fe, Mn, and Ni acquisition at and beyond the midway point of overexpression (= 9 h post-induction). These increases correlated with decreases in total cellular metal content for each metal, consistent with metal deficiency sensed by metal-responsive transcriptional regulators. These deficiencies have potential effects on translational efficiency (Mg), synthesis of non-natural amino acids that affect the fidelity of tRNA charging (Ni), and posttranslational processing of newly synthesized polypeptides (Fe/Mn). No evidence for Zn-deficiency, or Cu-stress, was detected based on transcript levels and metal content.
Start Year 2015
 
Description Metal utilisation in Clostridium microbial biocatalysts 
Organisation Green Biologics
Country United Kingdom 
Sector Private 
PI Contribution We funded and administered a Business Interaction Voucher in a collaboration with Peter Chivers of Durham University (BIVMiB035)
Collaborator Contribution Clostridium is an exemplar for microbial fermentations that convert biomass to renewable chemicals, such as butanol (with diverse uses including consumer fuels, paints and coatings and food additives) and acetone (used in cosmetics, plastics and numerous other markets). These fermentations depend upon metalloenzymes to convert starting material to product. The optimisation of metal supply is therefore critical for cost-efficiency. Currently, little is known about the metal requirements of industrial Clostridium species, or the metal circuitry important for maintaining metal supply to these pathways. We will identify the metal requirements during different fermentative processes and in different environments (batch vs. continuous; lab vs. industrial). Results will be applied to commercial operations for immediate outcomes including reducing waste and water use impacts. To further the development of Clostridium in a variety of IB processes, the metal sensor components of the metal circuitry will be identified to enable fine-tuning of metal supply pathways.
Impact Project on-going
Start Year 2017
 
Description Metallo-enzymes for production of nootkatol, a potential new citrus flavour 
Organisation Oxford Biotrans Ltd.
Country United Kingdom 
Sector Private 
PI Contribution We funded and administered a Business Interaction Voucher in a collaboration with Luet Wong, University of Oxford (BIVMiB008)
Collaborator Contribution The sought after grapefruit flavour compound nootkatone is biosynthesised by air oxidation of valencene catalysed by a haem enzyme, firstly to nootkatol and then nootkatone. Haem enzymes are involved in the biosynthesis of numerous natural products, including flavours such as menthol and nootkatone but also medicinal compounds such as antibiotics, the antimalarial artemisinin and the anticancer drug taxol. The industry partner is developing commercial scale biocatalytic synthesis of nootkatone from valencene. The academic partner will modify the haem enzyme used in this process to produce nootkatol, which is found in minute quantities in grapefruit, to explore it's potential as a novel flavour. This primer-project and the nootkatone process will underpin future collaborative work within the metals-in -biology community (1) to test strains optimised (by others) in haem production to further enhance nootkatone/nootkatol synthesis, (2) to manipulate the primary and secondary coordination sphere of the haem moiety to further enhance nootkatol synthesis.
Impact Samples of the pure nootkatol isomers were produced and delivered to the industry partner. The potential market of these novel flavours is being assessed. Enzyme variants that gave increased proportions of either nootkatol isomer were generated. Process optimisation also led to improved yields of the nootkatone production process. New strains from the NIBB MiB network can be applied to the systems and processes developed in this BiV project to benefit the UK industrial biotechnology sector.
Start Year 2015
 
Description Metalloenzyme system for Hydrogendriven NADPH recycling in pharmaceutical synthesis 
Organisation GlaxoSmithKline (GSK)
Country Global 
Sector Private 
PI Contribution Bacterial cells are miniature chemical factories, with enzymes as the key machinery for making molecules. Pharmaceutical companies have recognised the benefits of using enzymes instead of traditional chemical routes to make drug molecules. Enzymes generate less waste, make purer chemical products, and allow chemistry to be carried out in water rather than in toxic, polluting solvents. However, there are challenges to overcome before enzyme approaches can be widely adopted. Many enzymes only work in the presence of special helpermolecules called cofactors, which are used up during the production of chemicals. The cofactors are expensive and for enzyme processes to be economically viable, it is essential to have some way of recycling the cofactors. We have demonstrated a novel approach for recycling a common cofactor called NADH. In collaboration with GSK, this project extends the approach to a related cofactor, NADPH, and demonstrates NADPH recycling for enzyme synthesis of pharmaceutically-relevant molecules.
Collaborator Contribution We funded and administered a Business Interaction Voucher in a collaboration with Kylie Vincent of University of Oxford (BIVMiB004)
Impact Introduction. Biocatalysis is gaining increasing importance in synthesis of pharmaceuticals and other speciality chemicals, yet barriers remain - in particular the dependence of many redox enzymes on the expensive nicotinamide cofactor, NADH, and more costly NADPH. We have previously demonstrated a novel H2-driven NADH recycling system, and in this Business Interaction Voucher project we have extended this system to recycle NADPH. The project exploits genetic variants of an NAD+ reductase generated in the lab of Dr Oliver Lenz, Technical University of Berlin, which are designed to have higher affinity for the related cofactor NADP+. We have identified a suitable NADP+ reducing variant, and we have demonstrated NADPH recycling by coupling the cofactor recycling beads with a C=C bond reductase, PETNR, supplied by industrial collaborator GSK. Project outcomes: During the project we have recorded KM values for the most promising NAD+ reductase variants and identified one which functions as an NADP+ reductase with a KM of 1 mM NADP+, compared with >8 mM for the wild type. Data on the affinity constants for a series of variants will form the basis for the publication to be submitted in the coming months. The selected NADP+ reductase was then incorporated into cofactor recycling beads. A nickel-iron hydrogenase oxidises H2 and transfers electrons, via its internal relay chain of iron-sulphur clusters, to the electrically-conductive carbon bead. Electrons are then transferred through the bead to the co- immobilised NADP+-reductase which also possesses a relay chain of iron-sulphur clusters for fast electron transfer to its flavin active site. Thus H2 oxidation is coupled efficiently to NADP+ reduction. The imine reductase and ene reductase enzymes supplied by GSK were examined in biochemical assays with product detection by HPLC or GC. The C=C bond reductase PETNR was selected as the best enzyme for a first demonstration of NADPH recycling. In the presence of H2, NADPH was supplied to PETNR which was co-immobilised on the beads or handled in solution, for the reduction of cyclohexenone to cyclohexanone. Near-complete conversion (>99%) of the alkene to the alkane was observed by GC. Thus we have generated promising results on the feasibility of coupling H2-driven NADPH recycling to a NADPH-dependent hydrogenation. These results will be prepared for publication later this year as part of the demonstration of the modularity of the H2- driven cofactor recycling system which can be adapted for operation under different conditions by varying the hydrogenase or the NAD(P)+ reductase. Conference presentations: Dr Holly Reeve presented a Flash Talk and Poster that included these findings at the European Symposium on Biological and Organic Chemistry, Gregynog, Wales, 15th-17th May 2015. Prof Kylie Vincent gave an invited lecture at the Academic Day of GSK's Global Technologies Conference, Cambridge, 25 March 2015: 'Heterogeneous biocatalysts for H2-driven chemical synthesis', and included some of these results in her presentation.
Start Year 2014
 
Description Microbial recovery of metals from contaminated Miscanthus used in the industrial remediation of degraded landscapes. 
Organisation Terravesta Ltd
Country United Kingdom 
Sector Private 
PI Contribution We funded and administered a Business Interaction Voucher in a collaboration with Chris Chuck of University of Bath (BIVMiB026)
Collaborator Contribution Metal leaching from mining and further industrial activity has the potential to degrade landscapes across the globe. However, recently a range of techniques have been trialled and brought to market to restore the natural capital of these areas. One of the most promising is growing Miscanthus x giganteus, an energy crop that can remove metal contamination while being used as a biofuel feedstock. However, the processing of the contaminated Miscanthus x giganteus remains an issue. In this BIV we will explore using the oleaginous yeast M. pulcherrima, which produces metal chelators such as pulcherriminic acid, as a method of valorising the Miscanthus biomass into a range of products including a palm oil substitute and removing the metal waste into a smaller containable volume. This method will then be compared to hydrothermal processing of the Miscanthus waste
Impact The project demonstrated that the most suitable technique for metal recovery from MIscanthus grown on contaminated land was hydrothermal liquefaction (HTL). The HTL of the Miscanthus showed a reasonable bio-oil yield and in addition the majority of metals from the MIscanthus partitioned in the aqueous phase or the solid residue and could be recovered / recycled easily. Further work would need to be invested in increasing the bio-oil content, further optimisation to partition the metals into the solid residue while decreasing the carbon content.
Start Year 2016
 
Description New routes for expression of heme protein targets 
Organisation F. Hoffmann-La Roche AG
Department Roche Innovation Centre
Country Denmark 
Sector Private 
PI Contribution We funded and administered a Proof of Concept Award in a collaboration with Emma Raven of University of Leicester (POCMiB022)
Collaborator Contribution Heme-containing enzymes are a mainstay of industrial biotechnology, and the industry depends on fundamental improvements in methodology emerging from academic groups around the world to harness the potential of their investments in biopharmaceuticals, bioenergy, biocatalysis and drug design. For a number of complex reasons, the interactions between industry/biotechnology and academic laboratories are often less facile and less extensive than they could be, so that new (often specialist and/or unpublished) information is not transferred fluently to industrial partners. Our overall objective is to use this project to develop new refolding methodologies for expression of difficult (insoluble) heme protein targets, and to set up an on-going dialogue between industrial and academic partners with mutual cognate interests in specific heme enzyme targets. The methodologies that we develop will open up new avenues for industry partners in cases where they have intractable (insoluble) protein targets.
Impact In this project, new expression methods were developed for a range of different heme enzymes that are not readily expressed using conventional methodologies in E. coli. Targets from the Raven laboratory were used as a "test bed" for other heme systems. Human CLOCK protein was included amongst the targets, which is important in circadian control and thus an important drug target.
Start Year 2015
 
Description Novel disposable cell culture systems for microbial growth in metal-regulated environments 
Organisation Kirkstall Ltd
Country United Kingdom 
Sector Private 
PI Contribution We funded and administered a Business Interaction Voucher in a collaboration with Robert Poole of University of Sheffield (BIVMiB020)
Collaborator Contribution Growing cells and tissues in biotechnology requires a well-defined growth environment. Important factors include design of the culture apparatus and growth medium, which must provide all nutrients including metals in biologically accessible forms, but not in excess. Most growth vessels are metal or glass, but these can leach or adsorb metal ions. Synthetic materials, however, may be biologically inert and interact little with dissolved metals. This project will test the suitability for microbial growth of miniaturised growth chambers (Quasi-Vivo® by Kirkstall) that were developed for culturing mammalian cells. These chambers are made from biocompatible materials and, under flow conditions, mimic conditions in the body. We will grow bacteria in such chambers and test their ability to provide environments in which the metal concentrations available for growth will be varied from 'trace' to toxic. The work has potential for developing improved methods of cell culture in industrial biotechnology.
Impact This project aimed to test the suitability for microbial growth of miniaturised growth chambers (Quasi Vivo® by Kirkstall) that were developed for culturing mammalian cells. These chambers are made from biocompatible materials and, under flow conditions, mimic conditions in the body. We grew bacteria in such chambers and tested their ability to provide environments in which the metal concentrations available for growth could be varied from 'trace' to toxic (Hubbard et al., 1990; Hubbard et al., 1986). The work has potential for developing improved methods of cell culture in industrial biotechnology. We conclude that these vessels are suitable for growths involving metal-controlled conditions.
Start Year 2016
 
Description Optimisation of heme incorporation into a commercially important enzyme 
Organisation Biocatalysts Ltd
Country United Kingdom 
Sector Private 
PI Contribution We funded and administered a Business Interaction Voucher in a collaboration with Nick Le Brun of University of East Anglia (BIVMiB003)
Collaborator Contribution Metals as cofactors of proteins and enzymes are essential for all of life. Many metalloproteins have properties that are useful outside of the cell, for example in the synthesis of useful materials or medicines. However, to exploit this the metalloprotein must be purified with the metallo- cofactor fully incorporated, requiring a matching of protein and cofactor synthesis/insertion. In this project, the academic partners, who are experts in metalloprotein chemistry, will provide analyses of commercially generated metalloprotein samples to assist the commercial partner in solving a problem of highly variable cofactor insertion.
Impact The aims of the project were met in that it was determined that: • Cytochrome P450BM3 samples contained heme with properties characteristic of the P450 environment; • Samples were only ~50% loaded with heme. The poor protein expression and truncated form(s) of the protein were unexpected outcomes and point to some fundamental problems with the expression system.
Start Year 2015
 
Description Optimising metal acquisition by commercial metalloenzymes 
Organisation Biocatalysts Ltd
Country United Kingdom 
Sector Private 
PI Contribution We funded and administered a Business Interaction Voucher in a collaboration with Kevin Waldron of Newcastle University (BIVMiB002)
Collaborator Contribution Biocatalysts Ltd produce a number of metalloenzymes of commercial value through expression in bacteria and fungi. However, metal supply to these 'foreign' enzymes may not be optimal in these protein production hosts, so that a proportion of the commercial product is either bound to the 'wrong' metal, or lacks a bound metal ion altogether. Increasing the proportion of the enzyme that is correctly metal-loaded can directly increase profitability of metalloenzyme products. We will analyse the metal content of samples of the metalloenzymes produced by Biocatalysts. Where metal occupancy is found to be sub-optimal, we will work with the Biocatalysts technical team to test the effect of altering the bacterial/fungal growth conditions during enzyme production in order to increase the proportion of the target protein bound to the 'correct' metal ion. We will analyse the resulting enzyme samples to determine the effect of these changes on metal occupancy.
Impact The aim of the project was to perform analyses of the elemental composition of the enzyme products that are produced by Biocatalysts Ltd. for the commercial market to determine whether the metal occupancy of these enzymes was sub-optimal. If so, the expression conditions could be adjusted in an effort to improve the metal supply to the enzymes. A single class of enzyme was selected for analysis, a set of three variants of magnesium-dependent galactosidase. We hypothesised that magnesium's position at the bottom of the Irving-Williams series would make it likely that these enzymes could wrongly acquire a non-native metal ion from further up the Irving- Williams series, either during heterologous expression or during sample preparation. Such association with the non-native metal cofactor is likely to lead to inhibition of the enzyme activity. The project partner, Biocatalysts Ltd. supplied the PI (Newcastle University) with a set of samples of these three enzymes, manufactured over an ~18 month period, to enable a longitudinal comparison of the metal content of these preparations. Each sample was solubilised and then analysed for total metal content by inductively coupled plasma mass spectrometry (ICP-MS). As a positive control, this confirmed the presence of abundant magnesium in each of the preparations (the product is exchanged into a buffer containing elevated magnesium concentration prior to drying). Importantly, in addition all preparations were found to contain significant trace quantities of (in order of amount detected) iron, zinc, copper and nickel. As the target enzymes in these samples are not homogenous however, as demonstrated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis, further analysis was required in order to determine the amount of this trace metal detected in the preparations that was associated with the enzyme. Each sample was resolved by liquid chromatography, and the resulting fractions were again analysed for elemental composition by ICP-MS and for protein by SDS-PAGE. This method allowed accurate quantitation of the enzyme-associated metal, by separating it from unbound metal ions and from metals bound to contaminating proteins. The results of these analyses were that small amounts of copper, zinc and nickel (though not iron) were found to be present on the target enzyme in the preparations. The quantity of contaminating metals varied slightly between the longitudinal samples, and interestingly also varied between the three enzyme variants suggesting possible differences in their metal-binding properties. Importantly, though, the absolute quantity of these contaminating metals was low as a percentage of the total enzyme present (ranging from 3.8 - 12.8% occupancy), suggesting that they would make only a minor diminution of the total enzyme activity of the enzyme preparations.
Start Year 2015
 
Description Proline Hydroxylases for Biocatalysis 
Organisation UCB Pharma
Department UCB Celltech
Country United Kingdom 
Sector Private 
PI Contribution We funded and administered a Proof of Concept Award in a collaboration with Chris Schofield of University of Oxford (POCMiB016)
Collaborator Contribution Biocatalysis is a successful method for the industrial production of small- or macro-molecules and can enable production of molecules that are difficult, expensive or often impossible to synthesise. Metallo-enzymes (biological molecules that use metals to catalyse chemical reactions) are abundant in nature and are used by microbes for biosynthesis of small-molecules such as antibiotics or for the modification of biological macromolecules. The long term objective of this project is to develop and provide access to metallo-enzymes useful for biocatalysis in cells on an industrial scale. In collaboration with UCB Celltech we will focus on enabling discoveries that will support useful bio- catalysis and bio-transformations for the generation of end products or intermediates involved in the manufacture of biofuels, pharmaceuticals, fragrances, or flavours and also for the late stage diversification of tools for drug discovery (i.e. compound libraries) in a sustainable manner (i.e. Green Chemistry). There is a current lack of accessible libraries of oxygenase enzymes suitable for use in biocatalysis and little information on how their activity is limited by metal binding in cells. Our wide range of resources will allow us to efficiently explore and engineer novel methods for accessing industrial production of new chemical entities.
Impact Enzymes are incredibly powerful biological catalysts that already have widespread applications in industry and the home. In microorganisms they make an enormous range of natural products, some of which are incredibly useful medicines, e.g. the penicillins. One of the strategies nature uses in the biosynthesis of many natural products is to modify a common intermediate to give multiple other products, which may have very different biological functions. Harnessing the power of such late stage modification has potential to generate many molecules from a single drug candidate. Such a process could expedite drug discovery by enabling the efficient discovery of optimised molecules. However, the natural enzymes are often not suitable for such use, because they have evolved to catalyse the production of specific products. What is needed are less selective enzymes, that can be subsequently optimised to be made selective if need be. Our BBSRC NIBB work concerned studies on a family of metal dependent enzymes that add oxygen, or sometimes chlorine or bromine, atoms to drug-like small-molecules and proteins. To accomplish these reactions the oxygenases employ oxygen from the atmosphere and iron in its ferrous form, as well as a common small-molecule metabolite, 2-oxoglutarate (2OG). Our BBSRC NIBB project principally focused on a specific subfamily of these enzymes that catalyse the addition of oxygen, or hydroxylation, to proline, an amino acid with a 5-membered ring. In one line of investigation we explored the proline hydroxylases for its selectivity towards different ring sizes and substitutions. We found that they can catalyses the hydroxylation of an unexpectedly wide range of rings, including tricyclic ring structures; some of these products are precursors for conversion into potential antibiotics. We also explored how these hydroxylases bind iron, using both assays for product formation and by X-ray crystallographic analyses of 'mutant' enzyme structures. Interestingly, we found that the proline hydroxylases can work with only two, rather than the normal three, points of attachment (ligand) of iron to the protein. These results inspired us to study metal binding by enzymes in cells (employing mass spectrometry) and to study variations on iron binding by other types of hydroxylases. In one case we found the hydroxylase can work with only one protein ligand.
Start Year 2015
 
Description Site-specific bioconjugate chemistry for antibody-nanoparticle conjugates 
Organisation Aeirtec
Country United Kingdom 
Sector Private 
PI Contribution We funded and administered a Seeding Catalyst Proof of Concept Award with Lu Shin Wong of University of Manchester (ISCFPOCMiB032)
Collaborator Contribution Fluorescence-based immunosorbent assays have become a key technology for the detection and quantification of biomolecules, and have found application in a range of fields from the testing of microbial contamination (in water, chemical, food and drug production), biomarkers (in medical diagnostics and drug discovery) and in biomedical imaging. This project will develop production methods for metal nanoparticle-antibody conjugates that are robust and scalable, which would be needed for commercial implementation. These hybrid metal-biomolecule materials offer advantageous spectroscopic properties that could greatly increase detection sensitivity. It is envisaged that such nanoparticle-antibody conjugates will themselves be highly sought-after industrial biotechnology products for the applications noted above. In addition, they will be utilised in the development of new biologics and peptides. Indeed, the development of such products will be greatly enhanced and accelerated by incorporation of very sensitive and faster monitoring assays that these conjugates will enable.
Impact Project on-going
Start Year 2017
 
Description Studies into the Uptake and Distribution of Metal Oxide Nanoparticles in Plants 
Organisation Croda Europe Ltd
Country United Kingdom 
Sector Private 
PI Contribution We funded and administered a Business Interaction Voucher in a collaboration with Neil Bricklebank of Sheffield Hallam University (BIVMiB019)
Collaborator Contribution The uptake of metals is essential for the growth and development of healthy plants. Plants obtain the metals they need from soil or from fertilizers applied to the growing plant. One of the most important metals is zinc which is found in many metalloenzymes. Zinc is also essential for humans who gain it from the grains and vegetables which they eat. In this project we will study the effect of zinc, in the form of a formulation containing zinc oxide, on the growth of plants and use a new analytical tool, known as Laser Ablation-Inductively Coupled Plasma-mass spectrometry (LA-ICP-MS), to study the uptake and distribution of zinc in plants. This project fits within the remit of the BBSRC-NIB because it will investigate metal availability, uptake and assimilation into biomolecules required for bio-energy production. It will also enable us to develop new tools and technologies for studying metals in biological systems.
Impact Project on-going
Start Year 2016
 
Description Tailoring the in planta synthesis of metal nanoparticles for production of high-value catalysts 
Organisation Yorwaste Ltd
Country United Kingdom 
Sector Private 
PI Contribution We funded and administered a Proof of Concept Award in a collaboration with Neil Bruce of University of York (POCMiB024)
Collaborator Contribution Platinum group metals (PMGs) are used in an ever-expanding arrange of technologies and demand is spiralling upwards. PGMs are rare, exist in low concentrations and expensive to mine. It is essential that these metal reserves are utilised and recycled responsibly, not dispersed and lost into the environment. Plants can take up metals from their environment, and, in the case of PGMs, can deposit them as nanoparticles within their tissues. Nanoparticles have remarkable properties, when compared to the bulk of the same metal, which have been exploited. For example palladium nanoparticles are important catalysts for many pharmaceutical applications. Although currently synthesised chemically, we have shown that plants containing palladium nanoparticles can also be used to make efficient biocatalysts. These biocatalysts utilise carbon-neutral plant biomass, reduce processing steps by using the nanoparticles together with the plant material and concentrate valuable metals from waste sources. The addition of specific peptides (very small proteins) to solutions of metals increases nanoparticle formation, and alter size and shape; factors that can be used to optimise catalysts for different processes. This proposal is to investigate if expression of peptides in plants can be used to increase the formation, and control the size of, plant-derived biocatalysts.
Impact We have achieved our objectives to investigate NP formation using synthetic peptides and analyse catalytic activity in the subsequently pyrolysed NP-containing plant biomass. Our promising ICP-OES, TEM and catalysis results demonstrate that the expression of synthetic peptides in plants can be used to alter gold NP size and subsequent catalytic activity in planta. As part of our third objective, to determine if plants could be used to selectively take up PGMs from sweeper wastes, we have shown that sweeper wastes contain detectable levels of valuable metals. However, our studies show that further work is needed to understand the phytotoxicity behind these wastes so that they can be optimised to allow plant growth. Our wider studies indicate that synthetic biology could be used to develop plants that can selectively take up PGMs from sweeper wastes.
Start Year 2015
 
Description GCRF B12 Bioprocess Hub Social Sciences Scoping Workshop 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Outline proposal submitted to GCRF Research hub call.
Year(s) Of Engagement Activity 2017
 
Description Cafe Scientific Public Lecture entitled: The elements of life: A metal centered view of biology and biotechnology 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Members of the public attended the Cafe Scientific Lecture by Professor Nigel Robinson entitled: The elements of life: A metal centered view of biology and biotechnology. There was a 45-minute questions and answer session at the end including questions and the lecture was summarised on Cafe Scientific's facebook page: https://www.facebook.com/cafescientifiquestocktonontees
Year(s) Of Engagement Activity 2018
URL http://www.cafesci-stockton.org.uk/
 
Description Characterising and Utilising Rhodococcus Enzymes for Industrial Biotechnology 
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 We organised a workshop that brought together academics and industry to discuss funding opportunites: http://prospect.rsc.org/MiB_NIBB/charactering-and-utilising-rhodococcus/
A shared resource for moleuclar biology tools in Rhodococcus was set-up following the event: http://prospect.rsc.org/MiB_NIBB/wp-content/uploads/2017/01/Workshops-kickstart-Rhodococcus-molecular-toolkit.pdf
Year(s) Of Engagement Activity 2015
URL http://prospect.rsc.org/MiB_NIBB/wp-content/uploads/2016/10/Industrial-biotechnology-workshops-kick-...
 
Description EuroBIC bioinorganic chemistry conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Disseminated knowledge about the cell biology of metals which sparked questions and discussion.
Year(s) Of Engagement Activity 2018
URL https://www.birmingham.ac.uk/facilities/mds-cpd/conferences/eurobic/index.aspx
 
Description FASEB, Lake Tahoe, Trace Metals in Health and Disease 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Dissemination of knowledge about the cell biology of metals which sparked questions and discussion.
Year(s) Of Engagement Activity 2018
 
Description GCRF B12 Bioprocess Hub Pre-Planning Workshop 
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 To discuss potential scoping workshops for a GCRF pre-application
Year(s) Of Engagement Activity 2017
 
Description GCRF B12 Bioprocess Hub Scoping Workshop 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Outline proposal submitted to GCRF Research hub call
Year(s) Of Engagement Activity 2017
 
Description IBioIC 5th Annual Conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Opportunity for promotion of phase 2 Metals in Biology Network "The elements of Bioremediation, Biomanufacturing and Bioenergy (E3B): Metals in Biology (MiB), a BBSRC NIBB"
Year(s) Of Engagement Activity 2019
URL http://www.ibioic.com/news_and_events/annual_conference/d1137/
 
Description MRC/BBSRC GCRF Vaccines in Research and Development Networks 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact To encourage recently funded MRC/BBSRC Networks to identify commonalities across different Networks and to hear from PIs who have experience in running existing BBSRC Networks.
Year(s) Of Engagement Activity 2017
 
Description Metal Circuits, Synthetic Biology and C1 Gases Workshop 
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 We organised a workshop bringing together academics and industrialists to discuss research opportunities: http://prospect.rsc.org/MiB_NIBB/metals-synthetic-biology-c1-gas-scoping-workshop/

Durham University and Green Biologics Ltd submitted a business interaction voucher (BIVMiB035) following the workshop which was funded and othere collaborations have been developed.
Year(s) Of Engagement Activity 2015
URL http://prospect.rsc.org/MiB_NIBB/wp-content/uploads/2016/10/Researchers-come-together-to-push-green-...
 
Description Metal-Related Antimicrobials Workshop 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact We organised a workshop to bring together academics and industry to discuss opportunities in collaborative reseach: http://prospect.rsc.org/MiB_NIBB/wp-content/uploads/2015/11/Final-programme.pdf

Led to new collaborations and a volume on "Microbiology of Metals Ions" 2016 volume 70: https://www.elsevier.com/books/microbiology-of-metal-ions/author/978-0-12-812386-7
Year(s) Of Engagement Activity 2015
URL http://prospect.rsc.org/MiB_NIBB/wp-content/uploads/2017/01/Metals-in-Biology_Elements-of-the-bioeco...
 
Description Metals in Biology BBSRC NIBB twitter account and website 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact The Metals in Biology BBSRC NIBB websiite supports about 500 members with a third from outside academia, as does the linked twitter feed.
Year(s) Of Engagement Activity 2015,2016,2017,2018
URL http://prospect.rsc.org/MiB_NIBB/
 
Description Metals in Biology Community Event 
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 We organised a workshop involving academic and industry to show case funded projects as exemplars to instigate future collaborations.
Year(s) Of Engagement Activity 2016
URL http://prospect.rsc.org/MiB_NIBB/metals-in-biology-network-community-meeting/
 
Description NIBB phase 2 call 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 Expression of interest application submitted for NIBB phase II
Year(s) Of Engagement Activity 2017
 
Description NJR Bangalore 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Invited keynote speaker, 6th International conference on Metals in Genetics, Chemical Biology and Therapeutics, Bangalore, India.
Year(s) Of Engagement Activity 2016
 
Description NJR GRC Vermont 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Dissemination of knowldege about the Cell Biology of Metals which sparked questions and discussion.
Year(s) Of Engagement Activity 2017
URL https://www.grc.org/cell-biology-of-metals-conference/2017/
 
Description NJR Queen Mary University of London 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Undergraduate students
Results and Impact Delivered the Wills lecture. This sparked questions afterwards followed by requests for information including ICP-MS analyses (for example).
Year(s) Of Engagement Activity 2017
URL http://www.sbcs.qmul.ac.uk/research/researchseminars/speciallectureseries/#4
 
Description NJR San Diego ACS conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Invited speaker, American Chemical Society National Meeting Spring 2016, San Diego California.
Year(s) Of Engagement Activity 2016
 
Description NJR TUM Munich 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Delivered lecture and interacted with postgraduate students and staff at a series of meetings.
Year(s) Of Engagement Activity 2017
 
Description National Institutes of Health, Wshington DC, presentation 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Disseminated knowledge of the cell biology of metals which sparked questions and discussion.
Year(s) Of Engagement Activity 2018
 
Description Nature Microbiology Community Blog 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Invited blog linked to a paper in Nature Communications
Year(s) Of Engagement Activity 2017
URL https://naturemicrobiologycommunity.nature.com/users/71254-deenah-osman/posts/24964-sensing-the-diff...
 
Description Overseas visit to an industrial production facility 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact We funded a member to visit an industrial production facility in the USA There is an agreement that company will support of a BBSRC Link grant application.
Year(s) Of Engagement Activity 2017
 
Description Penn State Summer Symposium in Molecular Biology 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Disseminated knowledge of the cell biology of metals which sparked questions and discussion.
Year(s) Of Engagement Activity 2018
 
Description Tetrapyrroles GRC, Rhode Island 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Disseminated knowledge of the cell biology of metals which sparked questions and discussion.
Year(s) Of Engagement Activity 2018
URL https://www.grc.org/chemistry-and-biology-of-tetrapyrroles-conference/2018/