disco final public report · final report page 6 of 37 against market leaders and showed that the...

From DISCOvery to products:

A next generation pipeline for the sustainable generation of high-value

plant products

Final Report

Final Publishable Summary

Grant Agreement no: 613513

Project acronym: DISCO

Project title: From DISCOvery to products: A next generation pipeline for the

sustainable generation of high-value plant products

Funding scheme: Collaborative project

Period covered: 01/11/2013 to 31/10/2017

Coordinator: Prof. Paul D. Fraser

Organisation: Royal Holloway University of London

Tel: +44 (0)1784 443894

Fax: +44 (0) 1784 414224

E-mail: [email protected]

Project website: http://disco-fp7.eu/

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Table of Content

1. Executive summary ........................................................................................................................................ 3

2. Summary description of project context and the main objectives .............................................................. 4

3. Description of main Scientific &Technological results/foregrounds............................................................. 7

WP 2: Bioprospecting for bioactive identification .............................................................................................. 7

WP 3: Bioactivity assessment and valorisation ................................................................................................... 8

WP 4: The biosynthesis of bioactives; pathway elucidation, candidate gene isolation ................................... 10

WP 5: Enabling technologies for the optimisation of metabolic engineering .................................................. 12

WP 6: The production of high-value plant products by genetic intervention .................................................. 15

WP 7: Downstream processing and Biorefining ............................................................................................... 19

WP 8: Technical and economic feasibility of plant based renewable production ............................................ 22

WP 9: Demonstrate the processing of the bioactive containing biosources into effective products .............. 24

4. Description of the potential impact ............................................................................................................. 29

5. Address of project public website and relevant contact details ................................................................. 36

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1. 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.

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2. Summary description of project context and the main 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.

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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

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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.

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3. Description of main Scientific &Technological results/foregrounds

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).

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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.


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

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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.

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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.

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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.

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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.


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.

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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).

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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.

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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.


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.

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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

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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.

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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).

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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.


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.

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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.

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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.

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.

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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.


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.

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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.

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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.


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.

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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.

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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

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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

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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.

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4. Description of the 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).

Fig. 17 A

Fig. 17 B

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.

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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 Ameri

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