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From DISCOvery to products: A next generation pipeline for the sustainable generation of high-value plant products

Final Report Summary - DISCO (From DISCOvery to products: A next generation pipeline for the sustainable generation of high-value plant products)

Executive Summary:
DISCO functioned as an academic/industry alliance, consisting of pan-European and IPCP partners with complementary multidisciplinary expertise. The project represented a timely opportunity to translate innovation into commercial practice. The overall aim and concept of DISCO was the implementation of a generic pipeline from discovery to industrial valorisation, using the very latest enabling technologies, to deliver sustainable biosources of plant derived products.
A key feature of the DISCO project was its ability to build on existing and previous EU investments, rapidly and efficiently transferring the tools and strategies developed to new plant derived target molecules. The bioactives and high-value compounds targeted in DISCO were carotenoids, other terpenoids and tropane alkaloids. These targets required the development of new sustainable biosources and “greener” production chemistries.

The RTD and demonstration activities of DISCO were industry driven and directed towards:

- Exploiting existing and evolving biodiversity in Solanaceae and Iridaceae to perform bioprospecting with state of the art metabolomic approaches for the targeted molecules of interest. The project was successful in analysing diversity germplasm for Capsicum annuum, Iridaceae, potato, and medicinal Solanaceae such as Atropa belladonna, Brugmansia, Datura, Hyoscyamus, Duboisia, Scopolia spp., Prezewalskia and Latua. All activities were compliant with the Convention of Biological diversity.

- The utilisation of proprietary high-throughput bioassays to assess the bioactivities of extracts and enriched compounds derived from the biodiversity collections accessed proved to be a valuable strategy and provided several strong leads to be taken forward by our industrial consortium partners.

- The latest transcriptomics and network biology approaches were used to elucidate new biosynthetic and regulatory pathway components and their alleles involved in the formation of the DISCO targeted bioactives/high-value phytochemicals. The use of next generation sequence (NGS) was highly beneficial to the project and its scientific advances. Interestingly, in comparison with metabolomics it was obvious to see that the rapidity of advancements in metabolomics were not as great. This suggests that more resources are required to advance this technology especially in the cases of annotation and spatial metabolomics.

- Enabling technologies were developed and implemented into the discovery, application and translational pipelines. One of the highlights being the development of an inducible transplastomic system, which overcomes the detrimental loss of vigour associated with a system that delivers such high protein levels. This approach has generic application and extends to the production of pharma products in plants.

- New biosources of high-value carotenoids, terpenoids and tropane alkaloids by metabolic engineering and molecular breeding approaches has been achieved. A key aspect of this work being the use of sink organs in plants (food crops) to accumulate the desired products.

- Integrative biorefining strategies for co-product and biomass utilisation that reduce environmental impact were developed, the emphasis on minimal down-stream processing being a key factor.

- Demonstrate production feasibility and product effectiveness beyond the present state of the art.

- The consortium performed cost benefit and economic analysis of the processes to generate business models and marketability strategies for the translation of DISCO prototypes into commercial practice. It was clear that the new sources developed with the advantage of minimal bioprocessing could provide a 10-fold reduction in costs.

- Numerous training for staff and students was carried out, including intersectorial training between industry and academia and dry and wet laboratory activities to enhance the competence base of the European workforce.

The developments generated in DISCO will have real-life impacts reducing environmental impact, provide new material to benefit human activities and stimulate economic development as exemplified by the quality of the publication outputs, training provided and delivery of commercial products into the market place.
Project Context and Objectives:
To date chemical synthesis and/or refining remain the production method of choice for most chemical entities used in everyday life. Be it general household commodities, medicines or industrial raw materials. With the dwindling supply of fossil fuels and increased atmospheric carbon dioxide levels it is paramount that economies and society switch from existing chemical based non-renewable sources to biological raw materials. The DISCO project has been directed towards the provision of innovative research solutions to create sustainable bioresources for use in the emerging biobased economy.

The DISCO project has focused on plant derived natural products, which remain the most prolific source of industrially useful compounds known to date. The chemical complexity of these compounds restricts industrial production by chemical synthesis, as the procedures are often difficult, expensive and have a detrimental environmental impact. Alternative isolation from natural sources have not been straightforward as natural sources are typically low yielding and limited to a few plant species not readily amenable to agricultural production processes. Genetic engineering and the exploitation of natural biodiversity offer an alternative approach that will go beyond the present state of the art used to produce useful plant products that are traditionally generated by chemical synthesis. The DISCO project selected several classes of high-value natural products with known bioactivity, namely carotenoids (ketocarotenoids, colourless carotene, apo-carotenoids), terpenoids (solanesol) and tropane alkaloids (scopolamine) to apply its pipeline of activities directed towards the delivery of new or alternative products derived from innovative discovery. The RTD and demonstration activities addressed; (i) the bioprospecting of unique biodiversity collections, (ii) bioactivity assays for natural products against prevalent societal disease states; (iii) the implementation of the very latest technological advances to enhance discovery and translational biology; (iv) genetic intervention pipelines for new renewable biosources; (v) integrated biorefining cascades and (vi) techno-economic feasibility and product effectiveness.

The specific objectives of the DISCO project were:

Objective 1: Implement a pipeline that utilises generic tools and strategies, to take innovative discovery to plant derived high-value products. This objective was achieved by establishing a modular structure that utilises common resources to deliver quantifiable progress, through a pipeline of defined activities necessary to obtain a useful end-product. The key aspect of this approach was the ability to incorporate fundamental science across the pipeline (discovery to translation) to add value to the existing or emerging supply chains to generate new or better products. The delivery of the DISCO commercial products phytoene and phytofluene enriched formulation exemplifies this progress.

Figure 1. Showing the generic DISCO pipeline: Entry points of the target molecules and how fundamental science and innovation can add value to the supply chain are illustrated via the positioning of the WP activities.

Objective 2: Exploit existing and evolving biodiversity collections of Solanaceae and Iridaceae to perform bioprospecting for known and new bioactive entities and activities. To achieve this objective existing diversity germplasm collections developed through previous EU funding were screened for bioactives across a wide dynamic range using state of the art metabolomic approaches. Concurrently, a bank of high-throughput in vitro, in vivo and ex vivo SMART bioassays were used to screen for potential activities against prevalent disease states. Initially the project accessed existing diversity collections, but access to new germplasm following the convention on biological diversity was achieved, particularly potato and Capsicum collections from South America. The utilisation of both Solanaceae and Iridaceae genera indicated the potential medicinal and aromatic plant species.

Objective 3: Biosynthetic pathways, their components, diverse alleles and regulatory mechanisms will be elucidated for the targeted bioactives/high-value compounds targeted in the DISCO project. Conventional biochemical approaches were used, in conjunction with modern state of the art technologies and strategies. Differential RNA-seq approaches performed on tissues containing a low or high abundance of the molecules of interest as well as developmental tissue series across compound accumulation were used to achieve this objective. In addition, parallel “omic” studies enabled construction of biological networks from which biosynthetic and regulatory HUBs can be identified. This approach delivered candidates not only within the pathway of interest but also across metabolism per se. Fundamental knowledge of the targeted biosynthetic pathways is a pre-requisite for metabolic engineering and molecular breeding approaches to enhance/optimise levels of a given plant derived chemical product. Modern sequencing technologies were paramount in DISCO to ascertain the biosynthetic pathways for saffron derived apocarotenoids, terpenoid formation and tropane alkaloids.

Objective 4: The development and incorporation of enabling technologies into metabolic engineering and molecular breeding pipelines to deliver sustainable biosources of high-value bioactive and industrial phytochemicals. Activities towards this objective developed new tools for flux analysis, optimised modular vector construction and transient expression systems used to iteratively ascertain the best gene combinations. Stable transformations were carried out to engineer pathways, regulators and sequestration mechanisms for optimal/hyper production of DISCO targets. Key outputs highlighting the project’s achievements included the development and implementation of an inducible transplastomic system, which alleviated the unintended effects associated with plastid hyperproduction of metabolites or proteins. In addition, techniques to facilitate horizontal gene transfer were used to generate new species and conveniently transfer plastid engineering of astaxanthin formation across related species.
In the case of the DISCO project, marker assisted screening procedures were used to generate new genotypes with high yielding DISCO target molecules.

Objective 5: Develop efficient, rapid and “green”, down-stream processing and formulation processes, for the DISCO target molecules. Co-product generation and biomass utilisation will also be integrated into these pipelines. The high levels of target compounds produced facilitated non-solvent based extraction procedures and enrichment. Spray drying and encapsulation techniques were used to maintain chemical integrity and stability. Analysis of multifractional processes identified potential co-products compatible with the down-stream processing pipeline. The success of minimal bioprocessing relied on two factors (i) the high levels of target molecules produced and (ii) the use of plant host sink tissues that are Generally Regarded As Safe due to their use as food material.

Objective 6: Demonstrate the techno-economic feasibility and effectiveness of production for the DISCO targets and their associated processes. Production scale trials were performed under different cultivation conditions (glasshouse, polytunnel and field) and in different geographical locations. Tolerance of the genotypes, robustness of yields to environmental stresses were evaluated. Production scale down-stream processing, including co-product formation and biomass utilisation, was carried out. The effectiveness of the product, compared to existing alternatives was carried out to ascertain techno–economic feasibility and potential of the product. In DISCO, the consortium showed techno-economic feasibility for new sustainable sources of ketocarotenoids for the aquaculture and poultry industry. The materials were benchmarked against market leaders and showed that the DISCO product was superior and reduced production costs by 10-fold. The phytoene and phytofluene derived products have entered the market place as viable products.

Objective 7: The DISCO project will perform intersectorial training and dissemination activities as core project events.
The DISCO consortium had an active dissemination and outreach programme through the project. For example, dissemination of project results to the scientific community was achieved through peer-reviewed publications and presentations at scientific conferences. To potential industrial partners through existing networks of the SME partners and multinational industrial partner in DISCO. The public facing aspects of the project were disseminated via the website (http://disco-fp7.eu/) and other general dissemination activities such as press releases, regional open days, magazines and the internet. Project animations and interviews with senior and junior DISCO scientists were also generated (http://disco-fp7.eu/media/). The training activities contributed to the development of career prospects for trainees involved directly within the project, with other complementary EU networks and outside the immediate project both from Europe and non-European countries. DISCO organised and contributed to metabolite training schools. Unique features of DISCO outreach activities included; an industrial forum, workshop and early researcher session in Chile ensuring the project outcomes reached the very widest audience.

Following the completion of the DISCO objectives a number of future recommendations are proposed:

- Firstly fundamental science is essential to industrial innovation and the valorisation of outputs.
- Metabolomics has not progressed with the same rapidity as Next Generation Sequencing (NGS) or proteomics, coordinated investments are required to increase metabolite annotation and add a spatial aspect to field.
- Exploitation of existing natural variation and biodiversity remains untapped especially in Solanaceae and the technology transfer from model crops such as tomato to other related species such as Capsicum needs to be fully addressed.
- Use modern molecular breeding techniques to generate new functional alleles (e.g. TILLING) and their use in Marker Assisted Selection (MAS) to optimise production requires future activities.
- There is a need to implement the innovative inducible transplastomic system for the production of small molecules and protein products.
- Use new and emerging plant breeding techniques such as CRISPR-Cas9 and transient expression systems to produce small molecules is an important area to address and evaluate.
- The use of non-food crops is a major hindrance to the development of viable sustainable feedstocks.
- Develop and implement down-stream processing and biorefining strategies for co-product and biomass utilisation that reduce environmental impact.
- Minimal bioprocessing has great potential for the reduction of production costs to enable viable competition with the chemical synthesis platforms, but requires GRAS material.
- Demonstrate production feasibility and product effectiveness beyond the present state of the art.
- Perform social science surveys on the consumer perception of new breeding techniques for the production of natural products compared to those chemically synthesised from fossil fuel derived by-products.
- Acquire cost benefit and economic analysis of the processes to generate business models and marketability strategies for valorisation.
- Complement previous EU funded programmes in the area and act as an intersectorial training vehicle for industry and academia to enhance the competence base of the European workforce. The continuation of DISCO from METAPRO greatly contributed to the successful impact of DISCO, in effect a stage I and II process.
Project Results:
WP 2: Bioprospecting for bioactive identification

Objectives of WP2:
O2.1: Database creation and curation (bioactives and useful metabolites) for in silico bioprospecting in Solanaceae and Iridaceae.
O2.2: Access existing and new biodiversity sources for bioprospecting under the auspices of the convention on biological diversity (CBD).
O2.3: Identify new or under-utilised sustainable sources of Saffron related apocarotenoids, solanesol (and related terpenoids), tropane alkaloids (e.g. scopolamine) and extracts for bioactive screening.
O2.4: Perform a bioethical review of the projects activities.

Activities and results of WP2:
Activities in WP2 were directed towards harnessing the chemical diversity that exists within selected genera of the Solanaceae and Iridaceae families. The DISCO project employed literature mining and state of the art metabolomics. Activities were compliant with the convention on biological biodiversity. The following tasks were performed in WP2.

Literature mining and the acquisition of traditional knowledge was performed to establish and/or evolve existing bioprospecting databases for Solanaceae and Iridaceae families, highlighting the presence of known or potential bioproducts, and their uses in the health, food, feed, cosmetics, agrochemicals and industrial sectors. Execution of the task created a valuable resource that can act as a digital library/ repository for in silico mining of useful bioproducts and their sources (Figure 2).

Figure 2. Demonstrating how the database was formatted using the first 9 Iridaceae species (out of 124 species found) (A) and first 14 Solanaceae species (out of 117) (B). The data base lists the species, the associated bioactivity found, active molecule if known, references reporting this information and source of seeds if known. There is also a numerical scale to indicate how reliable the evidence for the bioactivity was (0=not available, 1=anecdotal, 2 =cell free, 3=cell based, 4=animal testing, 5=epidemiology, 6=human tested). The full database contains a total of 241 species with 714 supporting references.

Metabolomic analysis was carried out predominantly on the following collections;
(i) The Commonwealth potato collection, which is a major potato gene bank with 1300 accession.
(ii) The EUSOL tomato collection comprising of 7,000 accessions and wild species.
(iii) The medicinal Solanaceae species present in the plant genebank of the Volcani institute, which include accessions from Atropa belladonna L., Brugmansia spp., Datura spp., Hyoscyamus, Duboisia spp., Argyrea nervosa (BURM. F.) BOJ and Mandragora spp., Solandra spp., Scopolia spp.
(iv) The endogenous Solanaceae from the Romanian area.
In the case of Iridaceae, the Crocus Bank (www.crocusbank.org) has also been curated through EU-funding, which contains 246 accessions and wild species.
In all these cases, existing MTAs were in place for the transfer and/use of resources. DISCO activities also widened the biodiversity available by establishing bioprospecting agreements with Latin American partners (e.g. CIAT and CIP), industry and Botanical (e.g. Kew Gardens, UK). Through these activities we were able to screen Capsicum varieties and large potato collections as well as sweet potato.
Modern metabolomic approaches were used that used both polar and non-polar extracts, liquid phase partitioning and solid phase matrix procedures. Robust 2D NMR and DiMS un-targeted profiling, supported by GC-MS, HPLC/UPLC-PDA and LC-MSn metabolite profiling methods.

Conclusions:
- Access to existing and new biodiversity sources for bioprospecting under the auspices of the convention on biological diversity (CBD) is a key factor in future projects directed towards characterising genetic resources.
- An untapped biodiversity resources exits among the Solanaceae and Iridaceae genus.
- Metabolomics is an ideal method for capturing biochemical diversity in crop collections.


WP 3: Bioactivity assessment and valorisation

Objectives of WP3:
O3.1: Preparation of well characterised and traceable material in adequate quantity to enter the workflow for bioactivity and efficacy assessment.
O3.2: Primary screen: Target molecules (and derivatives), extracts from biodiversity collections and enriched/fractionated extracts will be screened for bioactivity/efficacy using cell-free assay platforms representing signalling pathways involved in prevalent disease states.
O3.3: Secondary screen: Those target molecules, extracts and fractions exhibiting cell-free activity will be evaluated using cell-based assays for disease prevalent signalling pathways.
O3.4: Tertiary screen: Activities identified in cell-free and cell-based assays will then be evaluated in ex vivo models.
O3.5: Construction of a biodiversity/bioefficacy databases for the target molecules and germplasm linked to the bioprospecting data repository created in WP2.

Activities and results of WP3:
WP3 is a step-change compared to many previous EU projects concerned with plant and crop bioactivity in that rather than focusing on known compounds this has as its focus the phytochemical diversity of Solanaceae and Iridaceae. WP3 implements extraction, fractionation and screening protocols on the existing and new biodiversity sources for bioprospecting under the auspices of the convention on biological diversity identified in WP2. WP3 was an industry-led activity that assessed the bioactivity and efficacy of the outputs generated in WP2 against prevalent disease states. Within WP3 state-of-the-art and proprietary cell-free, cell-based assays and ex vivo models were used. The high-throughput assays focused on signalling components known to be involved in disease states such as cardiovascular disease (CVD), cancer, and inflammation, metabolic and neurological disorders.

The approach employed was that of classical phytochemical activity testing with the incorporation of metabolomics as outlined schematically in Figure 3.

Figure 3. A schematic of germplasm screening and target optimisation against state of the art.

SOP was developed for plant extraction and primary bioactivity screening to ensure consistency of approach across the project was produced. In WP2 a database of bioactivities and useful chemicals in Solanaceae and Iridaceae species was constructed. This identified the widest operating limits for the germplasm in DISCO. The germplasm identified 80 genera/1500 species of Iridaceae and 98 genera/2700 species of Solanaceae. Where possible this material was acquired and extracts prepared and tested, in total over 2500 extracts/assays were conducted. The screens involved, Primary cell-free screening, Secondary cell-based assays, and Tertiary Assays. Collectively, a biodiversity/bioefficacy database for the target molecules/germplasm has been constructed and lead compounds/extracts have been identified.

Conclusions:
The ontological approach to; (i) databasing germplasm, (ii) extraction, (iii) fractionation and (iv) screening activities has proven successful. The approach has been established on the success of the existing databases generated in DISCO and the associated extraction, characterisation and bioactivity screening activities. A key component has been the establishment of viable bioactivities and many hits have been identified across several species, the chemical basis of which has, in some cases, been elucidated.


WP 4: The biosynthesis of bioactives; pathway elucidation, candidate gene isolation

Objectives of WP4:
O4.1: The elucidation of new biosynthetic pathways involved in (i) solanesol formation in Solanaceae ecotypes, (ii) saffron related apocarotenoids and (iii) scopolamine and other tropane alkaloids in high yielding ecotypes of Solanaceae.
O4.2: The identification and functional characterisation of biosynthetic and regulatory genes involved in the solanesol formation (ii) saffron related apocarotenoids and (iii) the formation of scopolamine and other tropane alkaloids.
O4.3: Construction of biological networks and flux models representing terpenoid (solanesol), saffron related apocarotenoids and tropane alkaloids (scopolamine) formation.

Activities and results of WP4:
The focus of WP4 was concerned with the elucidation of the biosynthetic pathways involved in the formation of the targeted bioactives, and the isolation of candidate genes encoding structural and/or regulatory components. The predetermined pathways to be addressed in the DISCO project were (i) the formation of apocarotenoids associated with Saffron products, (ii) terpenoid formation in particularly solanesol and (iii) tropane alkaloids biosynthesis e.g. Scopolamine. The activities of WP4 employed a modern systems level approach to pathway elucidation and candidate gene identification, utilising state of the art cell biology, transcriptomic, metabolomic, and in some cases proteomic approaches.
It was clear from WP4 activities that the use of RNA-Seq had a major contribution to the elucidation of pathways and identification of candidate genes. Metabolomic analysis in this case was more an augmenting role and more work is required to upgrade the communities’ metabolomics potential. The highlight of WP4 activities has been the elucidation the biosynthesis of high-value Saffron derived apocarotenoids (Figure 4B), which has been published in PNAS (Frusciante et al. 2014, PNAs, 111, 12246) and has attracted significant industrial third stream income for valorization. The work exploited, transcriptomic analysis using RNA-seq across the developmental window of production, in specialised organs/tissue or cell types involved in the production of the secondary metabolites targeted (Figure 4C). Metabolite profiling/metabolomics was also carried out concurrently with the transcriptomic approach. Bioinformatics using advanced homology searches with known genes and/or predicted gene functionality for gene ontologies was used to identify candidates and biological networks constructed from the metabolite and transcriptomic datasets incorporated. Clustering of up and down regulated transcripts was performed to elucidate those transcripts associated with the formation of the secondary metabolites of interest, co-regulation (Pearson coefficients) and other inference analysis was also be carried out. The networks were visualised in programmes such as Cytoscape (www.cyctoscape.org) and HUBs identified for future testing.

Figure 4. (A) The saffron apocarotenoid pathway. Crocus sativus flower at anthesis. The yellow arrowheads point at the three stigmas. (B) Proposed saffron apocarotenoid biosynthesis pathway. Zeaxanthin is cleaved at the 7,8 and 7′,8′ positions by a CCD activity. The C20 cleavage product, crocetin dialdehyde, is converted to crocetin by an aldehyde dehydrogenase, and then to crocins by at least two UDPG-glucosyltransferases. The C10 product, 3-OH-β-cyclocitral, is converted to picrocrocin by an UDPG-glucosyltransferase, and then to safranal. (C) The expression patterns of the putative dioxygenases deduced from RNA-Seq datasets.

The gene products of interest have been functionally characterised through expression in microbial and plant based systems. Sub-cloning of PCR products into expression cassettes containing different tags to ease purification, visualisation (GFP and RFP), and expression in plants has been carried out to ascertain sub-cellular localisation. On purification of the recombinant protein, precursor/product utilisation and kinetic parameters have be determined and product/precursor relationships. In the case of Carotenoid Clevage enzymes from saffron the E.coli colour complementation system has been used to determine the precursors for the respective enzymes (Figure 5).

Figure 5. The figure illustrates CCD2 expressed in E. coli cleaves zeaxanthin to yield crocetin dialdehyde. (A) E. coli cells accumulating lycopene, β-carotene, or zeaxanthin were transformed with the empty pThio vector, C-, or the same vector expressing CCD2 or ZCD, induced for 16 h at 20 °C with arabinose and pelleted. Note the discoloration of zeaxanthin in CsCCD2-expressing cells. (B) LC-HRMS analysis of zeaxanthin cleavage products. Zeaxanthin-accumulating E. coli cells expressing CsCCD2 were induced for 16 h at 20 °C with arabinose, extracted with acetone, and the extracts were run on a LC-HRMS system alongside authentic standards. The accurate masses of zeaxanthin, 3-OH- β-apo-8′-carotenal, and crocetin dialdehyde were extracted. Only crocetin dialdehyde is detectable and has an accurate mass and a chromatographic mobility identical to that of the authentic standard.

Similar approaches have been used to elucidate the tropane alkaloid pathway from Mandragora autumnalis, Mandragora officinarum, Datura inoxia, Hyoscyamus aureus (compound of interest: scopolamine) using RNA-Seq and then functional characterization in microbial hosts, this time in vitro. Littorine synthase, phenyllactyl-CoA synthase, putrescine N-methyltransferase, PMT, Tropinone reductase and Hyoscyamine-6β-hydroxylase have been identified.
In the case of Solanesol formation candidate gene testing has been described in WP6. Following RNA-Seq analysis a list of QTL affecting leaf solanesol content in segregating populations have been identified. The strong candidate gene present within a QTL interval on chromosome 7 has been transiently expressed in N.benthamiana resulting in a ca. 3 fold increase in leaf solanesol content compared with the mock inoculated plants.

Conclusions:
- The use of RNA –Seq data in conjunction with metabolomics has facilitated the identification of a plethora of candidate genes, both biosynthetic and regulatory involved in: Saffron derived apocarotenoids, (ii) solanesol and (iii) Scopolamine formation.
- Their genes represent essential tools and resources for future genetic intervention using conventional marker assisted selection and new breeding techniques.


WP 5: Enabling technologies for the optimisation of metabolic engineering

Objectives of WP5:
O5.1: Develop and extend the tools and strategies available for metabolic engineering including:
- Optimisation and modulation of transplastomic expression systems facilitating high level production.
- Adopt and contribute to the GoldenBraid platform for the rapid combinatorial assembly of multigene constructs.
- Further developments to transient expression systems for small molecule production.
- Procedures to reduce the impact of gene silencing.
- Utilise flux determination tools to evaluate rational design of bioengineering strategies.
- Create artificial metabolons, using isoprenoid enzymes as an example.

Activities and results of WP5:
WP5 activities built on previous EU funded projects focusing on new tools and optimised approaches for metabolic engineering and gene validation. The methodologies developed and implemented were designed to deliver greater tuning and control over production, the reduction of DNA methylation, flux analysis, improved vectors and promoter control and the construction of artificial metabolons.

Development and implementation of new transplastomic expression systems. Proof-of-concept studies for the RNA polymerase-based enhanced riboswitch (RAmpER) used Green Fluorescent Protein (GFP) as a visual reporter (Emadpour et al., Nucleic Acids Res., 2015) and the HIV antigen Nef as a known cytotoxic protein. Subsequently, vectors for RAmpER-inducible transplastomic expression of the astaxanthin and solanesol pathways were constructed and introduced into the tobacco plastid genome by stable transformation. Transplastomic lines with the astaxanthin operon under RAmpER control were isolated, purified to homoplasmy and fully characterized using molecular methods. Importantly, the growth phenotype of transplastomic lines constitutively expressing the astaxanthin pathway was fully remediated by placing the astaxanthin operon under RAmpER control. Northern blot analyses confirmed transcriptional activation of the astaxanthin operon. Induction experiments by watering with the inducer metabolite theophylline in low concentrations indicate that astaxanthin production can be further boosted under at least some conditions. Systematic induction experiments analysing time series and different tissues (leaves of different ages and developmental stages) have been conducted and metabolite measurements have been performed.
To transfer the astaxanthin pathway into a nicotine-free plant species, we used grafting, a procedure recently shown by us to facilitate horizontal transfer of plastid genomes between plants. The DISCO partner P2-MPG successfully moved transgenic chloroplast genomes (harboring the astaxanthin operon) across the graft junction from N. tabacum plants into plants of the nicotine-free tree species Nicotiana glauca (Figure 6). Transplastomic N. glauca trees expressing the astaxanthin pathway were recovered at high frequency and fully characterized. The results demonstrate that graft-mediated horizontal genome transfer provides a straightforward method for trait transfer and extension of the transplastomic technology to new species. This work was published in the October issue of Current Biology (Lu, Y., Stegemann, S., Agrawal, S., Karcher, D., Ruf, S. and Bock, R. 2017. Horizontal transfer of a synthetic metabolic pathway between plant species. Curr. Biol., 27, 3034-3041).

Figure 6. Transfer of the synthetic astaxanthin operon from N. tabacum to N. glauca by horizontal genome transfer. (A) Reciprocal grafting of the transplastomic astaxanthin-producing N. tabacum line (Nt-AXT-1) with a kanamycin-resistant N. glauca line (Ng-kan). (B) Detection of horizontal genome transfer on regeneration medium containing kanamycin and spectinomycin. The control explants (stem sections and leaf pieces) from the two graft partners are in the two left frames, the excised graft sites are in the right frame. Growing orange calli (indicated by arrows) indicate horizontal genome transfer events. Note that the Nt-AXT-1 control explants lost their orange color due to their sensitivity to kanamycin, a potent inhibitor of chloroplast translation. (C) Growth of regenerated transplastomic astaxanthin-synthesizing N. glauca plants (right) in comparison to a plant of the Ng-kan line used for grafting (left). (D) An astaxanthin-synthesizing N. glauca plant after transfer to the greenhouse. (E) Confirmation of horizontal genome transfer by RFLP analysis. RFLP analysis of 7 independent horizontal genome transfer lines of N. glauca (Ng-AXT lines) detects the same 7.3 kb fragment that is present in the transplastomic N. tabacum line used for grafting (Nt-AXT-1). Note that line Ng-AXT-8 is heteroplasmic and still contains copies of the resident N. glauca plastid genome, as evidenced by presence of the hybridization signal at 2.15 kb. (F) Flower phenotypes of an N. glauca wild-type plant (upper flower) and an Ng-AXT line (lower flower). Expression of the astaxanthin operon converts the yellow pigment of wild-type flowers (mainly lutein) into red astaxanthin (figure from Lu et al., 2017).

Modular vector resource for metabolic engineering. The GoldenBraid (GB) platform was used (Sarrion-Perdigones et al., 2011, 6(7), e21622), allowing rapid combinatorial assembly of multigene constructs for plant transformation, encoding multiple metabolic steps under the control of constitutive, tissue specific, and developmental promoters. In order to maximise biomass prior to secondary metabolite production new promoters controlling expression in senescing tissues, specific tissues and inducible systems were characterised. In addition, the DISCO consortium utilised and contributed to the development of parts using MoClo and Gibson cloning systems. In all cases partners have actively contribute to the community repository of vector and their components postulated through the COST ACTION FA1006 and patron et al, New Phytologist, 208, 13.

Plant based transient production systems. DISCO contributed to this task through the optimisation of transient Agrobacterium-mediated transformation systems in both homologous and heterologous plant systems. Solanaceae, tomato, tobacco and potato were the focus of activities. In these cases robust efficient protocols that permitted inter-lab transfer were established. To complement these activities transient expression systems for diverse Solanaceae identified in WP2 were attempted for example pepper (Capsicum annuun) and Eggplant (Solanum melongena) were tested, the latter being successful. The effectiveness of gene stacking in a transient manner was also evaluated, as a means of predicting metabolic engineering outcomes and assessing different gene combinations. This approach was used successful in WP6 when ascertaining the optimal gene product combinations for solanesol and crocin formation. Virus Induced Gene Silencing (VIGS) was also used as a production system to generate key intermediates for use as authentic standards and substrates.

Reducing gene silencing to maximize production. Multigene constructs and high-level nuclear expression can and often results in a degree of silencing, most commonly as a result of DNA methylation. In an attempt to reduce this occurrence it was postulated to (i) use TILLED tomato mutants (generated in EU-SOL) in which the DNA methylase genes have been knocked-down, (ii) use methylation inhibitors (e.g. 5-aza-2'-deoxycytidine and zebulerine) to determine the impact of reducing silencing and thus increased product levels. Unfortunately the TILLED mutants were lethal and the inhibitors were found to be non-specific which resulted in a number of unintended effects. As an alternative, in combination with Nottingham University (Prfo G. Seymour), a number of Epi-RUILs have been generated which will act as a valuable resource to ascertain potential methylation effects on heterologous transgenes.

Tools for determining pathway flux. Flux analysis of pathways has predominantly been performed on primary metabolism and required sophisticated apparatus and specialised expertise. In the DISCO project a convenient computational approach to the determination of pathway flux measurements (Liu et al. 2010,J. Med. Plants Res. 4, 1708) was implemented and used to ascertain the control points in scopolamine formation using a microbial platform with biotransformation features.

The creation of artificial metabolons. Metabolite channelling is a limiting factor associated with the engineering of most natural product pathways. Co-localizing pathway enzymes into complexes optimizes protein stoichiometries, reduces the accumulation of intermediates and decreases unintended interactions. In DISCO a number of short linker sequences were assessed to provide the resource for creating fusion and metabolons between target proteins. The utility of the system has been demonstrated with carotenoid biosynthetic enzymes using the colour screening capacity in microbial hosts.

Conclusions:
- An inducible transplastomic system has been developed and its utility demonstrated.
- The use of horizontal gene transfer to create new species and the transfer of industrial biotechnology traits have been shown.
- DISCO has used and contributed to the community resource of modular DNA parts for Synthetic Biology.
- Effective tools for the creation of synthetic metabolons have been developed.
- Computational approaches to flux analysis have been investigated and shown to offer potential in future rationale engineering.
- Methylation is an important level of regulation that requires further investigation in order to exploit and overcome it future engineering approaches.


WP 6: The production of high-value plant products by genetic intervention

Objectives of WP6:
The overall goal of WP6 is to generate plant derived sources of high value bioactive compounds.

O6.1: Systems level metabolic engineering of high level solanesol production.
O6.2: Production of saffron derived apocarotenoids through metabolic engineering.
O6.3: Scopolamine biosynthesis engineered for high level production of the end-products and intermediates.
O6.4: New Solanaceae varieties producing high-levels of colourless carotenes for the cosmetic industry.
O6.5: Generation of Solanaceae genotypes that can deliver solanesol as a by-product from waste vegetative material.

Activities and results of WP6:
WP6 implemented genetic intervention approaches to demonstrate and generate genotypes producing the DISCO targeted molecules/pathways which included; (i) the high-value plastidial terpenoid solanesol, (ii) carotenoids including the colourless carotenoids phytoene and phytofluene, (iii) saffron derived apo-carotenoids and (iv) new sources of tropane alkaloids.

(i) Solanesol biosynthesis. Tobacco hosts amenable to transformation, with high biomass properties, were used as hosts for engineering solanesol production by nuclear, transplastomic and transient transformation approaches. The hosts were also created with enhanced levels of the ubiquitous isoprenoid precursor, isopentenyl diphospahte (IPP) and geranylgeranyl diphospahte (GGPP). To achieve this goal enzymes involved in the plastid MEP pathway were co-ordinately amplified. Using Goldenbraid based alpha series constructs under control of the CaMV 35S promoter, plastid MEP pathway gene combinations were tested in N.benthamiana using Agrobacterium tumefacians mediated transformation. In total, 11 genes (CMK, CMS, DXR, DXS1, DXS2, GGPPS3, HDR, IDI, MCS, SDS and SIDPS) were expressed singly, or co-expressed in a multigene construct with the SDS gene over a period of 7 days. Leaf solanesol content was quantified by QQQ LC-MS analysis and the results are summarized in Figure 7.

Figure 7. Comparison of the relative fold changes in leaf solanesol content relative to the mock inoculated WT N. benthamiana plants, in plants (A) transiently expressing single MEP pathway genes and (B) co-expression of MEP pathway gene and SDS. Values shown are presented at 3 time points and are the mean of three biological replicates. Statistical analysis was performed using Student’s t-test and the significances are indicated (c) P <0.05 (d) P <0.01 (d) P <0.001.
In addition to pathway engineering, regulators of plastid parameters/core metabolism (DE-ETIOLATED (DET-1) and GOLDEN LIKE (GLK-1)) showed increased solanesol content. This could be due to improved supply of precursors from primary to secondary metabolism, CO2 fixation and sequestration properties. In tomato natural variation (alleles) were determined as high accumulator of (up to 4 fold) levels of solanesol in vegetative tissues. Natural variation in potato was also carried out to identify potentially alleles having a positive positive effect on solanesol content.

(ii) Carotenoids. A stable ketocarotenoid tomato line (ZWRI) was generated from the genetic crossing of a ketocarotenoid genotype (ZW) and high beta carotene (RI) background tomato. ZW lines overexpress the bacterial genes carotene hydroxylase (CrtZ) and carotene ketolase (CrtW), which are essential for the production of ketocarotenoids from endogenous plant carotenoids, primarily β-carotene (Figure 8).

Figure 8. Representative scheme of the ketocarotenoid pathway introduced in plant. Enzyme names are as follow: CRTR-B1, plant carotene beta-hydroxylase 1; CRTW, bacterial carotene ketolase and CRTZ, bacterial carotene hydroxylase. The purple and blue shadings depict the position of the newly added functional group (hydroxyl or ketone, respectively).

Interestingly, ZW expressing lines do not produce high levels of ketocarotenoids (e.g. ZWRIØ ~70 μg/g DW) due to the lack of the biosynthetic precursor β-carotene. RI are orange fruited recombinant inbred lines accumulating high levels of β-carotene. They derive from crossing the cultivated tomato Solanum lycopersicum with the wild S. galapagense accession. Analysis of this collection identified concurrent high β-carotene fruit content with the presence of high comparative expression of the fruit ripening enhanced lycopene β cyclase (β-Cyc).
Two ZW events were crossed with two RI lines. The best combination in terms of ketocarotenoids levels were selected and kept as a hemizygous state for ZW genes, in order to prevent detrimental effects on plant vigor, and a homozygous state for the S. galapagense lycopene cyclase (β-Cyc) gene. The greater supply of immediate precursor (β-carotene) in ZWRI overcame biosynthetic limitations to ketocarotenoid formation and high ketocarotenoid lines containing about 3 mg/g DW in the fruit material (40-fold increase compared to ZWRIØ) were generated. In addition to the ZWRI line, the double azygous control (ZWØRIØ), which lost both the CrtZ and CrtW genes (ZW) plus the S. galapagense β-Cyc promoter (RI), and the azygous controls (ZWØRI and ZWRIØ) were studied. ZWRIØ deep red fruit were defined by a high level of lycopene (77% of total carotenoids) and a small level of ketocarotenoids (2%). ZWØRIØ were red tomatoes predominantly accumulating lycopene (68% of total carotenoids), ZWØRI tomatoes had an orange color representative of their β-carotene content (66%) and the ZWRI tomatoes had a deep red color reflecting the presence of the ketocarotenoids (87%). Chromatographic analysis of the ZWRI line revealed a complex ketocarotenoid profile (Figure 9). The main ketocarotenoids found were phoenicoxanthin (in its free and esterified forms, ~45%) and canthaxanthin (~35%). The stereoisomer of phoenicoxanthin was determined as an S configuration (Figure 9). High resolution MS/MS was used to identify phoenicoxanthin esters (C14:0 and C16:0). No statistically significant differences of total fatty acid content of the tomatoes was observed.

Figure 9. Chromatographic profiles of ZWRI tomato carotenoids and phoenicoxanthin chirality. The chromatographic carotenoids profile was obtained by UPLC and recorded at 470 nm. The insert shows that the chiral carbon of the ZWRI phoenicoxanthin has an S configuration.

Phytoene and phytofluene are toxic to plants when they accumulate in vegetative tissues. In DISCO this was alleviated by generating novel varieties with high fruit phytoene and phytofluene contents. This resource has been generated using diverse alleles of the isomerase genes (CRTISO and Z-ISO) involved in carotene isomerisation. Gene specific molecular probes for these alleles are available. In order to enhance the phytoene and phytofluene contents these traits were further pyramided with synergistic genotypes. For example, genotypes containing impaired isomerases were crossed with HIGH PIGMENT Varieties HP-1 and HP-2 to enhance product levels.

(iii) Saffron derived apo-carotenoids. The carotenoids β-carotene and zeaxanthin are the biosynthetic precursors for saffron derived apo-carotenoid formation. Tobacco, Arabidopsis and maize genotypes producing these compounds have been used as the hosts for subsequent engineering (stable and transient) of the apo-carotenoid pathways. The optimal gene products elucidated from WP4 and WP5 have been integrated into vectors with enhanced translation and transcriptional properties and expressed in the high precursor tobacco genotypes. The apo-carotenoids present in these transgenic plants have been characterised.

(iv) Scopolamine biosynthesis. The feasibility of producing scopolamine through metabolic engineering has been demonstrated (Palazon et al. 2008. Molecules, 13, 1722-1742). In the DISCO project tobacco varieties producing precursors for scopolamine production have been generated. An alternative biotransformation approach has also been adopted by feeding cheap bulk precursors to engineered yeasts expressing key regulatory enzymes (e.g. putrescine N-methyltransferase, PMT, Tropinone reductase and Hyoscyamine-6β-hydroxylase).

Conclusions:
- The molecular tools are available to generate tomato genotypes producing ketocarotenoids at high levels.
- Optimised production of phytoene and phytofluene in tomato fruit has been achieved using marker assisted selected (MAS).
- A solanesol production platform has been created.
- Proof of concept studies have illustrated the potential to produce saffron derived apocarotenoids and scopolamine in plant and microbial hosts.

WP 7: Downstream processing and Biorefining
Objectives of WP7:
O7.1: Efficient down-stream processing approaches that minimise losses and maximise yields and have a low environmental impact.
O7.2: Formulation methods that improve chemical stability and enable versatile incorporation into commercial products for the feed, cosmetic, pharma and industrial sectors.
O7.3: The utilisation of resulting biomass and by-products into the production of fine chemicals, nutraceuticals, biofuels, and energy.

Activities and results of WP7:
WP7 addressed downstream processing and biorefining of the feedstocks generated. Integrated biorefining pipelines traditionally use harsh initial chemical/physical treatments. In the case of the plant derived bioactives targeted in the DISCO project, it was paramount that their structural integrity was maintained to ensure functionality. Therefore, given the high economic returns, WP7 placed the plant derived bioactives at the focus of the down-stream processing procedures and the remaining biomass used as feedstock for bioethanol, chemical and energy production. These developments ensured that, in addition to sustainable biosources, their production would utilise green chemistries in an attempt to secure a carbon neutral production process.

Downstream processing procedures for high-value bioactive/chemical recovery.
(i) Ketocarotenoids. In the DISCO project, optimised procedures have been developed for ketocarotenoid rich preparations for use in the feed sector (especially aquaculture), and as fine chemicals, ensuring compatibility with potential multifractional biorefining processes and colourless carotene production. Traditional methods, using organic solvents and chromatography over preparative adsorption stationary phases, have been adopted to supply fine chemical end users or for incorporation into feeds. Typically this involved; extracting several kilograms of frozen tomato material. The method (illustrated in Figure 10) typically used 1.5 kg of frozen tomatoes, 1.5 L of acetone was added and left for two hours. Then the mixture was homogenized using a blender and 1 L of 10% diethylether in petroleum ether was added. The mixture was then filtered through two layers of Whatman filter paper using an electric pump. Subsequently, 200 mL of water saturated with salt were added to the filtrate. A glass separatory vessel was used and two phases were observed and the upper phase (red colour) was collected. Anhydrous Na2SO4 was then added to the extract and left at -20°C for two hours. The supernatant was then collected and dried using nitrogen.

Figure 10. Illustrating the traditional extraction of carotenoids from the tomato based material.

For the incorporation into feedstuffs, admixes, oleoresins, encapsulation into microspheres and juices were also generated. These approaches have low environmental impact using no toxic solvents and minimal processing. For example, mechanical pressing, sonication, differential centrifugation and extraction into vegetable oils were all be employed. Figure 11 illustrates the preparation of the admix and extract incorporated into the prototype aquaculture feeds.

Figure 11. Workflow illustrating the minimal bioprocessing of the tomato derived material into effective aquaculture feed.

(ii) Colourless carotenes. To supply the fine chemicals market, the colourless carotenes (phytoene and phytofluene) were extracted from tomato fruit material using traditional solvent extraction. Preparative chromatography approaches were used to enrich and purify the products. To supply enriched extracts for the cosmetic industry oleoresins and/or other concentrated forms have been generated by solvent extraction, concentration, drying and/or mechanical pressing, as well as extraction into vegetable oils. The evaluation of supercritical CO2 biomass extraction processes was also performed. Partly, as a result of this work, IBR was able to market two new and improved versions of tomato-powder oral supplement active ingredient (Phytofloral®), with increased phytoene and phytofluene concentrations and varied particle size profiles for different end applications.

(iii) Solanesol. Waste vegetative material from tomato and potato crops, along with GM tobacco varieties was utilised. A disruption procedure was used to create a juice, from which traditional solvent procedures were used to extract non-polar molecules like solanesol. Following enrichment by preparative chromatography, enriched solanesol preparations were achieved. Minimal disruption procedures with non-toxic extraction methodologies were also used, this included the use of supercritical CO2 extraction processes.

(iv) Scolopamine. Dried leaves from Solanaceae species was subjected to continuous solvent extraction using process programmable logic controllers. The existing processes used by Boehringher proved difficult to improve.

Biorefining: Utilisation of biomass material for chemical, energy and biofuel production. Following optimal processes for DISCO target molecules, our aim was to develop workflows that are versatile to the feedstocks generated and can convert biomass into valuable/useful by-products. The production of a plant derived “Juice or oleoresins” was an initial step in down-stream processing. These preparations can be fractionated into polar and non-polar material. For example, Figure 12 illustrates the different multifractional approaches adopted by Proplanta to generated useful enriched extracts and product lines.

Figure 12. Initial down-stram processing and formulations followed by prototype products created with green chemistry.

Based on the experimental data generated by P1-RHUL, P9-Proplanta and P10-FPP a biorefinery model by using Superpro Designer v8.5 software has been created by P5-FCR. Three plant capacities have been selected at small (10 ha), middle (314 ha) and larger (1,900 ha) agronomic traits, so three scenarios were techno-economically evaluated. A simplified biorefinery scheme is presented in Figure 13.

Figure 13. Simplified process flowsheet proposed for biogas production using tomato plant residues.

Conclusions:
- Traditional methodologies can be used to purify the DISCO target molecules.
- “Green” minimal bioprocessing procedures have been developed for ketocarotenoid and phytoene/ phytofluene.
- The use of GRAS food crops using sink tissue material is highly advantageous for “green” minimal bioprocessing.
- A biorefining model has been developed for the production of Solanaesol, ketocarotenoids and associated co-products.


WP 8: Technical and economic feasibility of plant based renewable production
Objectives of WP8:
O8.1: Demonstrate the production feasibility for new sustainable plant based sources of (i) scopolamine, (ii) ketocarotenoids and (iii) phytoene/phytofluene supplements/materials in aquaculture and cosmetic products.
O8.2: Show the potential of new production approaches for solanesol.

Activities and results of WP8:
WP8 established a framework to assess the logistical and economic feasibility of the production platforms generated in the project. Performing these feasibility studies provided the data from which informed economic judgements were made on the viability of the processes, and the most effective business and marketing strategies to employ.

Plant based production systems for Scopolamine. P15-Boehringer has a long tradition as the producer of butyl scopolamine (Buscopan®). Their platform uses Solanacae hybrids grown on plantations globally. Modern metabolomics was carried out using HPLC-MS and 1H NMR based approaches to compare different genotypes of Solanaceae species and growth stages as well as different cultivation conditions (outdoor vs. indoor cultivation) and on different soils in various geographical locations. The hybrids developed and those presently used demonstrated good complementarity and robustness. However, it was clear that environmental factors such as the influence of light and temperature on scopolamine biosynthesis and biomass production were key factors. Thus, a clear environmental influence existed that appeared to have more influence that the genetics of the genotypes. One aspect that was not investigated, that the consortium believe will have a major influence, is the effect of the microbiota.

A terrestrial plant based source of ketocarotenoids. Prior to DISCO, no plant based sources of ketoacrotenoid existed that show amenability to agricultural production.
Tomato hybrids were evaluated, a canthaxanthin, astaxanthin and 4-ketozeaxanthin producer, concurrently with their closest comparator. The cultivation of 200 plants/crop (or 0.4 hectare) was carried out over two repeated life cycles, in two geographical locations using different cultivation approaches, contained glasshouse and polytunnel (See Figure 14). In addition, a comparison was made with a rootstock grafted crop, hydroponic cultivation and exposure to environmental stresses. The procedures used to assess the cultivation of the GM material adhered to national regulatory guidelines and approval acquired prior to the generation of the crop and transfer of germplasm. Agronomic parameters and environmental impact were recorded. Ripe fruit and vegetative material were harvested and analysed for carotenoids to determine the precise ketocarotenoid contents. Metabolomics analysis was carried out to provide a judgement on substantial equivalence as well as the effect of environment and cultivation conditions on chemical composition.

Figure 14. Polytunnel cultivation of DISCO prototypes; minimal resources input was used.

Table 1. Carotenoid content in ZWRI tomato line and azygous controls grown in greenhouse or polytunnel conditions. Carotenoid levels are represented as μg/g dry weight and in bold figures as percentages. Three representative fruits of N plants were used. The fruits were respectively pooled and three determinations were made. The mean data are shown as ± SD. Nq signifies that a compound has been detected but it is under the level of quantification. Polytunnel condition are defined by a polytunnel structure, without extra heating and lighting. Computed p-values for the comparison of ZWRI tomatoes grown in the greenhouse and the polytunnel are tabulated.

New varieties for the production of colourless carotenes. WP6 delivered new tomato genotypes producing high levels of phytoene/phytofluene (colourless carotenes). These varieties were compared when grown in the UK and Israel under glasshouse conditions as well as full industrial scale field trials in Akko, Israel.

Utilisation of a potato and tomato waste stream for solanesol production. Studies in WP6 and WP7 concluded that the most logistically effective source of solanesol was vegetative waste material from potato. Potato controlled material and material challenged by abiotic stresses cultivated on a pilot scale (e.g. 0.4 hectare) was used. In addition, under contained cultivation conditions, at a pilot scale production level a stable GM tobacco genotype developed in WP6 was used alongside as a comparator. The Commonwealth Potato Collection accession, S.tuberosum TBR5646 was identified as being high in leaf solanesol content in WP2 when grown under glasshouse conditions. This accession was evaluated for its potential as a dual crop by growing 100 plants using standard field practices at Balruddery Farm, Dundee, during 2015 and 16. The leaf solanesol content in this material was found to be lower than the levels previously observed in glasshouse grown plants (1.6 % DW vs < 0.1 % DW), thereby suggesting that environmental conditions have a significant impact on solanesol accumulation in potato leaves. Waste foliage from 2015 season TBR5646 plants was air dried and green extraction methods used for solanesol enrichment.

Conclusions:
- New genetic resources for the production of scopolamine have been shown to be fit for purpose and can withstand environmental fluctuations experienced in different geographical locations.
- The scalability of the new ketocarotenoid biosources has been demonstrated. The yield of the bioactive compounds ensures that enough material for an aquaculture season can be cultivated in a contained manner in polytunnels with low resource input.
- Accessions of potato have been identified that can deliver solanesol at 1 to 1.5% from vegatitive waste material.
- New genotypes for phytoene and phytofluene demonstrated economic viability at industrial field trails evaluation.


WP 9: Demonstrate the processing of the bioactive containing biosources into effective products
Objectives of WP9:
O9.1: Demonstrate how “green chemistry” can be used to deliver an effective aquaculture feedstuff and concurrent valuable products.
O9.2: Demonstrate the incorporation of “natural” phytoene/phytofluene into an effective new range of potential cosmetic products.
O9.3: The production of solanesol as a raw material for industrial and health sectors using an integrated process utilising Solanaceae waste as the feedstock.
O9.4: In comparison to existing processes, demonstrate that scopolamine can be produced in a competitive manner by the new biosources developed.

Activities and results of WP9:
In WP9 the sustainable biosources generated in previous WPs, containing the bioactive of interest were processed into products and the effectiveness of these products demonstrated. Comparisons to existing processes and products was performed and biomass utilisation for potential co-product production evaluated concurrently in order to impact upon the circular economy. The processes and end-products evaluated included; (i) ketocarotenoids in aquaculture feedstuffs, (ii) scopolamine as a pharmaceutical product, (iii) colourless carotenes for the cosmetic industry and (iv) solanesol for the health sector.

Aquaculture feeding trials:
The scalability and down-stream processing procedures developed in WP8 were carried out at an industrial scale to generate a “DISCO” prototype feed for aquaculture and poultry. These preparations were bench marked against existing market leaders.
The potential of the DISCO derived aquaculture material containing sustainable colorant as feed for the coloring of rainbow trout (Oncorhynchus mykiss) fillets was investigated in two geographical locations (Germany and Chile), where different conditions (fresh and brackish water, respectively) were used to assess the robustness of the platform. Four to five feed treatments were tested (basic, control tomato, ZWRI tomato, ZWRI extract and commercial feeds). The tomato feeds were made using freeze dried tomato powder. The ZWRI extract feed was based on an oily carotenoid extract of the ZWRI tomatoes. The synthetic BioMar supplement and carophyll pink® pigments were utilized for the commercial feed in the fresh and brackish experiment, respectively. Levels of total ketocarotenoids in the ZWRI and commercial feeds were targeted at 75-80 ppm and clarified after feed processing. Trout with a starting weight of 100 g and 40 g were fed with the different treatments for 50 days and up to 80 days under fresh and brackish conditions, respectively.
Following the feeding trials, a pink stripe along the lateral line of the fish (from gills to tail) was observed on the fish fed with the ZWRI tomato, ZWRI extract and commercial treatments. These same fish harbored coloured fillets with an orange to pink hue whereas the fish fed with the basic and control tomato feeds had white fillets (Figure 15). Fillet colour estimation using the DSM SalmoFanTM lineal showed that ZWRI feeds provided comparable fillet colour compared to the commercial feeds despite the commercial feed for the fresh water trial having a greater initial ketocarotenoid content. The ketocarotenoid composition in the feed and in the fillet remained the same for the commercial treatments and was predominantly astaxanthin (Figure 16). However, for the ZWRI treatments, the main change was the loss of the ketocarotenoid esters in the fillet compared to the feed (Figure 16). The ketocarotenoids found in the ZWRI fillets were phoenicoxanthin, canthaxanthin and some astaxanthin (Figure 16). Most of the endogenous tomato carotenoids such as lycopene and β-carotene were also not found in the trout fillet. The retention of (keto) carotenoid indicates quantitatively how these compounds were retained in the fillet, compared to their initial amount in the feed and is represented as a percentage. For the trout trial in fresh water, the retention of the coloring ketocarotenoids (phoenicoxanthin, canthaxanthin and astaxanthin) in the fillet was more than 2-fold greater in the ZWRI treatment compared to the commercial supplement. In particular, astaxanthin and phoenicoxanthin had exceptionally high retention while in the case of the brackish water experiment, the retention of the coloring ketocarotenoids was similar when comparing the ZWRI tomato, ZWRI extract and the commercial treatments. Carotenoids were also quantified in the feces of the fresh water trout. Levels of coloring ketocarotenoids were 7 times greater in the feces of trout fed with the commercial treatment compared to the ZWRI tomato one and the retention was 1.2-fold higher for the commercial treatment.
No significant difference was observed when comparing the weight of the fish across the experiments. Levels of carotenoids deposited in the eyes and livers of the trout obtained in the fresh water trial were minimal (~10 μg/g DW) and actually lower in ZWRI compared to the commercial treatment. No significant difference was detected in cholesterol contents in the fillets and livers of the fresh water trout from the various feed conditions. The retinoid, retinyl acetate and apocarotenal, β-apo-14´-carotenal were found at similar levels in the trout livers from the basic and control tomato conditions. However, their levels were both increased in livers of trout fed with the ZWRI tomato and commercial feeds (2.4 to 4-fold and 3.3-fold respectively). No significant difference in retinyl acetate and β-apo-14´-carotenal contents was noticed between the two latter treatments. Fatty acids were also quantified in the different feeds and fillets from the fresh water trial. No significant difference in total fatty acid content was observed between the different feeds. The main fatty acids in the feeds were C18:1, C18:2 and C16:0. In the fillets, these fatty acids were still predominant but C22:6 increased considerably in the fillets compared to the feeds. The fatty acid composition of the fillets reflected that of the feeds used. Moreover, a global analysis of the non-polar metabolites present in the fillets, derived from the different feed treatments tested, displayed no discernable clustering/separation following Principal Component Analysis (PCA) on the basis of the feed supplement used.

Figure 15. ZWRI tomatoes color trout fillets. Photographs of the trout fed with the basic, commercial, control tomato, ZWRI tomato and ZWRI extract feeds, taken at the end of the fresh and brackish water trials (50 and 80 days, respectively).

Figure 16. Chromatographic profiles of carotenoids in the feed and fillet corresponding to the commercial and ZWRI tomato treatments. 1, astaxanthin; 1#, unknown ketocarotenoid-1; 2, phoenicoxanthin; 3, canthaxanthin; 4, 3´-OH-echinenone; 5, 3-OH-echinenone; 6, echinenone; 7, phoenicoxanthin-C14:0; 8, adonixanthin-C14:1; 9, phoenicoxanthin-C16:0; 10, adonixanthin-C16:1; 11, β-carotene.

Effective cosmetic products containing colourless carotenes.
PhytoflORAL®, a lycopene-free tomato powder rich in phytoene and phytofluene, was formulated into two model oral supplement formulations, each of whose daily dose delivered ca. 5 mg of Phytoene and Phytofluene: a simple, powder-filled hard-shelled capsule; and a more sophisticated softgel capsule.
The sensorial / organoleptic properties of the supplements were evaluated and found acceptable for the purposes of the studies. The stability of the supplements was evaluated, and found to be sufficient for the immediate purposes of the study at hand. Finally, a model drink formulation comprising PhytoflORAL® was also developed on the laboratory scale for technical and commercial evaluation. IBR-TCLC®, a lycopene-free tomato extract rich in phytoene and phytofluene, was formulated.
The sensorial / organoleptic properties of the formulations were evaluated and found acceptable for the purposes of the proposed studies. The stability of the products was evaluated, and found to be sufficient for the purposes of the studies at hand as well as to qualify it as a preliminary model for a final product execution. Equivalent placebo formulations were also developed in parallel.
The photoprotective effect were assessed and found to have a positive impact on skin mechanics such as elasticity, firmness, anti-wrinkle properties and skin tone.

Solanesol production from waste material.
Potato vegetative waste from the optimal variety identified in WP8 was used. Solanesol was processed from this raw material and residue biomass utilised using procedures developed in WP7. As a measure of quality the physical, chemical and biological properties of the product were assessed and compared to that available in the market place. After purification the product was comparable to that on the market place. In addition, the solanesol present predominated as an ester which appeared to improve stability. Under bioactivity assays developed in WP3 the solanesol preparations displayed the same bioactive profiles as that obtained in the market place.

Production and effectiveness of scopolamine from the new biosources
Pharmaceutical grade scopolamine and its derivatives were prepared from the hybrids tested in DISCO. The production characteristics were compared to the present system. The cost of scopolamine circumvents market forces for added value products derived directly from a multifractional down-stream processing activity avoiding classical organic solvents. Thus, the utilisation of biomass was the focus in an attempt to improve the environmental impact and help “green” the process but was not deemed commercially attractive to the multinational partner producing scopolamine and would involve altering the capital infrastructure involved.

Conclusions:
A new source of colorants for aquaculture.
A new plant-based source of ketocarotenoids has been achieved and scalability has been demonstrated in a contained manner under a field-like environment with low resource input. The effectiveness of the tomato based material or its derived ketocarotenoid extract to act as an aquaculture feed supplement, responsible for coloring salmonid flesh, has been demonstrated and bench marked against two existing chemically synthesized products on the market. Over two trials in different geographical locations, in both brackish and fresh water conditions, the addition of the tomato material as an admix out performed existing industry products in terms of ketocarotenoid retention in the trout fillets. No adverse effects on animal husbandry or yield parameters were observed and chemical substantial equivalence was determined. The high ketocarotenoid tomato extracts also had the potential to color trout fillets. However, the tomato matrix seemed to improve the retention of the ketocarotenoids in the fillets by nearly 2-fold. Further work is required to ascertain the mechanism underlying this important phenomena.
The rudimentary approach to formulation used in this study and the results achieved suggest that further optimization of the process will deliver an improved product beyond the prototype used to date. The use of an admix also greatly improves the environmental impact of the process as no organic solvents are required in the down-stream processing or formulation process. In addition to its improved environmental credentials, the reduction in costs are significant. Although a full Life Cycle Analysis (LCA) is necessary, our estimates suggest that the keto tomato admix could provide an approximate 10-fold cost saving, as presently the production cost of the synthetic feed is in the range of US$1000 to 2000 per kg. Using the data generated in this study, the production costs for tomato material containing a kilogram of coloring ketocarotenoids are in the region of US$150. It is important to note that the reason such an admix can be effective is because of the levels reached in the selected tissues used. In this particular case, tomato fruit is the ideal sink tissue because it is intrinsically adapted to isoprenoid production. Although ketocarotenoids have been produced in lettuce, potato, maize, canola and soybean seeds, the levels are over two orders of magnitude lower than those achieved in tomato. These low levels make the vast amounts of seed material required for incorporation into feed formulation impractical. Presumably, a major reason why these sources are limited is because they have evolved and been selected for starch and oil accumulation. It is interesting that the non-endogenous ketocarotenoids produced existed, where chemically possible, in an esterified form. Precisely how this phenomena arises and their potential to facilitate sequestration awaits further elucidation. Tomato fruit is also an established food, which is readily digestible and regarded as Generally Recognized as Safe (GRAS). Non-food sources such as tobacco do not have these credentials, no sink organs are readily amenable for production, this means that pleotropic effects are likely to occur in vegetative tissues when high levels (above 3% dry weight) are reached and substantial down-stream processes essential.

Phytoene and phytofluene cosmetics products. New formulations including DISCO derived phytoene and phytofluene were demonstrated to be effective and have been commercialised as products, e.g. IBR-TCLC® and PhytoflORAL®.
Solanesol. The potential of utilizing certain plant varieties as sources of solanesol has been demonstrated. The use of vegetative non-food material necessitates extensive purification.
Scopolamine. The new hybrids were demonstrated as sources capable of delivering effective sample formulation.

The data generated by DISCO indicated that if production costs savings for economic competitiveness and low environmental impact are the objectives, then bioprocessing of food GRAS plant based sources is highly favorable. Thus, non-food sources as typically advocated contradicts the findings of DISCO.
Potential Impact:
The outputs from the DISCO project will impact directly on a number of key strategic areas including:

Scientific and technological quality.

The DISCO project has delivered scientific discovery with impact by furthering scientific advancement, public engagement to disseminate outputs and knowledge, education and training of the workforce and technology transfer to industries. The delivery of a commercial product from the project illustrates and supports the approach taken in DISCO to add value across all stages of the discovery pipeline.

Testimony to quality of the scientific advances are the number and quality of the publications in prestigious high impact peer reviewed scientific journals. For example, (Figure 17 A to C), the PNAS manuscripts describing the development and application of efficient and inducible transplastomic systems (WP5; Fig. 17 A), the isolation and characterisation of tools and resources associated with crocin and other apocarotenoid formation (WP6; Fig 17 B) and the technical, production and economic feasibility studies demonstrating new sustainable sources of ketocarotenoids for aquaculture (Fig. 17 C).

Figure 17. Examples of publications in high impact peer reviewed journals.

Besides the notable scientific advances in answering key biological questions, DISCO has had pure technological impacts. For example, the power of new generation sequencing (NGS) technologies in the form of RNA-Seq has proven invaluable to the progress of DISCO activities (P14-IGATS). These RNA-Seq technologies were essential in the elucidation of saffron derived apocarotenoid formation and of tropane alkaloid formation. Metabolomics protocols and platforms have also been advanced for utilisation across the scientific community. In several cases, new authentic standards and well annotated chromatograms have been delivered. It was, however, evident that in comparison to NGS technologies, metabolomics has not progressed with the same rapidity and in order to generate greater impact more activities in the field of metabolomics are required. These could include a step-change in metabolite annotation and incorporation of spatial and dynamic parameters into the analyses.
DISCO has generated valuable large-scale datasets, at the point of publication these data have and will be deposited in publicly available databases (ENA). All data will be released within 3 years of the project’s completion. All software and analytical pipelines have been openly shared to the community using GitHub and publicly released upon publication using open source licenses (e.g. GPL, MIT). Experimental datasets and accompanying metadata have also been deposited on the departmental and PIs website and within public databases. Metadata descriptions have also been included as outlined by the MIAMET. The metabolomics data have be submitted to databases such as MetaboLights (www.ebi.ac.uk) and Science data (www.nature.com/data) at the point of publication. In addition, experimental protocols have been made available via the bioprotocols website (www.bio-protocol.org/e1861). Furthermore, WP2 has created a new database of bioactives from Solanaceae.

Genotypes and molecular resources have been distributed under a Material Transfer Agreement (MTA), following communication with the Principle Investigators and their research offices. Resources have be deposited in community repositories such as addgene (www.addgene.org) Project outputs in peer reviewed journals have been published in preprint format (biorxiv) and in an open access format. The OpenAIRE (Open Access Infrastructure for Research in Europe) guidelines have been adhered to and surpassed. An archive copy of all data from DISCO will be securely retained for 10 years. The exploitation of IP has not impede delivery of the information to the wider scientific community.

In conclusion, we believe that the DISCO project has made a major contribution to the development and provision of genetic tools and resources as well as unique data sets that can be exploited by researchers from diverse disciplines in the future not only for the advancement of science but delivery of impact.

Public engagement wide participation.

DISCO conveyed its scientific advances and their potential impact through a number of avenues. These included digestible video animations and scientific interviews (http://disco-fp7.eu/media/) with Early Career Researchers (ECRs) and senior researchers.

The regional open days enabled the delivery of impact to lifelong learners and school pupils. One of the most rewarding activities was providing practical scientific activities to primary school children. Their appreciation is illustrated in the thank you card displayed in Figure 18.

A key objective of DISCO was to deliver a project that went beyond Europe and had global impact. This was clearly achieved through the number of invited lectures at prestigious conferences. However, a defining activity was taking the consortium to Chile, facilitated by our ICPC partner P5-FCR. To our knowledge this was the first time an EU integrated project had been taken to South America. The programme in Chile enabled interaction with companies in the region, via an industrial forum, resulting in potential licensing agreements, engagement with universities and research institutes in the region where agreement for postgraduate/teaching alliances where established bringing revenue to Europe. Also performed was a scientific workshop where ECRs presented their work. Similar activities were also carried out in China later in the project.

In summary, the dissemination tools and targeted activities of DISCO enabled the very widest project participation. The outcomes of DISCO were global and across all levels from primary school children to lifelong learners. Successfully performing these tasks will have a positive impact on the adoption of the technologies necessary for the development of the bioeconomy.

Figure 18. Thank you card for the practical activities provided for primary school children.

Training and education.

DISCO provided direct and indirect training for undergraduate science students via numerous internships, postgraduate studies at both MSc and PhD levels, postdoctoral research staff training and academic/industry staff via Continuous Professional Development (CPD). The consortium organised a dedicated training school for ECRs (WP11) and contributed to numerous other training schools typically organised via COST ACTION funding. It is our impression that DISCO delivered the same if not more training than typically associated with an ETN programme. The presence of academic partners responsible for the delivery of scientific curriculum to the next generation of scientists/workforce was fully exploited, with the very latest developments in the field being incorporated into the scientific curriculum across Europe. For example, the techniques and application of horizontal gene transfer for the development of new species (Figure 19; P2-MPG) are now an integral component of the University of London undergraduate syllabus.

Figure 19. An example recent scientific advances incorporated into the scientific curriculum in Europe.

The environment and sustainable development.

DISCO has created renewable biosources for high-value chemicals (bioactives) that have traditionally been chemically synthesised using petro-chemical derived precursors. The utilisation of cheap renewable bio-resources and eco-efficient processes will reduce environmental impact in comparison to existing processes. DISCO has generated data that clearly shows the utility of the approach with significant results capable of delivering translational impact in the field. In the case of ketocarotenoids the data generated has been compiled from two robust aquaculture and poultry trials. To add value, biomass utilisation has been performed to generate biofuels, reducing dependence on fossil fuels and contributing to a carbon neutral process.

P6-JHI has demonstrated how potato crop waste can be efficiently used to create high-value chemicals adding value for the grower and stimulating new market opportunities. An overarching theme of DISCO has been the development of biorefining concepts. The cascades developed by P9-PRO and P10-FPP have demonstrated that advantages of minimal bioprocessing (eliminating the use of solvents) and how useful GRAS starting material can be in this respect. Therefore, for downstream processing with low environmental impact, DISCO strongly advocates the use of GRAS material will minimal bioprocessing. These findings are contrary to the adoption of non-food material as a feedstocks to drive the bioeconomy.
DISCO outputs have demonstrated the production, technical and economic utility of sustainable feedstocks as direct replacements for fossil fuel derived precursors. The data, tools and prototypes generated will impact directly on the delivery of the circular economy to support the activities of the bioeconomy. One of the key objectives now is to create a societal and political framework that will facilitate large multinational adoption of sustainable feedstocks over existing traditional chemical synthesis.

Industrial policy, prosperity and employment.

The scientific and technological advances made in DISCO have impacted on European competitiveness creating new and increased markets leading to increased economic growth, prosperity and job creation. Industrial SMEs and multinational partners have been actively involved in the programme fostering cooperation and capacity building at a global level. During the course of the project it was evident from the industrial partners wanting to join the consortium that industrial awareness and capacity had been created. The DISCO project has been able to convert EUROs to products with a commercial product derived from DISCO innovation released by the SME partner (P12-IBR) in conjunction with an academic partner (P3-HUJ), (see Figure 20). This has resulted in increased economic prosperity and job stability as well as creation. The success of the approach has also created the opportunity to apply the methodology to other high-value compounds such as zeaxanthin, lycopene and beta carotene. Further funding has been sought to develop this experimental workplan.

Figure 20. DISCO derived innovation product development by P12-IBR and P3-HUJI.

In addition to the direct generation of prosperity through product commercialisation, a number of successful licensing agreements of DISCO derived patents were achieved for income generation.

The training and expertise developed in DISCO has enabled direct employment for many of the ECRs involved in DISCO, despite the less than favourable economic climate. Employment opportunities have been captured within the scientific community both in academia and industry, policy, management, teaching and marketing. This diversity illustrates the impact disco has had on delivering technically skilled individuals that are adaptable to multiple employment sectors.

Agriculture.

The cultivation of new and underutilised crops as biosources for high-value bioactives has stimulated agricultural development in both developed and developing countries. The short-term impact will be the development of improved varieties for the production of high-value bioactives from GRAS related material. For example, scalability is being assessed for the production of other bioactives in this manner. The incorporation of similar metabolite profiling techniques into the CGIAR centers for the assessment of biodiversity and breeding programmes has now been adopted following development by P1-RHUL. The DISCO consortium also benefited from the germplasm collections for Capsicum and potato maintained the CGIAR. Potential has been identified within these collections for the production of extracts containing enriched bioactives. The utilisation of genetic resources has also lead to further funding in the area via DISCO consortium members participating and coordinating the G-2-P network.

Quality of life and health.

The feasibility studies performed in DISCO have now provided the data on the new platforms for the production of bioactives that are cheaper, have increased accessibility, new activities as well as conferring quality and health properties. In this way, the quality of life and health of European populations will be improved. P13-SB have also fully exploited the potential of their high through-put bioassays and identified extracts with promising activities to known pharmaceutical targets. IPR has been captured to ensure optimal impact.

An output from DISCO was the identification of molecular mechanisms associated with increased beta carotene (provitamin A) content in plants. Also evident was the importance of carotenoid degeneration. Vitamin A deficiency (VAD) is the predominant cause of childhood blindness (500,000 cases per yr) and death (1-2 million) annually in Sub-Saharan Africa. Staple crops prevalent in developing countries such as cassava, banana and yam could be bred or engineered with higher beta -carotene, (provitamin A) contents. DISCO partners and tools could deliver a feasible strategy for the biofortification of provitamin A in staple crops found in low-income countries. Alleviating this problem is critical to achieving the UN Sustainable Development Goals. Consistent dialogue with organisations such as the CGIAR, HarvestPlus and BMGF has the potential to take DISCO outputs into projects that will have humanitarian impact in developing countries.

In addition to the DISCO target molecules, a number of “added value” by-products with health promoting properties were identified. Potential sustainable sources of these bioactives were also revealed. One such compound with the potential for great impact is Zeaxanthin a bioactive potent against age-related macular degeneration (AMD). This disease state is a chronic eye disorder, associated with the disturbance of the pigmented cell layer under the macula of the eye, especially during ageing. AMD accounts for 54% of all cases of blindness globally (Global Burden of Disease, 2015, Lancet, 386, 743), where in western societies, approximately 30% of the population, by the age of 65 years, are affected (Mehta et al. 2005, Primary Care, 42, 377 & Abel 2004, The Eye Care Revolution, N.Y. ISBN 0-7582-0622). Epidemiological and intervention studies, have demonstrated that the dietary intake of zeaxanthin/lutein rich foods can prevent the onset of this chronic eye disease (Brown et al. 1999, Am. J. Clin Nutr., 70, 517, Lyle et al. 1999, Am. J. Epidemiol., 801 & Seddon et al. 1994, J. Am. Med. Assoc., 272, 1413).
The vast economic losses and welfare costs associated with diet related disease states are estimated to be $2 trillion or a 3% global GDP loss. For AMD specifically it is estimated that by 2020 the resulting economic losses will be in the region of $150 to 250 billion. DISCO has developed the tools for the identification of high zeaxanthin/lutein solanaceae crops.

European cohesion.

DISCO is truly pan-European, including active industrial partners (30% of the total budget), an ICPC partner and a number of globally based associates. Through the transfer of technology and joint efforts of selected experts with complementary skills, industrial cooperation and capacity building on a global scale will be achieved. The training opportunities provided through DISCO have made a tangible impact on the professional and cultural development of staff and students, performing scientific and complementary activities. It is hoped that future twining projects will evolve from the interaction.

Rural development.

The project promoted investment and development of rural economies at both a European and global scale, the latter addressing Sustainable Millennium Developmental goals. The DISCO project provided opportunities for rural development (e.g. Romania and north east Scotland). Expertise developed will remain local, allowing higher wages and promote investment and development of rural economies. This approach is in line with the policy of involving rural areas into the globalisation process by ensuring an active economic role.

Therefore, in summary the DISCO project impacted directly on European policies relating to:

- Environment and sustainable development. The utilisation of cheap renewable bio-resources and eco-efficient processes will reduce environmental impact in comparison to existing processes.
- Quality of life, health and food. Through the development of platforms for the production of materials that are beneficial to human and animal health, as well as conferring quality to food and feedstuffs.
- Agriculture. Developing and implementing strategies for the plant based production of high value plant derived products.
- Industrial policy and employment. The scientific and technological advances impact on European competitiveness creating new and increased markets leading to increased economic growth and job creation. Industrial partners including SMEs were actively involved in the programme fostering industrial cooperation and capacity building at a European level.
- European cohesion. The project was truly pan-European and included active industrial partners. Through the transfer of technology and joint efforts of selected experts with complementary skills industrial cooperation and capacity building at a European level was achieved.
- Rural development. The project promoted investment and development of rural economies.
- Scientific and technological quality. The project delivered scientific excellence with impact by furthering scientific advancement, public engagement to disseminate outputs and knowledge, education and training of the workforce and technology transfer to industries.

Figure 21: DISCO interconnectivity of programme activities, outcomes and delivery of impact.
List of Websites:
Website: http://disco-fp7.eu/

Contact: Prof. Paul D. Fraser
ROYAL HOLLOWAY UNIVERSITY OF LONDON
Tel: +44 (0)1784 443894
Fax: +44 (0) 1784 414224
E-mail: P.Fraser@rhul.ac.uk