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AMBIOCAS Informe resumido

Project ID: 245144
Financiado con arreglo a: FP7-KBBE
País: United Kingdom


Executive Summary:

AMBIOCAS was a FP7 Cooperation Project that ran from 01-01-2010 to 30-06-2013. The key objective was to demonstrate the use of biotechnological enzymatic cascades in the synthesis of target chemical molecules that were of industrial relevance. Previous to this work few enzymatic cascades had been demonstrated using redox enzymes because co-factor recycling to achieve the high conversions was not available. This was of particular importance for industrial use of enzymatic cascades. The successful development and implementation of this technology would then lead to a more economic and less environmentally damaging European chemical manufacturing industry.

Amines are important chemical intermediates and are used in polymers, pharmaceuticals and fine chemical products and found in many pharmaceuticals and household products. One product target that demonstrated the overall value of this approach was 6-aminocaproic acid that is used by industry in the production of polyamide Nylon type polymers which are produced in very large quantities in Western Europe. These versatile materials are used in numerous applications, such as fibres for apparel and furnishing textiles, industrial yarns, floor coverings, and resins for engineering plastics as well as films for food packaging. The conventional chemical production route involves conversion of phenol into cyclohexanone, formation of cyclohexanone oxime, Beckman Rearrangement with strong acid to give caprolactam followed by thermal polymerization to Nylon-6 a route which has a significant energy and waste footprint.

The application of transaminase enzymatic synthesis of amines was made the core of the AMBIOCAS project. These would then be used as the key element in the synthetic enzymatic cascades that were the end target in these studies.

The initial objective focussed on the engineering of transaminase enzymes and creation of libraries of new mutants. These would then have the desired properties to be applied as the main biotechnology element to deliver synthetic routes to the amine functionality in the commercially valuable products of both chiral and bulk amine targets. Then to be able to meet the overall objective further objectives were defined in all the disciplines that had been identified as required to develop this technology to a level required. These were in molecular enzymology, enzyme supports, bioprocess engineering and process analysis.

Initially the required transaminase enzymes that that could convert the required inexpensive ketones and aldehydes into amines were identified. Once the proof of principle was established the enzymes of interest were then incorporated into enzymatic cascades for developing extra chemical functionality.

To exemplify the power of this approach a six step enzymatic cascade to convert the readily available commodity chemical cyclohexanol into 6-aminocaproic acid was achieved on demonstration reactions.

The novel method of biotransformation with co-factor balanced alcohol oxidation and transamination of the intermediate carbonyl compound route was developed. This technique is of great interest to the pharmaceutical industry where conventional routes use potential genotoxic intermediate electrophilic alkylation agents that are tightly regulated.

Elegant high throughput screening methods for detecting activity of enzymes to both enantiomers of amine products were developed in this project. These were combined with the molecular enzymology that used several crystal structure studies to provide powerful tools for enzyme improvement.

Engineering analyses of process conditions was used to identify obstacles associated with the implementation of this core transaminase technology and to guide the discovery of solutions to overcome these. One early key analysis of bottlenecks in the transaminase reaction identified thermodynamic and kinetic obstacles within the key transaminase step. This allowed clear operating windows to be defined and engineering option choices to be made. Improved supported enzyme formulations were further developed.

Life cycle, Environmental Impact and Economic analyses identified the metrics that need to be met to have advantages over conventional chemical synthesis in an objective manner. This gave some very clear metrics that defined the advantages of the new technology that had been developed.

Hence the overall supra-disciplinary technology chain was developed for application of enzymatic cascades centred on transaminase enzymes for the synthesis of organic molecular entities.

The contribution of the development of the science of transaminases has made a large impact in biocatalysis and this project has kept Europe as a lead in this arena. The improved technology will extend the range of the future application of enzymes and lead to complex multi-step chemical syntheses eventually in industry. This will make a major contribution to the development of industrial biotechnology using synthetic biology. Furthermore the contribution to the strengthening of the “Network of Excellence” will help keep Europe in a leading position in biotechnology. The highly trained personnel with a new skill set and has made a major contribution to the European trained workforce

Project Context and Objectives:


The AMBIOCAS project was conceived to develop enzymatic cascades that could later be used for industrial biotechnology solutions that could complement or replace classical chemistry to develop safer and cleaner methods for sustainable and cost-effective synthesis of amine chemical molecular building blocks. Industrial biotechnology had demonstrated that fermentative and enzymatic methods have applicability in chemical manufacture for economic and environmental reasons. Enzymatic methods offer the advantages of high activity and chemo, regio and stereo selectivity. Prior to the AMBIOCAS project enzymatic cascades and the emerging subject of synthetic biology for chemical synthesis was not well exemplified especially for redox pathways. However use and preparation of enzymes by recombinant microbial production strains was cost efficient compared to chemical catalysis methods that nearly always involved the use of expensive and rapidly globally depleting precious metals and expensive chiral ligands. Also these were used in hazardous high pressure reactions and the need for high environmental impact chemical solvents.

At this time another major obstacle to implementation of redox enzyme technology was the need to use expensive cofactors. The most exploited groups of enzymes were lipases, proteases and hydrolases that require no co-factor recycling. The major exception to this was the introduction of the platform technologies that produce chiral secondary alcohols by ketone reduction using ketoreductases. However there were still some structural limitations to the substrates and for all known examples in these categories sacrificial redox partners were used that were being added to the reaction mix in excess creating significant waste streams, poor equilibrium positions and often resulting in poor volume efficiencies. All of this could only be acceptable for very high value products. Hence these techniques could only make marginal contributions to society by reducing environmental impacts and the use of hazardous production plants. A further limitation to the application of enzymatic processes to more complex molecule synthesis was the lack of compatibility of the operation conditions of one enzymatic step with another.

Amines are very important chemical products. Most pharmaceuticals contain amine functional groups. In household goods amines are used in polymers for electronic and mechanical goods. Also a wide range of consumer products need amines for their production.

Chiral amines are a very important family of compounds that are components of a significant number of pharmaceutical targets which often have high added value as they are expensive to synthesize. Also in pharmaceutical production application these targets often contain further functionality that requires expensive protecting group strategies for chemical production.

Polyamides are widely used in bulk lower value products and most households will use products containing these. The introduction of the nitrogen into molecules to create amines is a relatively demanding chemical process in terms of overall economic and environmental costs. Preliminary calculations had demonstrated significant advantages were possible by applying industrial biotechnology solutions to amine synthesis.

One other aspect of this project was that the issues around true value of biotechnology to society were being questioned by some sectors. Environmental “greenness” was being claimed for all aspects of industrial biotechnology without the necessary proofs but this approach was leading to a reduction of societies’ confidence in this technology. Therefore it was important to include and disseminate Life Cycle Analysis and Environmental Impacts into the project to demonstrate the true advantages of IB. These were coupled to economic analysis to satisfy the needs of industry.

Transaminases were a class of enzymes identified as a group of enzymes that could be used to synthesize amines but were relatively underdeveloped. This family of enzymes had been found in a variety of bacteria. Mainly active for the synthesis of the (S)-enantiomer of the amine with only one enzyme with (R)-amine enantiomer specificity had been described. This provided the initial starting point to develop libraries of transaminase enzymes.

However there were several problems that needed to be overcome including the synthesis of an amine from a ketone had an unfavourable equilibrium. Therefore conditions had been devised to shift the equilibrium toward transamination of the ketone which used large excess of amine donor but this added cost and waste problems. A further problem was substrate and product inhibition of the transaminase had been identified. The use of transaminases for primary amine synthesis was almost unknown but these were interesting targets for bulk chemical synthesis. Further very few uses of redox enzymes in cascade reactions were known and the biocatalytic amination of alcohols was not known


The main overarching objective of AMBIOCAS was to engineer transaminase enzyme libraries from which suitable members could be applied as the core of enzymatic cascades to deliver the amine functionality in a range of chemical targets. The two main approaches were to develop alcohol to amine cascade or the cascade approach to use further enzymatic steps to remove amine products from the transaminase reaction which would also add value by developing extra functionality.

These targets were divided into the following main arenas.

• Amine syntheses by using integrated multi-enzyme cascades to be developed for complex multistep chemical syntheses that could lead to chemical methods being replaced by biocatalytic processes.

The evolution and identification of new transaminases with desired properties by using high throughput methods to give new libraries of transaminase enzymes with high activity would be the first step. Using directed evolution the enzyme properties to work at synthetically useful conditions would give industrial biotechnology a novel and valuable toolbox.

A key objective to achieve these outcomes would be the development of a range of high throughput assays (HTS) for novel transaminase enzymes. These would lead to the identification of colonies of mutants with high activity novel enzymes that could be visually identified using solid-phase colorimetric screens. Parameters including substrate specificity, enantioselectivity, thermal stability and substrate binding would be targeted.

• Process engineering solutions to implement multistep multi-enzyme enzymatic cascades using a highly multidisciplinary approach to synthesize industrially relevant amines was a significant objective of the project.

There was a clearly identified challenge with the thermodynamic equilibrium for the transformation of a ketone into the corresponding secondary amine. A strategy involving mutants of the transaminase was not expected to be sufficient to achieve the high conversions into the target amines required Therefore a suite of engineering tools including reaction analysis, enzyme support development and process engineering were all to form key roles in the technologies for process improvement.

Detailed analysis of the thermodynamics and kinetics of the reaction would be used to identify bottlenecks and allow the definition of operating window conditions. From that analysis scaled down reactor for reaction condition screening would be designed and desirable enzyme properties would then be defined. Reactor configurations would then have to be identified to allow enzymatic cascades to be applied to chemical synthesis.

Information for the scale-up from laboratory bioreactors to industrial scale of these cascades would be transferred to industrial partners.

One other engineering approach to enhance the applicability of the enzymes being used would be the preparation of supported enzyme formulations suitable for the target synthetic sequences.

Combination of enzymes would need to be prepared on supports to be able to carry out multi-step enzymatic cascades reactions with co-factor recycling.

• Co-factor regeneration needed to be engineered into multi-enzyme formulation and reactor design that would allow multi-step redox reactions to proceed without intermediate isolation.

Investigation and optimization of redox cofactor recycling in a balanced manner was the key innovative step that would need to be addressed and allow cost effective use of alcohols as starting materials in amine synthesis.

• Molecular Biology and Enzymology to Improve Enzyme Applicability

To match the specific requirements of the biocatalysts enzyme engineering techniques based on X-ray structures of active sites, directed evolution and homology modelling would be applied. This would involve the elucidation of the three-dimensional structures of several transaminases by protein X-ray crystallography. 3D-Structures of these transaminases would then allow the creation of improved designer mutants by molecular engineering. The overall goal would be to develop enzymes with broader substrate specificity and hence deliver more efficient catalysis methods. These engineered enzymes would be incorporated into the catalytic cascades designed in the project.

• Life Cycle Analysis and Environmental Impact analysis of processes would lead to more objective analysis of the advantages of these processes and allow sustainability of IB to be implemented and disseminated with more confidence.
• Networking of research infrastructures between European CoEs would be built into the programme plan.

Many excellent European research groups have expertise that can contribute to the challenge addressed in this project but individually they are unable to provide the supra-disciplinary expertise and the resources required to implement this ambitious project. Therefore this project will help provide the trans-national context to perform a complex project that will exploit national areas of excellence that has been built on traditional industrial focus.

• Innovation at the three industrial partners will be supported by the technology development and lead to a competitive edge will be given to Europe’s industrial biotechnology industry.

To unify these concepts a major example of 6-aminocaproic acid had been chosen for investigation. This method based on lower cost feedstock of cyclohexanol and would exemplify the use of a clean, cheap biocatalytic cascade alternative to conventional chemical processes.

Hence an overall integrated program would deliver a range of enzymatic cascades incorporating improved transaminase amine synthesis engineered to be of interest to industry.

Project Results:

Please see attached pdf.

Potential Impact:

In the development of a European knowledge based bio-economy (KBBE) the use of enzymes as biocatalysts is a key aspect for the sustainable production of better and purer products with concomitant less exposure of both the workforce and the community to toxicity, flammability and explosive hazards associated with the traditional chemical industries. To effectively deliver these objectives and maximise impacts the multidisciplinary research between academia and industry to deliver new cost-effective processes, new manufacturing approaches and communicate these to the stakeholders was an objective of AMBIOCAS.

Technology Overview

The AMBIOCAS project now expects to deliver the following innovative technical results that will contribute to the development of the application of transaminases and of enzymatic cascades.

• New high-throughput screens for transaminases
• Libraries of transaminases that can be screened for synthesis of new targets
• Synthetic cascades using these enzymes as core step for synthesis of targets
• Balanced alcohol oxidation-transaminase cascade that can recycle cofactors
• Enzymatic cascade that can convert cyclohexanol into bio-Nylon intermediate
• Co-factor multi-step redox reactions applied in enzymatic cascades
• Engineering scaled-down test rigs that can deliver insights into reaction bottlenecks
• Transaminase crystal structures that can be used as models to design improved enzymes
• Supported enzyme formulations that deliver cofactor recycling needed for applications
• Supported enzyme formulations suitable for enzymatic cascades
• Economic models that can identify full process economics
• LCA analysis that can identify greenhouse gas advantages
• Ability to identify best commercial applications of transaminases

Expected Technology Impacts of AMBIOCAS

The improved technology described above will extend the range of the future application of enzymes and lead to complex multi-step chemical syntheses being replaced by biocatalytic processes with high efficiency. The demonstration of the proof of principle that artificial enzymatic cascades with redox enzymes that can mimic natural product synthesis with generic methods to develop enzymatic cascades for use with redox enzymes and co-factor recycling incorporated into the reaction steps that can be used in a balanced manner will be a major contribution to the development of industrial biotechnology. The impacts of this foundation where industrial biotechnology can move from the one step chemical bio-transformation or the simple bulk scale fermentation to yield basic metabolites is where the impact can be expected to greatest and should help to keep Europe at the forefront of industrial biotechnology.

One important principle has been demonstrated is the necessity of the world class leading players in Europe need to work in a collaborative manner as the supra-disciplinary skills and equipment needed to deliver this project are not to be found in a single country.

Research Infrastructural Impacts

Further collaboration between CoEs in Europe are needed to bring about the SUSCHEM goal of a pan-European CoE in industriale biotechnology and this project has identified key skills and expertise that will help make this happen. The funding of this project has built on the informal exchanges of skills and people that was already on-going. On the wider European scale AMBIOAS has contributed to the longer term collaborations between the CoE’s and leading experts in Europe. This will extend after the project and will have a broad impact on future academic research and industrial applications of industrial biotechnology in Europe. This has been clearly demonstrated by partners UNIMAN, UNIGRAZ, RUG and DTU that are already leading CoEs in their aspects of industrial biotechnology with industrial affiliate group structures. The project AMBIOCAS has enhanced the relationships between these centres with their independent focus and skills with a demonstrated collaborative model that is necessary to deliver the big gains. It is now expected these relationships will deliver momentum to collaborative European Industrial-Academic technology development and help build a future virtual CoE in European Industrial Biotechnology with chemists, biologists, fermentation scientists and engineers working together. The project has also demonstrated the value of innovative collaborating SMEs as a model for SME company development in industrial biotechnology that can enhance the value of the individual SME effort and help develop relationships with the major companies in this arena. These are all significant impacts if Europe is to maintain its leading position in industrial biotechnology and compete with the very large investments in this area in countries such as the USA, Brazil and Japan.

The enhanced skills base targeting this supra-disciplinary interface is needed to move from fine chemicals to platform chemicals and biorefineries which will develop the new products and sectors for industrial biotechnology. Biotechnology has always been a great strength in Europe and it will be important to maintain this lead and to expand the market segments where this technology is applied.

These supra-disciplinary applied skills will therefore impact beyond the technological boundaries of the project by bringing a new skill set to the European labour pool.

SME Importance

One important impact area was contributing to SME development. Although a small project with only one SME partner the AMBIOCAS project has contributed to the concept on “innovative networks” of SMEs which are increasingly important for the development of SMEs in key technology s arenas. It has recently become apparent that current public innovation support has often failed to activate a significant group of SMEs and as a result, the innovation infrastructure is not utilized to its true value and capacity by a key constituency – small and growing companies. The model initiated in AMBIOCAS that collaboration between innovative SMEs with large companies through publically funded projects organised by CoEs is a very powerful tool and this has led to further projects using this model and the contribution of this lead project should not be underestimated.

On an individual basis the AMBIOCAS project has allowed CLEA to gain knowledge of the cascade system involved in converting ketones into chiral amines through the transaminase cascade. Through the development of this cascade system, particularly through the use of transaminase CLEAs, we have been able to develop CLEAs for a commercial partner who is testing these CLEAs in their own systems. CLEA have developed partnerships with the other participants of the project and through this been able to produce the transaminase CLEA and transaminase/alanine dehydrogenase CLEAs for a potential market application. Without the exchange of enzymes and procedures this possibility may not have been accomplished so readily and as such AMBIOCAS has played an important role in helping CLEA potentially commercialise these CLEAs for application in enzymatic amination cascades.

Wider Societal Benefits to EU Community

Developing New Researchers

By bringing a highly trained personnel group with a new skill set and network established at the highest level to the European labour pool AMBIOCAS has made a major contribution to the trained workforce that is necessary to keep the KBBE development on track. It will also improve the careers of scientists who worked in AMBIOCAS by the development of advanced research skills and networking in a supra-disciplinary consortium of internationally recognized leading scientists operating at the highest scientific level. It was considered particularly important to support younger researchers who enter this field and the need to get access to ever more expensive complex tools, materials and equipment and this programme offered the research fellows an outstanding opportunity for developing advanced research skills and networking in a supra-disciplinary consortium of internationally recognized leading scientists operating at the highest scientific level. This was coupled to major CoE’s in Europe and with major industrial partners. This developed skills and contacts that will make the fellows attractive future employment targets in both industry and academia and allow their careers to develop whilst using the skills to further contribute to the European KBBE. Therefore the maximum impact of this skills diffusion will need the research workers to be employed across a broad base in academia and industry after the project is completed and that process is now on-going. AMBIOCAS is therefore expected to produce European added value as a result of optimized use of these networking of research infrastructures and by the development of world class scientists experienced in working in a supra-disciplinary manner with an understanding of the needs of industry.

Cleaner Safer Environment

The technology developed will support the introduction of sustainable production techniques for the synthesis of better and purer products with less exposure of both the workforce and the community to toxicity, flammability and explosive hazards from the chemical industries.

The impact analysis that defines these advantages is being published in the scientific literature as both life cycle analysis and environmental impacts. This means that the wider benefits of industrial biotechnology will be fully understood by the wider community and we would expect this to lead to further uptake of the general methods.

Strong commitments and investments from industry are necessary for Europe to maintain a vibrant chemical and biotechnology industry and this project has contributed to development in this investment.

The Image of Biotechnology

Also by creating an understanding in the wider community of the advantages of these techniques will lead to better support for industrial biotechnology.

AMBIOCAS will make important contributions and impacts on the European industrial biotechnology sector. Biotechnology is considered as a key technology for the 21st century. Industrial biotechnology is contributing to the sustainable production of chemicals for diverse applications in chemical, pharmaceutical, detergent, paper, polymer, textile and biofuel industries and it is important that the image of biotechnology is explained to society in general and especially in an educational context.

Dissemination and Exploitation

In the early stages of the project a record of all developing results was set up so all members were aware of the potential of the project results. Confidential website material was regularly updated so that the information exchanged at project meetings was always available for review and extra information such as sources of enzymes was added. This was considered necessary to prevent duplication of effort and loss of integrity of results through time.

Publications A continual stream of academic publications were produced during the course of the project and continue to be prepared based on the results obtained. These have also been supported by poster and verbal presentations at major conferences. This combined high profile presentation of the project to the professional science community has resulted in a high profile for the project and the results obtained. The selection of journals has always been aimed at the high profile publications and also the journal of Organic Process Research and Development was used on several occasions to target the industrial community.

Conference Events

In the original project it was envisaged that the AMBIOCAS project would organise an event to promote transaminase and enzymatic cascades. These areas moved forward quickly and UNIGRAZ and DTU were both involved with organising conferences in these areas. It was the project management decision that joining these pertinent conferences being organised by AMBIOCAS members would reduce costs and engage a bigger audience especially from industry. UNIGRAZ organized MECP12 (Multi-Enzyme Catalysed Processes), April 10-13 2012 in Graz, Austria with presentations and posters from the AMBIOCAS team. These included “Redox-self-sufficient biocatalytic cascade for the amination of primary alcohols (Johann H. Sattler, Michael Fuchs, Katharina Tauber, Francesco G. Mutti, Kurt Faber, Jan Pfeffer, Thomas Haas, Wolfgang Kroutil, University of Graz, Austria), “Process engineering tools to guide implementation and scale-up of transaminase cascades (Pär Tufvesson, Krešimir Janeš, Naweed Al Haque, Joana Lima-Ramses, Paloma de Gracia Andrade Santacoloma, Watson Neto, John M. Woodley, DTU, Denmark, “High-throughput screening of transaminases” (Simon Willies, Kirk Malone, Rehanna Aslam, Nicholas Turner, University of Manchester, UK) and “Conversion of renewable raw materials into fine chemicals” (Jan Pfeffer, Evonik Industries, Germany). These were also supported by poster presentations from the project.

The 1st International Symposium on Transaminase Biocatalysis Stockholm, Sweden Thursday February 28th – Friday March 1st 2013 which had John Woodley from DTU as one of the main organisers was a similar golden opportunity to present and promote AMBIOCAS. This conference attracted considerable interest from industry and presented a major opportunity for us to disseminate AMBIOCAS. Three oral presentations were based on AMBIOCAS and included “Application of ω-transaminases in cascades and organic solvents” by Wolfgang Kroutil, University of Graz, Graz, Austria, “Transaminases: From discovery to application” by Nicholas Turner, University of Manchester, Manchester, UK and “Triple Combi CLEA for the synthesis of chiral amines” by Jo-Anne Rasmussen, CLEA Technologies BV, Delft, The Netherlands. Again these were supported by posters from the project.

As well as managing to make AMBIOCAS a high profile contributor to these directly relevant smaller conferences other major conferences were targeted by the AMBIOCAS management as directly relevant to the project and as such were used to present project material. These included Biotrans 2011, a major international biotransformation conference with over 700 attendees with a cluster of four posters being presented. The Gordon conference on biocatalysis, Rhode Island, July 11-16, 2010 which was a highly prestigious international conference in Biocatalysis where unpublished results are preferentially presented and everything presented at a Gordon conference is confidential and considered as not published even by presentation. Additionally this leads to the benefit that companies speak freely about their results. In 2010 the Gordon conference was partly dedicated to the use of -transaminases, the group of enzymes which was the focus of the AMBIOCAS project. Therefore it was of high importance for the AMBIOCAS project to be able to interact with companies present exemplified by Codexis, DSM, BRAIN (and attendees from academia) to ensure state of the art work was communicated and implemented.

In addition to this an outline of the project was made to CoEBio3 Affiliates at the CoEBio3 meeting on Thursday 27th October 2011. This led to Chirotech Ltd taking an opportunity to join the project.

In line with the objective to provide good opportunities for young professional researchers to develop their networking and presentation skills in the project dissemination opportunities and these were taken for major conferences in Simon Willies, a post-doctoral researcher from UNIMAN to give an oral presentation on AMBIOCAS results at the Zing Biocatalysis conference (4-9 December 2012) in Mexico and Jo-Anne Rasmussen at the ICOS conference, July 2012 in Melbourne.

Community Engagement

RUG attended the event “Nacht van Kunst en Wetenschap” (Night of Arts and Science )* on 4 June 2011 in Groningen Netherlands which was a public event featuring music, dance, theatre and science. Images of protein structures to explain protein crystallography and its applications to the general public, with AMBIOCAS as an example were used to present this biotechnology ( ).

At UNIMAN 29 Oct 2011 displays of biotechnology including AMBIOCAS were made in conjunction with Manchester Museum to present to general public and schoolchildren in a science spectacular within Manchester Science Week.


AMBIOCAS will have a direct impact on the implementation of the clearly identified underpinning societal needs and the industrial drivers to the achievement of the Lisbon strategy to make Europe the most competitive and dynamic knowledge-based economy (KBBE) in the world. It directly meets the key requirement for European investment in training to underpin the future development and health of the KBBE. This KBBE will encourage sustainable, eco-efficient development of products and processes that will protect and extend employment and expertise whilst leading to a better quality of life for a highly trained workforce and stimulating significant growth and wealth creation across several key biotechnology chemical sectors.

This role has been recognized in Japan and the USA where large consortia have been assembled to provide more complex bio-refinery technology tools. It is now essential that there is a strong focus in Europe in this area and AMBIOCAS will help to ensure that European Scientists can work as equal partners with international consortia.

List of Websites:


Nick Turner
Principal Investigator
University of Manchester
131 Princess Street
Manchester M1 7DN, UK
Tel: +44(161)306 5100
Fax: +44(161)275 1311

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