Author Archives: Jen Vanderhoven

Pseudomonas stutzeri as an alternative host for membrane proteins

Pseudomonas stutzeri as an alternative host for membrane proteins


Studies on membrane proteins are often hampered by insufficient yields of the protein of interest. Several prokaryotic hosts have been tested for their applicability as production platform but still Escherichia coli by far is the one most commonly used. Nevertheless, it has been demonstrated that in some cases hosts other than E. coli are more appropriate for certain target proteins.


Here we have developed an expression system for the heterologous production of membrane proteins using a single plasmid-based approach. The gammaproteobacterium Pseudomonas stutzeri was employed as a new production host. We investigated several basic microbiological features crucial for its handling in the laboratory. The organism belonging to bio-safety level one is a close relative of the human pathogen Pseudomonas aeruginosaPseudomonas stutzeri is comparable to E. coli regarding its growth and cultivation conditions. Several effective antibiotics were identified and a protocol for plasmid transformation was established. We present a workflow including cloning of the target proteins, small-scale screening for the best production conditions and finally large-scale production in the milligram range. The GFP folding assay was used for the rapid analysis of protein folding states. In summary, out of 36 heterologous target proteins, 20 were produced at high yields. Additionally, eight transporters derived from P. aeruginosa could be obtained with high yields. Upscaling of protein production and purification of a Gluconate:H+ Symporter (GntP) family transporter (STM2913) from Salmonella enterica to high purity was demonstrated.


Pseudomonas stutzeri is an alternative production host for membrane proteins with success rates comparable to E. coli. However, some proteins were produced with high yields in P. stutzeri but not in E. coliand vice versa. Therefore, P. stutzeri extends the spectrum of useful production hosts for membrane proteins and increases the success rate for highly produced proteins. Using the new pL2020 vector no additional cloning is required to test both hosts in parallel.

Read the full here. 

Government Publishes paper on ‘Collaboration on science and innovation: A FUTURE PARTNERSHIP PAPER’

Government Publishes paper on ‘Collaboration on science and innovation: A FUTURE PARTNERSHIP PAPER’

The United Kingdom wants to build a new, deep and special partnership with the European Union. This paper is part of a series setting out key issues which form part of the Government’s vision for that partnership, and which will explore how the UK and the EU, working together, can make this a reality.

Each paper will reflect the engagement the Government has sought from external parties with expertise in these policy areas, and will draw on the very extensive work undertaken across Government since last year’s referendum. Taken together, these papers are an essential step towards building a new partnership to promote our shared interests and values.

Read the paper here.

CBMNet awarded – Industrial Biotechnology Catalyst Seeding Funding

CBMNet awarded Industrial Strategy Challenge Funding

We are pleased to announce that CBMNet has been awarded £150,000 from the Industrial Strategy Challenge Fund in the form of an Industrial Biotechnology Catalyst Seeding Award.


£100,000 of this has just been awarded to Dr Alan Goddard, Aston University, in the form of a CBMNet FLAGSHIP award – Consolidation, Integration and Critical Mass Building- Optimizing membrane function in the Clostridial ABE process. This project involves collaboration between Dr Goddard, Dr Robert Fagan (University of Sheffield), Professor Gavin Thomas (University of York), Dr Peter Chivers (Durham University) and Green Biologics Limited.

The overall objective is to consolidate GBL-CBMNet interactions facilitated through five CBMNet Business Interaction Vouchers, one academic-industrial exchange and one Proof-of-Concept award, along with a Metals In Biology Business Interaction Voucher, to obtain a more holistic picture of the role of membrane dysfunction in restricting n-butanol yields.  The project will synergise the independent research of four PIs, who have not previously worked together, and GBL to build critical mass and generate new data to underpin substantial funding applications in the near future e.g. BBSRC-LINK, IPA, and Catalyst-type projects.  GBL achieved a significant milestone in 2016 by commissioning the first new ABE plant in the US since 1938.  This success has been achieved despite limited understanding of the physiology of Clostridia used in the process, especially the cell membrane that is critical for the stability and robustness of the strain in industrial fermentations, in particular nutrient uptake and metabolite efflux. This project will contribute to fundamental understanding of the membrane and identify gene targets for performance improvements.

Over the past few years, GBL have collaborated with each of the academic partners to developing a better understanding of the solventogenic Clostridia in a number of important areas.  GBL have recently opened their first large scale n-butanol plant, Central Minnesota Renewables in the US and have patented a highly efficient proprietary genome editing technology. By combining the academic expertise with GBLs technology, this project will provide novel routes for strain development that can impact on a number of process parameters. For example, higher butanol concentrations in the fermentation may reduce the likelihood of contamination, lead to a more efficient cell separation process, and reduce the significant costs associated with distillation, as well as improve the water balance of the plant (which has an environmental impact).  By understanding the butanol stress response at a number of levels (membrane lipids, proteins, transporters, metalloenzymes), strain improvement strategies can be better targeted.


£25,000 has been awarded to Dr Hoiczyk,  University of Sheffield, and GlycoMar, for their project ‘Use of Membrane complexes for the production of microalgal polysaccharides’. 

The utilisation of microalgae for production of high value chemicals has seen major advances in the last decade. A key limitation is the yield of target products, which can restrict their commercial viability. This is particularly the case with exopolysaccharides (EPS), which are produced by many microalgae and represent a large biochemically diverse resource. GlycoMar Ltd has developed pilot scale production of a microalgal EPS, which has been patented for use in healthcare and skin care. Although methods for the production of the EPS exist, increased yield would greatly improve its industrial potential. The EPS appears to be secreted through the decapore apparatus in the cell’s envelope although its precise role in synthesis, maturation, and secretion are currently unclear. The proposed project aims to isolate and purify the membrane pore complex with the goal to identify its protein components. Our working hypothesis is that the complex multi-layered decapore complex is more than a simple secretion portal and is crucial for the synthesis, maturation, and derivatisation of the exported polymer. Therefore, we expect that once the membrane protein components of the decapore are known, future work could address the deletion and/or overexpression of genes coding for these individual components to influence polymer production. This strategy should provide the basis for the identification of overproducing strains that would open up the route for larger-scale application of the identified EPS product.

The outcomes of the project have commercial potential, in terms of the application of the product, but also potentially as a platform technology utilising membrane pore complexes as production systems for microbial polysaccharides. This has the capacity to open the production and utilisation of a very wide range of polysaccharide products, which are currently limited by yield, culturing or handling issues.


£25,000 has been awarded to Dr Vincent Postis, Leeds Beckett University, and English Spirit Distillery, for their project ‘Bioenergy production from biorefineries waste using super yeasts’.

Due to the constant increase in energy prices, demand for cheap/renewable energy has escalated. The bioethanol sugarcane raffineries generates large amounts of wastes: bagasse (solid) and vinasse (liquid). For every litre of distilled ethanol, 10-15L of vinasse, are generated. While bagasse is used as carburent in electric generators, vinasse is disposed in agricultural fields or at sea leading to major environmental issues.

Vinasse main component is glycerol. Glycerol is also accumulated as a by-product in diverse types of biorefineries. Therefore such by products can be recycled at low costs for fermentation processes by yeast.

To reduce this environmental negative impact, this project therefore proposes to generate yeasts which will be able to transform this industrial waste into a green biofuel. Our aim is then to select/generate yeast as efficient ‘’cell factories’’ capable of converting glycerol-based products, such as vinasse, into large amounts of added-value products, namely free fatty-acids/neutral-lipids. Those can then be converted in biofuel of second generation or used for the anti-foam agents production.

Due to the accumulation of vinasse as a by-product in diverse types of biorefineries, this strategy would emerge as instrumental and cost-effective for the yeast- based fermentation industries. In addition,  it will also reduce the severe environmental impact of bioethanol sugarcane raffineries. In conclusion, yeast fermentation of vinasse (in this case) besides contributing to the recycling of waste, will also reduce the toxicity of this by-product.

Information Days on 2018-2020 Horizon 2020 Calls for Proposals

Information Days on 2018-2020 Horizon 2020 Calls for Proposals

The European Commission will organise a number of information days in Brussels on the upcoming 2018-2020 calls for proposals in the last Work Programme of Horizon 2020 (to be published in October). These events will provide information on the content of the calls and will often be combined with dedicated brokerage events to support prospective applicants with finding partners for projects. The following events are planned in the coming months

Furthermore, a series of national events is also planned by Innovate UK and the Enterprise Europe Network. A list of planned and confirmed events can be found on the Innovate Knowledge Transfer Network’s website.

Synthetic Biology Start-up company survey results

Synthetic Biology Start-up company survey results

A survey of synthetic biology company start-ups in the UK is published today by SynbiCITE, the UK’s national centre for the commercialisation of synthetic biology. The study reveals a vibrant ecosystem sustaining a thriving and rapidly growing sector of the bioeconomy.

Highlighting sources of innovation and entrepreneurship, and exploring R&D, technology transfer, investment and growth, the survey covers UK starts-ups between 2000 and 2016. A close look at 146 synthetic biology companies shows that the number of start-ups has doubled every five years during the survey period. Looking ahead, it seems likely that with the right support, the UK synthetic biology ecosystem will be able to model itself on the self-sustaining clusters found in the US in Silicon Valley, CA, and Cambridge, MA.

“Confirming the arrival of a new innovation ecosystem demands evidence: proof that variables ranging from investment, pipeline infrastructure, to talent and education are established and stable”, said Dr. Stephen Chambers, CEO, SynbiCITE. “We believe the industry has reached a critical mass of companies, showing a healthy churn of attrition and creation. Roughly 76% of all the start-ups founded in the survey period are still active and with the continuation of an effective national strategy in the future, this ecosystem will undoubtedly thrive, creating jobs and wealth while sustaining the UK’s leading role in the field.”

Professor Richard Kitney, Co-Director, SynbiCITE commented: “As you’d expect of an industrial sector at a relatively early stage, synthetic biology in the UK will continue to require public as well as private investment. This will be essential to translate today’s research into the exciting industrial products of the future that promise to make such a positive difference to our world in health, energy, materials and the environment.”

“Patience will be crucial,” added Professor Paul Freemont, Co-Director, SynbiCITE, “the UK government has shown great support for synthetic biology, investing £300m in between 2009-2016. We have no doubt this industry has a bright future, and look forward to expanding our work with world leaders in synthetic biology in the USA and Asia for example where so much exciting work is being done”.

Take a look at the survey UK Synthetic Biology Start-up Survey 2017

JRC Bioeconomy Knowledge Centre Launched

​The European Commission has launched a new Joint Research Centre (JRC) Bioeconomy Knowledge Centre (BKC). The BKC is the fourth Knowledge Centre to be launched by the JRC in recent years and aims to bring information and data from a wide variety of sources together in one place, in an open format. In doing so, the BKC aims to help provide a more coherent picture of knowledge on the bioeconomy across Europe, as well as identify gaps and bring information closer to the general public and policy makers. Following on from the work of the JRC Bioeconomy Observatory, it is hoped that the BKC will play a key role in the development of EU policies in the short, medium and long term.

WE NEED TO TALK ABOUT GENE TECH: CBMNet Management Board Member Prof Susan Molyneux Hodgson


CBMNet Management Board Member Professor Susan Molyneux Hodgson spoke at the Hay Litery festival last night in the session ‘We Need to talk about Gene Tech’.

Why does public debate and policy treat the application of genetic technology differently when we are discussing medicine and food? Why is our concept of what is ‘natural’ so controversial and the idea of GM food so alarming? Scientists and sociologists come together with Daniel Davis to discuss what’s being ventured and how it is perceived.


CBMNet Proof-of-Concept and Vacation Scholarship projects awarded

CBMNet Proof-of-Concept and Vacation Scholarship projects awarded

Following on from our latest Proof-of-Concept and Vacation Scholarship funding calls we are pleased to announce that we have funded 6 projects.

Proof-of-Concept: Newcastle University and Ingenza – L-form technologies: a novel platform for therapeutic protein production.

Many human proteins are used for the treatment of a wide variety of diseases and many more have the potential to be developed as drugs if they can be produced in sufficient quantities. To avoid contamination with agents (prions and viruses) that can cause serious diseases, these proteins are produced in bacterial or animal cells, rather than extraction from human tissue. There is therefore a need to produce sufficient amounts of novel human proteins for preliminary analyses and, if see to have therapeutic potential, for clinical trials. Production systems based on the bacterium, Escherichia coli, is the first choice system for producing such proteins. However, about one third of all human proteins are not capable of being synthesised in E. coli production systems and alternative systems have to be used. This particularly applied to proteins that are secreted from human cells and that have disulphide bonds in their final structure. Disulphide bonds are formed after synthesis and secretion from the cell and involve the formation of bonds between two amino acid residues (cysteine) in the protein. The collaboration between Newcastle University and Ingenza is aimed at relieving known bottlenecks in the production of therapeutic proteins by using a bacterium, Bacillus subtilis, that can be switched to a wall-less state that removes a major bottleneck to protein secretion. The project involves generating such strains and evaluating their performance under commercial biomanufacturing conditions. If successful, the strains could expand the range of therapeutic proteins available for the treatment of specific diseases.


Proof-of-Concept: University of Sheffield and Excivion – Viral antigen production for diagnosis and vaccine-mediated disease prevention by rational glycoengineering-mediated protein secretion in a mammalian cell factory.

The global incidence of mosquito-borne flavivirus induced disease such as dengue has increased exponentially over the last four decades. Fuelled by conditioning factors such as rapid urbanisation, demographic change, large-scale migration, and travel, dengue is now endemic in most countries of the tropics, and about 925 million people now live in urban areas that are at high risk of dengue infection. More recently Zika, another mosquito-borne virus, has been implicated in the causation of microcephaly in infants, and encephalitis in adults. In conjunction with concerns about expanding mosquito habitats, and global movement of humans on significant scales, there is a compelling need to find new solutions for the prevention this family of diseases, as well as diagnoses of extent and type of exposure, as this information has significant implications for subsequent treatment and strategies for prevention. A particular challenge in this family of diseases concerns the fact that while initial exposure gives rise to mild disease, subsequent exposure to other related viral strains can result in severe illness and death, as a consequence of known mechanisms associated with the immune system, which act to make the disease symptoms considerably worse. It follows that both specific diagnosis and new vaccine designs will be required to control these diseases effectively. This project aims to exploit specific cell factories, together with our understanding of key protein structures in flaviviruses, to generate novel non–natural proteins which will have the capability both of enabling strain-specific diagnosis, as well as inducing protection in vaccination programs of carefully screened individuals, without predisposing to haemorrhagic fever (which is a recognised risk for existing vaccine designs).

Vacation Scholarship: University of Kent – Functional characterisation of a putative succinate efflux pump from Corynebacterium glutamicum.

Succinate is a key precursor in the production of biodegradable plastics and fabrics. The majority of industrially produced succinate is derived from petrochemical precursors. However, several microbial species have been engineered to maximise succinate production during fermentation. A succinate efflux pump, SucE, was recently identified in C. glutamicum, which substantially increases succinate production when overexpressed. However, the structure, mechanism, energetics and substrate specificity of this transporter remain unknown. A comprehensive understanding of SucE’s transport mechanism could allow us to manipulate this transporter and/or it’s energy source to make succinate (and possibly other dicarboxylic acid) efflux more efficient, potentially increasing the succinate yield of C. glutamicum. This project fits perfectly within the remit of CBMNet as it is centred on understanding how an industrially important chemical is transported across the bacterial membrane. The aims of this project are to; 1) clone sucE from C. glutamicum into an E. coli expression vector, 2) optimise the expression and purification conditions, 3) assay SucE function using in vivo succinate accumulation assays, and 4) reconstitute SucE into liposomes for in vitro transport assays.

Vacation Scholarship: University of Leeds – Durable vesicles for stabilisation of membrane proteins in biotechnology

Hybrid vesicles, which combine the biofunctionality of phospholipids with the stability of block copolymer membranes, can enhance the functional durability of membrane proteins. We have demonstrated that hybrid vesicles extend the functional half-life of cytochrome bo3 from 1-2 weeks in proteoliposomes to 4-6 months in hybrids (Chem. Commun. 52, 11020, 2016). The student will aim to:
1. Successfully reproduce functional reconstitution of cytochrome bo3 into hybrid vesicles (training).
2. Characterise proton pumping by cytochrome bo3 in hybrid vesicles using a pH-sensitive fluorophore.
3. Test whether (i) using a different triblock copolymer, or (ii) protein reconstitution using SMALPs has advantages over our existing materials and protocols.
4. (If time permits) co-reconstitute cytochrome bo3 with F-ATPase to create a proto-organelle to generate ATP.

Vacation Scholarship: University of Nottingham – Purification of membrane transporters to identify topology and binding sites by mass spectrometry.

Multidrug (MDR) pump show an unusually broad substrate specificity, which is poorly understood. This lack of knowledge undermines their potential use in IBBE applications where this polyspecitify may be harnessed in engineered microorganisms. Our group has an excellent track record in working with MDR pumps and now wish to harness recent advances in protein labelling and mass spectrometry (MS) to see if we can map the polyspecific binding sites of MDR pumps using MS.

Vacation Scholarship: University of Kent – Vitamin B12 into yeast.

We will determine if Saccharomyces cerevisiae can transport vitamin B12 (cobalamin) into the cell. A recent paper concerning the production of butanol isomers suggests that this is possible (see Curr Opin Biol 2015, 33: 1-7) but we would like to provide definitive evidence and quantify the levels of cobalamin that can be accumulated. We will investigate the ability of S. cerevisiae to absorb B12 using two complementary approaches. Firstly, we will grow the yeast in the presence of a range of different concentrations of vitamin B12. After growth, the cells will be thoroughly washed and then lysed. The supernatant from the lysed cells will then be applied to a very sensitive B12-dependent microbial plate assay. This will allow the student to compare B12 uptake against external concentrations of added B12. Secondly, we have made a number of B12 fluorophores, whereby we have attached a fluorophore to the B12 mainframe. These fluorescent probes are taken up by bacteria, algae and worms. With our new confocal microscope system we will be able to follow the movement and accumulation of the fluorophore within the S. cerevisiae also.


£25,000 Proof-of-Concept Available

£25,000 Proof-of-Concept Available

As a result of our last Proof-of-Concept call, and some under-spend, we have £25,000 of PoC funding remaining (@80%fEC). Therefore, we are inviting applications from our members for a final PoC project.

Details: To allow consortia to generate the preliminary information required to establish the feasibility of their proposed approaches, with the target of generating competitive bids to other relevant funding calls.
Important dates: CALL OPEN UNTIL 12 NOON MAY 31st 2017
Amount available: £25,000 (@80% fEC) to fund ONE project.
Further information

CBMNet goes International

We are pleased to announce that we have successfully been awarded funding from the BBSRC to expand CBMNet activities across the globe!

We are headed to New Zealand for a workshop aimed at ‘Exploiting Algae and marine biomass for Industrial Biotechnology and Bioenergy’

In August 2017, CBMNet and PHYCONET members are heading to the Cawthron Institute, Nelson, New Zealand. The workshop focuses on a deeper understanding of the bottlenecks in producing polysaccharides, other bioactives and functional food ingredients from marine biomass. We will identify the challenges in characterisation, production and commercialisation, with the aim of generating project ideas to overcome yield-restricting bottlenecks in this process. The workshop will provide a forum for leading academic and industrial practitioners to establish a strong evidence-based assessment of our current understanding of the challenges and these will be carried forward by establishing new partnerships and collaborations.

CBMNet Co-Director, Dr Gavin Thomas, is planning a UK-Taiwan exchange to understand the structure & function of bacterial transporters for Industrial Biotechnology & Bioenergy.

The transport of small molecules across bacterial membranes via active transport is an underexploited component of metabolic engineering and has great potential in improving processes in industrial biotechnology and bioenergy (IBBE). To be able to rationally improve transporter function, knowledge of the structure/function relationships within these proteins is crucial. In this network we seek support to build a UK-Taiwan network of researchers sharing expertise in the study of transporters relevant for IBBE. The main component of the collaboration is the exchange of knowledge, in terms of understanding the function and structure of membrane transport proteins, in particular in scoping new research into IBBE-relevant targets and developing new techniques and expertise using TRAP transporters. This will be supported through three main activities; A Kick-off meeting July in York, several research exchanges and a grant writing and future perspectives meeting.

We’re welcoming Canadian and European Colleagues to Establish International Partnerships in Industrial Biotechnology and Bioenergy in improved glycoform-based biopharmaceutical production in plants.

This September we are hosting a 3 day workshop which will provide a forum to facilitate collaboration between international partners in developing ‘non-traditional’ expression systems, focusing on plant and yeast cell factories, developing capabilities that can translate to existing and future platform technologies for production of biopharmaceuticals. A key goal of this meeting is to explore opportunities for collaboration and funding within the BBSRC priority funding area ‘New approaches to industrial biotechnology’. Specifically, this workshop will focus on a deeper understanding of the native glycosylation machinery and the manipulation thereof for the production of biopharmaceutical products with enhanced or even novel functions.  The programme will draw on the extensive experience in plant and yeast-based systems and approaches developed to achieve predictive modification of glycoform. Invited speakers have been chosen based on their expertise in different areas of plant and yeast biology, glycosylation, protein biochemistry and cell trafficking.  A key aim of the workshop is to develop a new network of research groups interested in industrial biotechnology and identify common research goals for responsive mode funding opportunities.


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