Final Report Summary - TRIFORC (A pipeline for the discovery, sustainable production and commercial utilisation of known and novel high-value triterpenes with new or superior biological activities.)
Executive Summary:
The TriForC consortium has reached its goal and developed a pipeline for the discovery, sustainable production and commercial utilization of known and novel high-value triterpenes with new or superior biological activities (Figure 1). The integrative and innovative pipeline has been used for the exploitation of plant triterpenes, one of the largest classes of plant bioactive compounds with an astonishing array of structural diversity and spectrum of biological activities. Triterpenes are synthesized from the common 30-carbon intermediate squalene by a large number of plant taxa spanning the plant Kingdom and a few marine organisms. Over 20,000 triterpene compounds are known, representing a variety of basic skeletons that are further permutated by enzymes to produce more elaborated structures and diversity. Triterpenes’ known roles in nature are in defense against pathogens, pests and predators. As triterpenes are structurally highly diverse and multifunctional, they have a wide range of commercial applications in the agriculture, food, cosmetics and pharmaceutical sectors as pesticides, drugs, adjuvants, antimicrobials, anticancer agents, surfactants, preservatives, etc. Through six research and technological development (RTD) work packages (WPs), the TriForC partners have brought the consortium the necessary tools, resources, and methods to assemble the pipeline components from the beginning of the project (figure 2).
The five ambitious scientific and technical objectives have all been achieved:
• New bioactive triterpenes for commercial development for pharmaceuticals and agrochemicals have been identified.
• Structure-Activity-Relationships (SAR) of triterpenes through activity screenings have been determined.
• A genetic toolbox that captures triterpene diversity in the laboratory has been generated.
• A metabolic engineering platform for rational design of triterpenes for production in bioreactors has been established.
• Plant-based bioreactors for sustainable commercial production and biorefining of high-value triterpenes have been developed and upscaled.
In particular, TriForC has generated a unique library of extracts and purified triterpenes from a selected range of plants known to be rich in triterpenoids to unleash the potential of triterpenes (WP1). A range of semi-synthetic modifications of triterpenoids has been generated to increase the structural diversity of the TriForC triterpenoid library. This rich library has been screened for biologically active triterpenoids with insecticidal and fungicidal potential for agrochemical applications and/or as drug medicinal targets to treat cancer, inflammation, obesity, diabetes, and HIV-1 infection (WP2). At the same time TriForC has established a unique collection of genes encoding for enzymes catalyzing this vast structural diversity, thereby capturing the potential of triterpene diversity in the TriForC toolbox (WP3). Based on the information acquired through TriForC we have constructed a public database that allows for searches in triterpenoid structures, genes and pathways (WP3). The TriForC toolbox has been successfully used in our metabolic engineering platform for bio-based production of known triterpenes and new-to-nature structures by combining genes from different species, even across Kingdoms of Life, in a combinatorial manner (WP4). Extracts and purified compounds from these production platforms have been fed back into our screening platform (WP2). A range of biobased production platforms have been developed in TriForC, including yeast cells, plant hairy root cultures, plant shoot cultures, whole tobacco plants and various salt and fresh water algae, which has allowed us to scale-up biobased sustainable production of triterpenes (WP5). A prediction tool into which known costs can be input for the specific production platforms has been generated, and a development plan for identified lead TriForC compounds has been produced (WP6). Several triterpene hits have been formulated and their biosafety, efficacy and positioning by data screening and field tests has been evaluated.
TriForC has had wide-reaching socio-economic impact through delivery and dissemination of science, filing of a patent, training of participating researchers and through innovative strategies for engagement with broad audiences, in particular through the SAW (Science, Art and Writing) initiative (WP7).
Project Context and Objectives:
The TriForC goal: To develop a pipeline for the discovery, sustainable production and commercial utilisation of known and novel high-value triterpenes with new or superior biological activities.
Mankind is continually screening low-molecular-weight compounds from a plethora of synthetic and natural sources in the search for molecules with novel or superior pharmaceutical or biological activities. Various bioprospecting, synthetic and biotech strategies to produce and diversify natural products are being exploited to provide new pipelines for bioactive molecules, e.g. for use as drugs or agrochemicals. Plants are a potential rich source of such molecules. However, because of their extreme diversity and complex chemistry, plant metabolism is still under-explored. Consequently, the full potential of plant-derived, low-molecular weight, bioactive compounds is still largely untapped.
The TriForC consortium has tackled this issue by establishing an integrative and innovative pipeline (Figure 1) for the exploitation of plant triterpenes, one of the largest classes of plant bioactive compounds with an astonishing array of structural diversity and spectrum of biological activities. The consortium has deployed and developed state-of-the-art technologies to achieve its ambitious aims.
The ambitious goal of TriForC can be divided into five scientific and technical objectives:
• Identify new bioactive triterpenes for commercial development as pharmaceuticals and agrochemicals.
• Elucidate Structure-Activity-Relationships (SAR) of triterpenes through biological activity screenings.
• Constitute a genetic toolbox that will allow mimicking and expanding triterpene structural diversity in the laboratory.
• Develop a metabolic engineering platform for rational design of triterpenes for production in bioreactors.
• Develop and upscale plant-based bioreactors for sustainable commercial production and biorefining of high-value triterpenes
The TriForC partners have each brought to the consortium the necessary tools, resources, methods and production systems required to assemble the pipeline and to reach the five scientific and technical objectives. Although the TriForC pipeline is sequential in structure, all individual TriForC activities have been operational from the beginning of the project where each partner delivered their initial components.
Figure 1 The TriForC pipeline for discovery and production of known and novel bioactive
triterpenes for pharmaceutical and agrochemical development. From bioprospecting, bioactivity screens, gene discovery to sustainable and industrially exploitable supply by in planta production systems.
The TriForC pipeline has specifically addressed three of the major challenges for European bioindustry in the efficient exploitation of triterpenes with novel bioactivities:
• Sustainable access to triterpene-plant source material, by establishing platforms that allow bioreactor-based production of triterpenes from plants that are endangered or difficult to cultivate.
• Bottlenecks in triterpene metabolic engineering, by using cutting-edge gene mining concepts, a genetic toolbox has been established that enables a synthetic biology platform for the versatile production of designer triterpenes in organisms amenable to bioindustry-scale cultivation.
• Optimal use of triterpene producing plant biomass, by consciously assessing different production sources and investigating downstream processing, separation and biorefinery possibilities.
Why Focus on Triterpenes
Numerous specialised metabolic pathways have evolved within each plant species, leading to complex mixtures of hundreds to thousands of compounds that contribute to a plant’s development and survival in different ways. It is estimated that the number of metabolites is over 200,000 for the whole plant kingdom and, as such, matches the metabolic richness of the microbial world. In fact, the great diversity of specialised metabolites produced by plants (and microbes) derives from a limited number of building blocks that are ubiquitous in the majority of living organisms. These building blocks are combined and/or extensively ‘decorated’ to form a multitude of diverse structures that have highly divergent biological activities, despite their common biosynthetic origin. Among the classes of identified natural products, terpenes are the most numerous and structurally diverse plant natural products. Triterpenoids are synthesized by an array of plant families from isopentenyl pyrophosphate through the 30-carbon intermediate squalene. Over 20,000 members are known representing a variety of skeletons (such as cycloartanes, dammaranes, quassinoids, lupanes, oleananes, friedelanes, ursanes, hopanes) that are further permutated to produce more elaborated skeletons, including steroidal structures and glycosylated saponins. Their role in nature has been mainly reported to be in defence against pathogens, pests and predators although unrecognized biological functions may also exist. Triterpenoids have been studied intensively for their immense structural diversity and wide variety of biological activities. The bioactive properties and levels of bioactivity depend on the nature of the triterpenoid or steroidal skeleton and on the modifications and different degrees of glycosylation that are made to such skeletons.
Triterpenoids are highly multifunctional and, thus, have a wide range of commercial applications in the agriculture, food, cosmetics and pharmaceutical sectors as pesticides, drugs, adjuvants, antimicrobials, anticancer agents, surfactants, preservatives, etc. Similarly, the structurally-related, sterol-based molecules known as steroidal triterpenes have also well-established uses. Triterpenes are generally regarded as stable secondary metabolites. Because of their stability, and their wide range of industrial and medical applications the development of novel metabolic engineering approaches to increase their yields and their structural variability is of great interest.
Given their structural similarity to membrane sterols, it is perhaps not surprising that triterpene and steroidal molecules can influence membrane-related processes within cells. Moreover, the ability of triterpenoid saponins to complex with sterols and cause membrane permeabilisation and pore-formation at relatively high concentrations is well known (Figure 1.2).
In recent years it has become obvious from numerous bioassays that these molecules can also have a variety of other effects on cells that are mediated through specific interactions with metabolic processes, cellular receptors and structural proteins.
However, despite the enormous potential in structures and bioactivities, there are currently clear limitations that have led to only a restricted arsenal of natural triterpenes being commercially exploited.
Therefore, the objectives of WP1 is to provide TriForC a selection of bioactive triterpenoids suitable for further development, leveraging on: 1) the isolation and characterization of structurally unique triterpenoids from selected plant sources, 2) the semi-synthetic modifications of bioactivity leading to increase of the structural diversity of the library of natural plant triterpenoids obtained in the first point, and 3) the identification of natural or “new-to-nature” triterpenes generated in yeast, tobacco or algal expression systems in WP4 using synthetic biology/combinatorial-biochemistry approaches.
Triterpenes in pharmacology
For centuries extracts from plants have been the main source of folk and modern medicines, and ~70% of the current drugs on the market are of plant origin, and ~25% of prescription drugs in clinical use today contain one or more active principles of plant origin. Recent evidence from tissue culture, animal, and clinical studies demonstrate that triterpenoids, along with their close chemical relatives, the steroids, are potent anti-inflammatory, anticarcinogenic and antihyperglycemic molecules. The non-exhaustive list of examples in the litteratureserves to exemplify the vast array of triterpenoid variability in terms of structures and pharmaceutical bioactivities. The aim of WP 2 in TriForC is to screen for triterpenoids leads for future pharmaceutical development, with focus on screening using a range of in-house cell lines for new medicinal targets to treat cancer, inflammation, obesity, diabetes, and HIV-1 infection.
Triterpenes in agriculture (as biological pesticides)
Plants produce numerous chemicals for defence and communication, and can elicit their own form of chemical control against pests and pathogens. The exploitation of these chemicals, which may exhibit general or specific activity, for the protection of crops and stored products from pests and pathogens is probably as old as crop protection itself. Plant material or crude plant extracts have traditionally been used to serve these purposes. Recently, documented issues of widespread environmental contamination, toxicity to non-target organisms and negative effects on human health by synthetic agrochemicals has led to a resurgence in the interest of ‘natural’ means of pest and pathogen control, including intensified searches for new sources of botanical pesticides. Natural products are considered to have stronger prospects for environmental safety, biodegradability and renewability. Another impetus for the discovery of natural pesticides from plants has been their putative value as lead compounds with novel bioactivities for use by the agrochemical industry. Co-evolution of plants, pests and pathogens combined to niche biogeography has shaped a wide range of plant secondary metabolites with selective “warfare” properties against pests and pathogens (and minimal environmental disturbance traits). The high structural diversity of natural products, as compared to the limited molecular scaffoldings used in organic chemistry, may provide useful structures in developing new classes of natural product-based pesticides, often via novel mechanisms of action. The latter is particularly important since new pesticides are also needed to combat the evolution of resistance to synthetic pesticides. The 2nd aim of WP2 in TriForC is to screen for triterpenoids that can be leads for future development of biopesticides with focus on insects, fungi and bacteria.
Bioengineering in plant and microalgal production systems
TriForC has built a unique toolbox (WP3) to enable the synthesis and regio-selective modification of triterpenes through metabolic engineering (WP4) and provide novel enzyme catalysts for chemistries derived from the biochemical versatility of plant triterpene biosynthesis. There are four main objectives in the toolbox: 1) Mining genomes and transcriptomes for candidates for new triterpene biosynthetic enzymes and pathways; 2) Functional analysis of the candidate triterpene enzymes to build the TriForC toolbox; 3) Identiy a set of regulators involved in regulating when a selected set genes in triterpenoid biosynthesis is turned on or off; 4) construction of a public database that allows for searches in triterpenoid structures, genes and pathways.
As plants and microalgae naturally accumulate the triterpene precursor 2,3-oxidosqualene for sterole biosynthesis they are directly amenable to triterpene metabolic engineering. Using the TriForC toolbox (WP3), plant bioreactor yeast cultures, tobacco leaves, cucumber and legume hairy roots and microalgae systems have been used to metabolically engineer enzymatic steps in triterpene biosynthesis to produce novel and new-to-nature triterpenes by stacking triterpenoid pathway genes in this expression platforms (WP4). This offers a potent complementary approach to our exploitation of 3rd generation temporary immersion bioreactors that will secure a stable and sustainable production of rare or endangered triterpene-producing plant tissue.
From ‘Triterpene bioactives platform’ to upscaling and commercialisation.
TriForC has selected a few cases for upscaled supply of simple and novel target triterpenes. This will be achieved through the development of small- and large-scale bioreactors for production in differentiated plant biomass from source species, and in engineered biomass as a proof of concept (WP5).
Finally, TriForC has as objective to address the techno-economic potential of the different production platforms, in terms of production, exploitation and regulation. This includes i) benchmarking (economic analysis) of production systems; ii) the process of (pre)-clinical trials (Pharma) and field trials (Agro); iii) biorefinery/cascade approaches; and iv) toxicity and safety testing of formulated products, including identification and consideration of any regulatory issues associated with the intended use of products and co-products within specific markets. This completed the TriForC pipeline and established the ‘Triterpene commercialisation platform’.
The interactions between the TriForC Work Packages are shown in Figure 2 below. The RTD Work Packages 1-5 were initiated in month 1 and WP6 in month 18.
The objectives of WP7 was to implement the TriForC dissemination and engagement strategy and to ensure a productive and fertile training environment for the participating researchers. WP7 involves:
• Engagement with other consortia and research groups working in complementary areas to ensure optimal delivery of the programme.
• Dissemination of TriForC research and outputs to stakeholders, the international scientific community and society.
• Training of researchers in TriForC science and technologies, and in engagement and dissemination activities that will enhance awareness and appreciation of EU-funded science.
Project Results:
The TriForC goal: To develop a pipeline for the discovery, sustainable production and commercial utilisation of known and novel high-value triterpenes with new or superior biological activities.
Mankind is continually screening low-molecular-weight compounds from a plethora of synthetic and natural sources in the search for molecules with novel or superior pharmaceutical or biological activities. Various bioprospecting, synthetic and biotech strategies to produce and diversify natural products are being exploited to provide new pipelines for bioactive molecules, e.g. for use as drugs or agrochemicals. Plants are a potential rich source of such molecules. However, because of their extreme diversity and complex chemistry, plant metabolism is still under-explored. Consequently, the full potential of plant-derived, low-molecular weight, bioactive compounds is still largely untapped.
The TriForC consortium has tackled this issue by establishing an integrative and innovative pipeline (Figure 1) for the exploitation of plant triterpenes, one of the largest classes of plant bioactive compounds with an astonishing array of structural diversity and spectrum of biological activities. The consortium has deployed and developed state-of-the-art technologies to achieve its ambitious aims.
The ambitious goal of TriForC can be divided into five scientific and technical objectives:
• Identify new bioactive triterpenes for commercial development as pharmaceuticals and agrochemicals.
• Elucidate Structure-Activity-Relationships (SAR) of triterpenes through biological activity screenings.
• Constitute a genetic toolbox that will allow mimicking and expanding triterpene structural diversity in the laboratory.
• Develop a metabolic engineering platform for rational design of triterpenes for production in bioreactors.
• Develop and upscale plant-based bioreactors for sustainable commercial production and biorefining of high-value triterpenes
The TriForC partners have each brought to the consortium the necessary tools, resources, methods and production systems required to assemble the pipeline and to reach the five scientific and technical objectives. Although the TriForC pipeline is sequential in structure, all individual TriForC activities have been operational from the beginning of the project where each partner delivered their initial components.
Figure 1 The TriForC pipeline for discovery and production of known and novel bioactive
triterpenes for pharmaceutical and agrochemical development. From bioprospecting, bioactivity screens, gene discovery to sustainable and industrially exploitable supply by in planta production systems.
The TriForC pipeline has specifically addressed three of the major challenges for European bioindustry in the efficient exploitation of triterpenes with novel bioactivities:
• Sustainable access to triterpene-plant source material, by establishing platforms that allow bioreactor-based production of triterpenes from plants that are endangered or difficult to cultivate.
• Bottlenecks in triterpene metabolic engineering, by using cutting-edge gene mining concepts, a genetic toolbox has been established that enables a synthetic biology platform for the versatile production of designer triterpenes in organisms amenable to bioindustry-scale cultivation.
• Optimal use of triterpene producing plant biomass, by consciously assessing different production sources and investigating downstream processing, separation and biorefinery possibilities.
Why Focus on Triterpenes
Numerous specialised metabolic pathways have evolved within each plant species, leading to complex mixtures of hundreds to thousands of compounds that contribute to a plant’s development and survival in different ways. It is estimated that the number of metabolites is over 200,000 for the whole plant kingdom and, as such, matches the metabolic richness of the microbial world. In fact, the great diversity of specialised metabolites produced by plants (and microbes) derives from a limited number of building blocks that are ubiquitous in the majority of living organisms. These building blocks are combined and/or extensively ‘decorated’ to form a multitude of diverse structures that have highly divergent biological activities, despite their common biosynthetic origin. Among the classes of identified natural products, terpenes are the most numerous and structurally diverse plant natural products. Triterpenoids are synthesized by an array of plant families from isopentenyl pyrophosphate through the 30-carbon intermediate squalene. Over 20,000 members are known representing a variety of skeletons (such as cycloartanes, dammaranes, quassinoids, lupanes, oleananes, friedelanes, ursanes, hopanes) that are further permutated to produce more elaborated skeletons, including steroidal structures and glycosylated saponins. Their role in nature has been mainly reported to be in defence against pathogens, pests and predators although unrecognized biological functions may also exist. Triterpenoids have been studied intensively for their immense structural diversity and wide variety of biological activities. The bioactive properties and levels of bioactivity depend on the nature of the triterpenoid or steroidal skeleton and on the modifications and different degrees of glycosylation that are made to such skeletons.
Triterpenoids are highly multifunctional and, thus, have a wide range of commercial applications in the agriculture, food, cosmetics and pharmaceutical sectors as pesticides, drugs, adjuvants, antimicrobials, anticancer agents, surfactants, preservatives, etc. Similarly, the structurally-related, sterol-based molecules known as steroidal triterpenes have also well-established uses. Triterpenes are generally regarded as stable secondary metabolites. Because of their stability, and their wide range of industrial and medical applications the development of novel metabolic engineering approaches to increase their yields and their structural variability is of great interest.
Given their structural similarity to membrane sterols, it is perhaps not surprising that triterpene and steroidal molecules can influence membrane-related processes within cells. Moreover, the ability of triterpenoid saponins to complex with sterols and cause membrane permeabilisation and pore-formation at relatively high concentrations is well known (Figure 1.2).
In recent years it has become obvious from numerous bioassays that these molecules can also have a variety of other effects on cells that are mediated through specific interactions with metabolic processes, cellular receptors and structural proteins.
However, despite the enormous potential in structures and bioactivities, there are currently clear limitations that have led to only a restricted arsenal of natural triterpenes being commercially exploited.
Therefore, the objectives of WP1 is to provide TriForC a selection of bioactive triterpenoids suitable for further development, leveraging on: 1) the isolation and characterization of structurally unique triterpenoids from selected plant sources, 2) the semi-synthetic modifications of bioactivity leading to increase of the structural diversity of the library of natural plant triterpenoids obtained in the first point, and 3) the identification of natural or “new-to-nature” triterpenes generated in yeast, tobacco or algal expression systems in WP4 using synthetic biology/combinatorial-biochemistry approaches.
Triterpenes in pharmacology
For centuries extracts from plants have been the main source of folk and modern medicines, and ~70% of the current drugs on the market are of plant origin, and ~25% of prescription drugs in clinical use today contain one or more active principles of plant origin. Recent evidence from tissue culture, animal, and clinical studies demonstrate that triterpenoids, along with their close chemical relatives, the steroids, are potent anti-inflammatory, anticarcinogenic and antihyperglycemic molecules. The non-exhaustive list of examples in the litteratureserves to exemplify the vast array of triterpenoid variability in terms of structures and pharmaceutical bioactivities. The aim of WP 2 in TriForC is to screen for triterpenoids leads for future pharmaceutical development, with focus on screening using a range of in-house cell lines for new medicinal targets to treat cancer, inflammation, obesity, diabetes, and HIV-1 infection.
Triterpenes in agriculture (as biological pesticides)
Plants produce numerous chemicals for defence and communication, and can elicit their own form of chemical control against pests and pathogens. The exploitation of these chemicals, which may exhibit general or specific activity, for the protection of crops and stored products from pests and pathogens is probably as old as crop protection itself. Plant material or crude plant extracts have traditionally been used to serve these purposes. Recently, documented issues of widespread environmental contamination, toxicity to non-target organisms and negative effects on human health by synthetic agrochemicals has led to a resurgence in the interest of ‘natural’ means of pest and pathogen control, including intensified searches for new sources of botanical pesticides. Natural products are considered to have stronger prospects for environmental safety, biodegradability and renewability. Another impetus for the discovery of natural pesticides from plants has been their putative value as lead compounds with novel bioactivities for use by the agrochemical industry. Co-evolution of plants, pests and pathogens combined to niche biogeography has shaped a wide range of plant secondary metabolites with selective “warfare” properties against pests and pathogens (and minimal environmental disturbance traits). The high structural diversity of natural products, as compared to the limited molecular scaffoldings used in organic chemistry, may provide useful structures in developing new classes of natural product-based pesticides, often via novel mechanisms of action. The latter is particularly important since new pesticides are also needed to combat the evolution of resistance to synthetic pesticides. The 2nd aim of WP2 in TriForC is to screen for triterpenoids that can be leads for future development of biopesticides with focus on insects, fungi and bacteria.
Bioengineering in plant and microalgal production systems
TriForC has built a unique toolbox (WP3) to enable the synthesis and regio-selective modification of triterpenes through metabolic engineering (WP4) and provide novel enzyme catalysts for chemistries derived from the biochemical versatility of plant triterpene biosynthesis. There are four main objectives in the toolbox: 1) Mining genomes and transcriptomes for candidates for new triterpene biosynthetic enzymes and pathways; 2) Functional analysis of the candidate triterpene enzymes to build the TriForC toolbox; 3) Identiy a set of regulators involved in regulating when a selected set genes in triterpenoid biosynthesis is turned on or off; 4) construction of a public database that allows for searches in triterpenoid structures, genes and pathways.
As plants and microalgae naturally accumulate the triterpene precursor 2,3-oxidosqualene for sterole biosynthesis they are directly amenable to triterpene metabolic engineering. Using the TriForC toolbox (WP3), plant bioreactor yeast cultures, tobacco leaves, cucumber and legume hairy roots and microalgae systems have been used to metabolically engineer enzymatic steps in triterpene biosynthesis to produce novel and new-to-nature triterpenes by stacking triterpenoid pathway genes in this expression platforms (WP4). This offers a potent complementary approach to our exploitation of 3rd generation temporary immersion bioreactors that will secure a stable and sustainable production of rare or endangered triterpene-producing plant tissue.
From ‘Triterpene bioactives platform’ to upscaling and commercialisation.
TriForC has selected a few cases for upscaled supply of simple and novel target triterpenes. This will be achieved through the development of small- and large-scale bioreactors for production in differentiated plant biomass from source species, and in engineered biomass as a proof of concept (WP5).
Finally, TriForC has as objective to address the techno-economic potential of the different production platforms, in terms of production, exploitation and regulation. This includes i) benchmarking (economic analysis) of production systems; ii) the process of (pre)-clinical trials (Pharma) and field trials (Agro); iii) biorefinery/cascade approaches; and iv) toxicity and safety testing of formulated products, including identification and consideration of any regulatory issues associated with the intended use of products and co-products within specific markets. This completed the TriForC pipeline and established the ‘Triterpene commercialisation platform’.
The interactions between the TriForC Work Packages are shown in Figure 2 below. The RTD Work Packages 1-5 were initiated in month 1 and WP6 in month 18.
The objectives of WP7 was to implement the TriForC dissemination and engagement strategy and to ensure a productive and fertile training environment for the participating researchers. WP7 involves:
• Engagement with other consortia and research groups working in complementary areas to ensure optimal delivery of the programme.
• Dissemination of TriForC research and outputs to stakeholders, the international scientific community and society.
• Training of researchers in TriForC science and technologies, and in engagement and dissemination activities that will enhance awareness and appreciation of EU-funded science.
Potential Impact:
The major scientific impact of TriForC will lie in an improved understanding of plant secondary metabolism in general- and in the synthesis and the diversity therein for triterpenes. TriForC has enhanced our understanding of the structure-activity-relationships (SAR) of triterpenes. TriForC has also set new new standards for use of plant bioreactors and use of microalgae for bioproduction of triterpenoids. TriForC has performed basic research that has upgraded our current views of metabolic pathways in plants and how these pathways are structured, activated, controlled and how they might have evolved in time. This basic knowledge has provided novel tools that have facilitated (and will continue to do so in the future) rational metabolic engineering of plant metabolic pathways in general towards enhanced and diversified synthesis of novel, new-to-nature or existing bioactive molecules in plant, microalgal or yeast-based systems. Until now, more than 22 peer reviewed papers, more than 100 scientific talks at international meetings and a public TriForC database (http://bioinformatics.psb.ugent.be/triforc/#/home) has been disseminated to the wider scientific community, demonstrating the impact TriForC has had scientifically. Extensive training of early career scientists has been achieved through workshops and mentoring, to shape future scientists within academia and industry. TriForC has not only had wide-reaching socio-economic impact through delivery and dissemination of basic science, filing of a patent, training of participating researchers, but has also reached out further to broader audiences, in particular through the SAW Trust initiative, capturing the interest in science for the public. The SAW Trust practice has been disseminated by TriForC partners in different countries, including the UK, Greece, Israel, France, China and the USA. A reflective article reporting on the successes and challenges of TriForC project “Triterpene messages from the EU-FP7 project TriForC” has recently been submitted to the leading international science journal Trends in Plant Science. TriForC has also been disseminated to stakeholders thought journal ”Impact” (https://impact.pub/January2017digitaledition/ page 38-39) to promote the TriForC project and in particular the SMEs. Finally, gender issues were particularly addressed and special events were organized accordingly (i.e. Committee for Gender issues, Round table meeting, information on website etc)
Third grade elementary school pupils exploring plant and microbial natural products in Greece
Commercial impact: the complex biochemistry of triterpenoids makes their direct chemical synthesis extremely difficult, and hence extremely expensive, or impossible. TriForC has developed a fractionation protocol that allows for generation of extracts from plant material for systematic screening for bioactivities. TriForC has used this protocol for producing ~750 extracts for screening for activities related to triterpenoids. As the protocol is generic it can be used for other classes of compounds outside of TriForC. To help disseminate the protocol a video has been produced (https://drive.google.com/file/d/1WvWyuMJS5vOWCFEv7Ar68leIOjNbyheB/view). TriForC has also demonstrated ways to increase bioactivity by taking a semi-synthetic approach creating ~100 new-to-nature triterpenoid. This strategy has been patented (EP16139684), and can be used for future studies within triterpenoid research, but also other compound classes, and therefore has a potential impact on the ongoing exploitation in the pharmaceutical and agrochemical markets. The substantial IP generated throughout the project has been handled by our exploitation board headed by the Exploitation Manager, Andrew Spicer, in agreement with the Consortium Agreement. The Exploitation board has pro-actively screened the foreground coming out of TriForC for possible IP protection (summarized below).
The exploitable foreground from the TriForC project is varied and wide-ranging and expected to have an impact in the short term to support and catalyse world-class scientific research & development; in the mid to long-term to 1) drive commercial development opportunities around the exploitation of specific triterpenes and terpene/sterol/sapogenin related chemicals, 2) create job opportunities and 3) feed into educational and social policies; in the long-term to 1) drive substantial economic development, 2) improve agricultural outputs, 3) reduce our over-reliance on oil-based agricultural chemicals, 4) feed the world of the future and 5) care for our environment through the implementation of lower carbon footprint and sustainable technologies.
Within human health and medicine, new hits have been identified that may eventually find their way into the clinic in the form of new medicines to treat various medical conditions. New sustainable supply chains for existing pharmaceutical bioactives could be realised – indeed GSK has already been in active discussions with Algenuity around access to specific triterpenes produced through a synthetic biology supply chain as opposed to from biomass harvested from nature. These type of ongoing discussions highlight the growing realisation that we are and will be faced with limited natural resources from which we can continue to draw the raw materials we need to maintain or improve our standard of living. This problem is only expected to get worse with time and increasing global population.
Research & development findings range from furthering our understanding of structure function activity relationships (SAR) between enzymes and their ultimate biosynthetic product, an understanding of how combinations of heterologous enzymes can be used together to create novel or even new-to-nature compounds and a discovery of the enzymes responsible for the production of chemicals where no known biosynthetic pathway had been previously defined. Exploitable resources range from libraries of chemicals and plant extracts (~750 extracts/ ~100 semi synthetic analogs/~80 pure compounds), a diverse TriForC genetic toolbox encoding for enzymes in involved in triterpenoid biosynthesis, a library of gene cassettes encoding biosynthetic and/or regulatory enzymes important for combinatorial production of triterpenes in any one of three synthetic biology platforms that were the subject of this project, and an online database relating the project outputs in such a way that structure activity relationships can be identified and used as the basis to drive further cutting edge research on a wider, more global research canvas.
The exploitation routes for the key identified foreground that falls within a commercial sphere are quite specific and will be considered individually:
HIF-1alpha agonists for pharma
It has been reported in WP2 that several NCEs (TFC10-152, TFC10-153, TFC10-156 and TFC10-157) derived from oleanolic and betulinic acid presented as new biological activities as activators and stabilizers of the hypoxia inducible factor 1 alpha (HIF1-α). A European patent (EP16193684.4) describing these novel pentacyclic triterpenoid derivatives (triterpenoid derivatives) that show capacity to bind prolyl hydroxylase domain-containing protein (PHD2), stabilize HIF-1 and HIF-2 proteins, and activate the HIF pathway in different cell types, induce angiogenesis in human endothelial vascular cells, show neuroprotective activity in vivo, and increase the plasma levels of erythropoietin in vivo.
In March 2017 a consideration of the examiner’s response to the patent application led the inventors to devise an alternate approach whereby a new PCT application would be filed. The ongoing development of the target compounds is moving at a significant pace. Two lead compounds were identified as key considerations. Both were shown to have similarly potent inhibitory activity against PHD2. The compound derived from oleanolic acid was considered to be the more attractive based on raw material availability of the triterpene scaffold versus potential supply chain inconsistencies for betulinic acid.
Additional structure activity relationship studies have defined further the basis for the bioactivity towards PHD2. Plans are moving forward now to include manufacturing, non-clinical development (animal testing) and to enter into regulatory phase prior to any clinical trials. This includes a 4 year R&D and preclinical/nonclinical timeline; this has been described at length within the WP6 Final report.
In 2016-2017 controlling shareholding in VivaCell Technologies was acquired by Emerald Health Research Inc (Canada) – the continued development of the hits described above will be supported by an ongoing commitment from Emerald Health to pursue these leads at this time. It should be pointed out that the majority of hits or leads that enter into this process will not end up in the clinical. The average cost of taking a chemical through to the clinic represents is about $1 billion and the process can take as long as 10-15 years. The potential impact of successful launch of one of these chemicals into the clinic would be considerable. Advanced treatment of conditions ranging from stroke and wound healing to various other more specific diseases and conditions. Economic impact would be substantial and not simply due to sales or the final drug but the revenues and taxes drawn from all associated industries and disciplines that would need to be engaged along the drug development process. Social impact would be significant from a health perspective as well as a job creation perspective.
Triterpenes with pesticidal activity for agriculture
Twenty two TriForC compounds and extracts with potential pesticidal activity for potential use as biopesticides in agriculture were discovered in the WP2 screening activity. These compounds were considered “Hits” which are candidates for scaling up and further work in WP5 and WP6.
• Seven Hits were further produced by P6 in WP5 in small quantities (2- 15 gr), enabling small scale experiments in planta in growth chambers.
• One Hit, (euphol- euphorbol mixture) was obtained from P10 in 50 & 500 gr batches, which was sufficient for formulation and testing it in field and greenhouses trials.
• In parallel, four Hits were also obtained from vendors outside TrifoC in 1 kg batches, and formulated for further testing in field and greenhouse trials.
• The formulated Hits were evaluated for biosafety and regulatory potential (by data screening), and tested for phytotoxicity, pesticidal efficacy and positioning in field and greenhouse trials on a variety of crops and insect pests.
Exploitable foreground:
All Hits showed biological activity but the Euphol-euphorbol mixture, protopanaxatriol and betulinic acid ranked highest and will be further tested for development as new commercial biopesticide products at the end of TriForC.
The need for biopesticide solutions in agriculture is increasing due to restrictions on use of chemical pesticides as well as increasing customer awareness.
Biopesticides development is a 10-year process that requires investments of 5-7 million Euros per candidate. The process includes scaling up production to tonne scale and establishing industrial biorefineries. Bio- production and biorefineries are our next major challenges and solving them may require additional research. However, it is reasonable to assume that P5 (STK) will develop at least one TriforC Hit as a biopesticide product. IP will be generated where appropriate.
Extraction process for vegan cholesterol (from microalgae)
In the course of developing microalgal strains for triterpene production, Algenuity screened a large panel of commercially produced microalgae including freshwater and marine strains, to assess the baseline sterol productivity and profiles. One microalgal strain, a marine microalgal strain currently cultivated in the high 10’s to low 100’s of tons globally primarily to support aquaculture markets, emerged as a strain of significant interest based upon an extremely high cholesterol content and extremely simple sterol profile.
Within WP6, Extrasynthese and Algenuity worked together to develop this opportunity further both in technical and commercial terms. At present, negotiations are quite mature with a commercial client identified in the USA within the nutritional supplements sector; a pilot scale production process has been proposed to deliver a first 100g pilot batch to this client followed by a commitment to deliver 10kg in year 1 and 10’s of kilos up to a projected 100 kg in subsequent years. IP Protection is not possible with regard to the process or the target. Trade secret and stable supply chain for the biomass plus access and knowhow around new strains that could yield higher levels of the target compound as well as an exclusive agreement to supply and purchase all create barriers to entry for competitors.
Algenuity and Extrasynthese are negotiating a commercial agreement to exploit this opportunity. In the event that the client agrees with the terms of the pilot scale process development and commitment to supply product in the 10’s to 100’s of kilos, the pilot production phase will start immediately through a projected 3 month timescale. Subject to successful transition through the stage gate at 3 months, 10 kg of purified product would be supplied in the 1st year. This has the potential to bring in revenues in the low millions of Euros per year with profit sharing agreed between Algenuity and Extrasynthese. The impact of the successful commercial exploitation of this vegan cholesterol supply chain will be economic and environmental. At present, natural sources of vegan cholesterol as well as its desirable higher value derivatives are harvested from nature through a capped (limited) annual harvest. Removal of this plant material from the environment does have an impact on the relevant ecosystems.
The commercial opportunity could be substantially increased by identification of customers for other chemicals (biorefinery model) produced by the microalgal biomass in question including long-chain polyunsaturated fatty acids (omega-3’s). The commercial value of this fraction will largely be impacted by the extraction process being used and the purity of any resultant product – particularly with regard to the presence of any solvent traces in the final product.
Summary
It is difficult to quantify the long-term impacts of the foreground developed by this project. With only the three commercial opportunities outlined above as examples, these alone could bring 100’s of millions to billions of Euros in sales revenues globally over the next 25 years. There is little doubt that the fundamental research advances that have been made will provide a strong springboard from which further commercial opportunities will arise. The three commercial opportunities also highlight social and environmental impacts – job creation, development of linked businesses within supply chains, and reduced impact on the environment through development of new sustainable supply chains. Lastly, the potential impact of the strong and enthusiastic commitment within the consortium to participate in public-facing educational activities particularly spearheaded by the SAW Trust link provided by P8 cannot be overestimated. By taking time to invest in younger potential scientists, engineers, business developers, artists, writers, policymakers, and end-users – to pass on ideas and plant seeds of knowledge and inspiration – the return on investment of the TriForC project is expected to be staggering and longstanding.
List of Websites:
www.triforc.eu
Søren Bak Bak@plen.ku.dk
The TriForC consortium has reached its goal and developed a pipeline for the discovery, sustainable production and commercial utilization of known and novel high-value triterpenes with new or superior biological activities (Figure 1). The integrative and innovative pipeline has been used for the exploitation of plant triterpenes, one of the largest classes of plant bioactive compounds with an astonishing array of structural diversity and spectrum of biological activities. Triterpenes are synthesized from the common 30-carbon intermediate squalene by a large number of plant taxa spanning the plant Kingdom and a few marine organisms. Over 20,000 triterpene compounds are known, representing a variety of basic skeletons that are further permutated by enzymes to produce more elaborated structures and diversity. Triterpenes’ known roles in nature are in defense against pathogens, pests and predators. As triterpenes are structurally highly diverse and multifunctional, they have a wide range of commercial applications in the agriculture, food, cosmetics and pharmaceutical sectors as pesticides, drugs, adjuvants, antimicrobials, anticancer agents, surfactants, preservatives, etc. Through six research and technological development (RTD) work packages (WPs), the TriForC partners have brought the consortium the necessary tools, resources, and methods to assemble the pipeline components from the beginning of the project (figure 2).
The five ambitious scientific and technical objectives have all been achieved:
• New bioactive triterpenes for commercial development for pharmaceuticals and agrochemicals have been identified.
• Structure-Activity-Relationships (SAR) of triterpenes through activity screenings have been determined.
• A genetic toolbox that captures triterpene diversity in the laboratory has been generated.
• A metabolic engineering platform for rational design of triterpenes for production in bioreactors has been established.
• Plant-based bioreactors for sustainable commercial production and biorefining of high-value triterpenes have been developed and upscaled.
In particular, TriForC has generated a unique library of extracts and purified triterpenes from a selected range of plants known to be rich in triterpenoids to unleash the potential of triterpenes (WP1). A range of semi-synthetic modifications of triterpenoids has been generated to increase the structural diversity of the TriForC triterpenoid library. This rich library has been screened for biologically active triterpenoids with insecticidal and fungicidal potential for agrochemical applications and/or as drug medicinal targets to treat cancer, inflammation, obesity, diabetes, and HIV-1 infection (WP2). At the same time TriForC has established a unique collection of genes encoding for enzymes catalyzing this vast structural diversity, thereby capturing the potential of triterpene diversity in the TriForC toolbox (WP3). Based on the information acquired through TriForC we have constructed a public database that allows for searches in triterpenoid structures, genes and pathways (WP3). The TriForC toolbox has been successfully used in our metabolic engineering platform for bio-based production of known triterpenes and new-to-nature structures by combining genes from different species, even across Kingdoms of Life, in a combinatorial manner (WP4). Extracts and purified compounds from these production platforms have been fed back into our screening platform (WP2). A range of biobased production platforms have been developed in TriForC, including yeast cells, plant hairy root cultures, plant shoot cultures, whole tobacco plants and various salt and fresh water algae, which has allowed us to scale-up biobased sustainable production of triterpenes (WP5). A prediction tool into which known costs can be input for the specific production platforms has been generated, and a development plan for identified lead TriForC compounds has been produced (WP6). Several triterpene hits have been formulated and their biosafety, efficacy and positioning by data screening and field tests has been evaluated.
TriForC has had wide-reaching socio-economic impact through delivery and dissemination of science, filing of a patent, training of participating researchers and through innovative strategies for engagement with broad audiences, in particular through the SAW (Science, Art and Writing) initiative (WP7).
Project Context and Objectives:
The TriForC goal: To develop a pipeline for the discovery, sustainable production and commercial utilisation of known and novel high-value triterpenes with new or superior biological activities.
Mankind is continually screening low-molecular-weight compounds from a plethora of synthetic and natural sources in the search for molecules with novel or superior pharmaceutical or biological activities. Various bioprospecting, synthetic and biotech strategies to produce and diversify natural products are being exploited to provide new pipelines for bioactive molecules, e.g. for use as drugs or agrochemicals. Plants are a potential rich source of such molecules. However, because of their extreme diversity and complex chemistry, plant metabolism is still under-explored. Consequently, the full potential of plant-derived, low-molecular weight, bioactive compounds is still largely untapped.
The TriForC consortium has tackled this issue by establishing an integrative and innovative pipeline (Figure 1) for the exploitation of plant triterpenes, one of the largest classes of plant bioactive compounds with an astonishing array of structural diversity and spectrum of biological activities. The consortium has deployed and developed state-of-the-art technologies to achieve its ambitious aims.
The ambitious goal of TriForC can be divided into five scientific and technical objectives:
• Identify new bioactive triterpenes for commercial development as pharmaceuticals and agrochemicals.
• Elucidate Structure-Activity-Relationships (SAR) of triterpenes through biological activity screenings.
• Constitute a genetic toolbox that will allow mimicking and expanding triterpene structural diversity in the laboratory.
• Develop a metabolic engineering platform for rational design of triterpenes for production in bioreactors.
• Develop and upscale plant-based bioreactors for sustainable commercial production and biorefining of high-value triterpenes
The TriForC partners have each brought to the consortium the necessary tools, resources, methods and production systems required to assemble the pipeline and to reach the five scientific and technical objectives. Although the TriForC pipeline is sequential in structure, all individual TriForC activities have been operational from the beginning of the project where each partner delivered their initial components.
Figure 1 The TriForC pipeline for discovery and production of known and novel bioactive
triterpenes for pharmaceutical and agrochemical development. From bioprospecting, bioactivity screens, gene discovery to sustainable and industrially exploitable supply by in planta production systems.
The TriForC pipeline has specifically addressed three of the major challenges for European bioindustry in the efficient exploitation of triterpenes with novel bioactivities:
• Sustainable access to triterpene-plant source material, by establishing platforms that allow bioreactor-based production of triterpenes from plants that are endangered or difficult to cultivate.
• Bottlenecks in triterpene metabolic engineering, by using cutting-edge gene mining concepts, a genetic toolbox has been established that enables a synthetic biology platform for the versatile production of designer triterpenes in organisms amenable to bioindustry-scale cultivation.
• Optimal use of triterpene producing plant biomass, by consciously assessing different production sources and investigating downstream processing, separation and biorefinery possibilities.
Why Focus on Triterpenes
Numerous specialised metabolic pathways have evolved within each plant species, leading to complex mixtures of hundreds to thousands of compounds that contribute to a plant’s development and survival in different ways. It is estimated that the number of metabolites is over 200,000 for the whole plant kingdom and, as such, matches the metabolic richness of the microbial world. In fact, the great diversity of specialised metabolites produced by plants (and microbes) derives from a limited number of building blocks that are ubiquitous in the majority of living organisms. These building blocks are combined and/or extensively ‘decorated’ to form a multitude of diverse structures that have highly divergent biological activities, despite their common biosynthetic origin. Among the classes of identified natural products, terpenes are the most numerous and structurally diverse plant natural products. Triterpenoids are synthesized by an array of plant families from isopentenyl pyrophosphate through the 30-carbon intermediate squalene. Over 20,000 members are known representing a variety of skeletons (such as cycloartanes, dammaranes, quassinoids, lupanes, oleananes, friedelanes, ursanes, hopanes) that are further permutated to produce more elaborated skeletons, including steroidal structures and glycosylated saponins. Their role in nature has been mainly reported to be in defence against pathogens, pests and predators although unrecognized biological functions may also exist. Triterpenoids have been studied intensively for their immense structural diversity and wide variety of biological activities. The bioactive properties and levels of bioactivity depend on the nature of the triterpenoid or steroidal skeleton and on the modifications and different degrees of glycosylation that are made to such skeletons.
Triterpenoids are highly multifunctional and, thus, have a wide range of commercial applications in the agriculture, food, cosmetics and pharmaceutical sectors as pesticides, drugs, adjuvants, antimicrobials, anticancer agents, surfactants, preservatives, etc. Similarly, the structurally-related, sterol-based molecules known as steroidal triterpenes have also well-established uses. Triterpenes are generally regarded as stable secondary metabolites. Because of their stability, and their wide range of industrial and medical applications the development of novel metabolic engineering approaches to increase their yields and their structural variability is of great interest.
Given their structural similarity to membrane sterols, it is perhaps not surprising that triterpene and steroidal molecules can influence membrane-related processes within cells. Moreover, the ability of triterpenoid saponins to complex with sterols and cause membrane permeabilisation and pore-formation at relatively high concentrations is well known (Figure 1.2).
In recent years it has become obvious from numerous bioassays that these molecules can also have a variety of other effects on cells that are mediated through specific interactions with metabolic processes, cellular receptors and structural proteins.
However, despite the enormous potential in structures and bioactivities, there are currently clear limitations that have led to only a restricted arsenal of natural triterpenes being commercially exploited.
Therefore, the objectives of WP1 is to provide TriForC a selection of bioactive triterpenoids suitable for further development, leveraging on: 1) the isolation and characterization of structurally unique triterpenoids from selected plant sources, 2) the semi-synthetic modifications of bioactivity leading to increase of the structural diversity of the library of natural plant triterpenoids obtained in the first point, and 3) the identification of natural or “new-to-nature” triterpenes generated in yeast, tobacco or algal expression systems in WP4 using synthetic biology/combinatorial-biochemistry approaches.
Triterpenes in pharmacology
For centuries extracts from plants have been the main source of folk and modern medicines, and ~70% of the current drugs on the market are of plant origin, and ~25% of prescription drugs in clinical use today contain one or more active principles of plant origin. Recent evidence from tissue culture, animal, and clinical studies demonstrate that triterpenoids, along with their close chemical relatives, the steroids, are potent anti-inflammatory, anticarcinogenic and antihyperglycemic molecules. The non-exhaustive list of examples in the litteratureserves to exemplify the vast array of triterpenoid variability in terms of structures and pharmaceutical bioactivities. The aim of WP 2 in TriForC is to screen for triterpenoids leads for future pharmaceutical development, with focus on screening using a range of in-house cell lines for new medicinal targets to treat cancer, inflammation, obesity, diabetes, and HIV-1 infection.
Triterpenes in agriculture (as biological pesticides)
Plants produce numerous chemicals for defence and communication, and can elicit their own form of chemical control against pests and pathogens. The exploitation of these chemicals, which may exhibit general or specific activity, for the protection of crops and stored products from pests and pathogens is probably as old as crop protection itself. Plant material or crude plant extracts have traditionally been used to serve these purposes. Recently, documented issues of widespread environmental contamination, toxicity to non-target organisms and negative effects on human health by synthetic agrochemicals has led to a resurgence in the interest of ‘natural’ means of pest and pathogen control, including intensified searches for new sources of botanical pesticides. Natural products are considered to have stronger prospects for environmental safety, biodegradability and renewability. Another impetus for the discovery of natural pesticides from plants has been their putative value as lead compounds with novel bioactivities for use by the agrochemical industry. Co-evolution of plants, pests and pathogens combined to niche biogeography has shaped a wide range of plant secondary metabolites with selective “warfare” properties against pests and pathogens (and minimal environmental disturbance traits). The high structural diversity of natural products, as compared to the limited molecular scaffoldings used in organic chemistry, may provide useful structures in developing new classes of natural product-based pesticides, often via novel mechanisms of action. The latter is particularly important since new pesticides are also needed to combat the evolution of resistance to synthetic pesticides. The 2nd aim of WP2 in TriForC is to screen for triterpenoids that can be leads for future development of biopesticides with focus on insects, fungi and bacteria.
Bioengineering in plant and microalgal production systems
TriForC has built a unique toolbox (WP3) to enable the synthesis and regio-selective modification of triterpenes through metabolic engineering (WP4) and provide novel enzyme catalysts for chemistries derived from the biochemical versatility of plant triterpene biosynthesis. There are four main objectives in the toolbox: 1) Mining genomes and transcriptomes for candidates for new triterpene biosynthetic enzymes and pathways; 2) Functional analysis of the candidate triterpene enzymes to build the TriForC toolbox; 3) Identiy a set of regulators involved in regulating when a selected set genes in triterpenoid biosynthesis is turned on or off; 4) construction of a public database that allows for searches in triterpenoid structures, genes and pathways.
As plants and microalgae naturally accumulate the triterpene precursor 2,3-oxidosqualene for sterole biosynthesis they are directly amenable to triterpene metabolic engineering. Using the TriForC toolbox (WP3), plant bioreactor yeast cultures, tobacco leaves, cucumber and legume hairy roots and microalgae systems have been used to metabolically engineer enzymatic steps in triterpene biosynthesis to produce novel and new-to-nature triterpenes by stacking triterpenoid pathway genes in this expression platforms (WP4). This offers a potent complementary approach to our exploitation of 3rd generation temporary immersion bioreactors that will secure a stable and sustainable production of rare or endangered triterpene-producing plant tissue.
From ‘Triterpene bioactives platform’ to upscaling and commercialisation.
TriForC has selected a few cases for upscaled supply of simple and novel target triterpenes. This will be achieved through the development of small- and large-scale bioreactors for production in differentiated plant biomass from source species, and in engineered biomass as a proof of concept (WP5).
Finally, TriForC has as objective to address the techno-economic potential of the different production platforms, in terms of production, exploitation and regulation. This includes i) benchmarking (economic analysis) of production systems; ii) the process of (pre)-clinical trials (Pharma) and field trials (Agro); iii) biorefinery/cascade approaches; and iv) toxicity and safety testing of formulated products, including identification and consideration of any regulatory issues associated with the intended use of products and co-products within specific markets. This completed the TriForC pipeline and established the ‘Triterpene commercialisation platform’.
The interactions between the TriForC Work Packages are shown in Figure 2 below. The RTD Work Packages 1-5 were initiated in month 1 and WP6 in month 18.
The objectives of WP7 was to implement the TriForC dissemination and engagement strategy and to ensure a productive and fertile training environment for the participating researchers. WP7 involves:
• Engagement with other consortia and research groups working in complementary areas to ensure optimal delivery of the programme.
• Dissemination of TriForC research and outputs to stakeholders, the international scientific community and society.
• Training of researchers in TriForC science and technologies, and in engagement and dissemination activities that will enhance awareness and appreciation of EU-funded science.
Project Results:
The TriForC goal: To develop a pipeline for the discovery, sustainable production and commercial utilisation of known and novel high-value triterpenes with new or superior biological activities.
Mankind is continually screening low-molecular-weight compounds from a plethora of synthetic and natural sources in the search for molecules with novel or superior pharmaceutical or biological activities. Various bioprospecting, synthetic and biotech strategies to produce and diversify natural products are being exploited to provide new pipelines for bioactive molecules, e.g. for use as drugs or agrochemicals. Plants are a potential rich source of such molecules. However, because of their extreme diversity and complex chemistry, plant metabolism is still under-explored. Consequently, the full potential of plant-derived, low-molecular weight, bioactive compounds is still largely untapped.
The TriForC consortium has tackled this issue by establishing an integrative and innovative pipeline (Figure 1) for the exploitation of plant triterpenes, one of the largest classes of plant bioactive compounds with an astonishing array of structural diversity and spectrum of biological activities. The consortium has deployed and developed state-of-the-art technologies to achieve its ambitious aims.
The ambitious goal of TriForC can be divided into five scientific and technical objectives:
• Identify new bioactive triterpenes for commercial development as pharmaceuticals and agrochemicals.
• Elucidate Structure-Activity-Relationships (SAR) of triterpenes through biological activity screenings.
• Constitute a genetic toolbox that will allow mimicking and expanding triterpene structural diversity in the laboratory.
• Develop a metabolic engineering platform for rational design of triterpenes for production in bioreactors.
• Develop and upscale plant-based bioreactors for sustainable commercial production and biorefining of high-value triterpenes
The TriForC partners have each brought to the consortium the necessary tools, resources, methods and production systems required to assemble the pipeline and to reach the five scientific and technical objectives. Although the TriForC pipeline is sequential in structure, all individual TriForC activities have been operational from the beginning of the project where each partner delivered their initial components.
Figure 1 The TriForC pipeline for discovery and production of known and novel bioactive
triterpenes for pharmaceutical and agrochemical development. From bioprospecting, bioactivity screens, gene discovery to sustainable and industrially exploitable supply by in planta production systems.
The TriForC pipeline has specifically addressed three of the major challenges for European bioindustry in the efficient exploitation of triterpenes with novel bioactivities:
• Sustainable access to triterpene-plant source material, by establishing platforms that allow bioreactor-based production of triterpenes from plants that are endangered or difficult to cultivate.
• Bottlenecks in triterpene metabolic engineering, by using cutting-edge gene mining concepts, a genetic toolbox has been established that enables a synthetic biology platform for the versatile production of designer triterpenes in organisms amenable to bioindustry-scale cultivation.
• Optimal use of triterpene producing plant biomass, by consciously assessing different production sources and investigating downstream processing, separation and biorefinery possibilities.
Why Focus on Triterpenes
Numerous specialised metabolic pathways have evolved within each plant species, leading to complex mixtures of hundreds to thousands of compounds that contribute to a plant’s development and survival in different ways. It is estimated that the number of metabolites is over 200,000 for the whole plant kingdom and, as such, matches the metabolic richness of the microbial world. In fact, the great diversity of specialised metabolites produced by plants (and microbes) derives from a limited number of building blocks that are ubiquitous in the majority of living organisms. These building blocks are combined and/or extensively ‘decorated’ to form a multitude of diverse structures that have highly divergent biological activities, despite their common biosynthetic origin. Among the classes of identified natural products, terpenes are the most numerous and structurally diverse plant natural products. Triterpenoids are synthesized by an array of plant families from isopentenyl pyrophosphate through the 30-carbon intermediate squalene. Over 20,000 members are known representing a variety of skeletons (such as cycloartanes, dammaranes, quassinoids, lupanes, oleananes, friedelanes, ursanes, hopanes) that are further permutated to produce more elaborated skeletons, including steroidal structures and glycosylated saponins. Their role in nature has been mainly reported to be in defence against pathogens, pests and predators although unrecognized biological functions may also exist. Triterpenoids have been studied intensively for their immense structural diversity and wide variety of biological activities. The bioactive properties and levels of bioactivity depend on the nature of the triterpenoid or steroidal skeleton and on the modifications and different degrees of glycosylation that are made to such skeletons.
Triterpenoids are highly multifunctional and, thus, have a wide range of commercial applications in the agriculture, food, cosmetics and pharmaceutical sectors as pesticides, drugs, adjuvants, antimicrobials, anticancer agents, surfactants, preservatives, etc. Similarly, the structurally-related, sterol-based molecules known as steroidal triterpenes have also well-established uses. Triterpenes are generally regarded as stable secondary metabolites. Because of their stability, and their wide range of industrial and medical applications the development of novel metabolic engineering approaches to increase their yields and their structural variability is of great interest.
Given their structural similarity to membrane sterols, it is perhaps not surprising that triterpene and steroidal molecules can influence membrane-related processes within cells. Moreover, the ability of triterpenoid saponins to complex with sterols and cause membrane permeabilisation and pore-formation at relatively high concentrations is well known (Figure 1.2).
In recent years it has become obvious from numerous bioassays that these molecules can also have a variety of other effects on cells that are mediated through specific interactions with metabolic processes, cellular receptors and structural proteins.
However, despite the enormous potential in structures and bioactivities, there are currently clear limitations that have led to only a restricted arsenal of natural triterpenes being commercially exploited.
Therefore, the objectives of WP1 is to provide TriForC a selection of bioactive triterpenoids suitable for further development, leveraging on: 1) the isolation and characterization of structurally unique triterpenoids from selected plant sources, 2) the semi-synthetic modifications of bioactivity leading to increase of the structural diversity of the library of natural plant triterpenoids obtained in the first point, and 3) the identification of natural or “new-to-nature” triterpenes generated in yeast, tobacco or algal expression systems in WP4 using synthetic biology/combinatorial-biochemistry approaches.
Triterpenes in pharmacology
For centuries extracts from plants have been the main source of folk and modern medicines, and ~70% of the current drugs on the market are of plant origin, and ~25% of prescription drugs in clinical use today contain one or more active principles of plant origin. Recent evidence from tissue culture, animal, and clinical studies demonstrate that triterpenoids, along with their close chemical relatives, the steroids, are potent anti-inflammatory, anticarcinogenic and antihyperglycemic molecules. The non-exhaustive list of examples in the litteratureserves to exemplify the vast array of triterpenoid variability in terms of structures and pharmaceutical bioactivities. The aim of WP 2 in TriForC is to screen for triterpenoids leads for future pharmaceutical development, with focus on screening using a range of in-house cell lines for new medicinal targets to treat cancer, inflammation, obesity, diabetes, and HIV-1 infection.
Triterpenes in agriculture (as biological pesticides)
Plants produce numerous chemicals for defence and communication, and can elicit their own form of chemical control against pests and pathogens. The exploitation of these chemicals, which may exhibit general or specific activity, for the protection of crops and stored products from pests and pathogens is probably as old as crop protection itself. Plant material or crude plant extracts have traditionally been used to serve these purposes. Recently, documented issues of widespread environmental contamination, toxicity to non-target organisms and negative effects on human health by synthetic agrochemicals has led to a resurgence in the interest of ‘natural’ means of pest and pathogen control, including intensified searches for new sources of botanical pesticides. Natural products are considered to have stronger prospects for environmental safety, biodegradability and renewability. Another impetus for the discovery of natural pesticides from plants has been their putative value as lead compounds with novel bioactivities for use by the agrochemical industry. Co-evolution of plants, pests and pathogens combined to niche biogeography has shaped a wide range of plant secondary metabolites with selective “warfare” properties against pests and pathogens (and minimal environmental disturbance traits). The high structural diversity of natural products, as compared to the limited molecular scaffoldings used in organic chemistry, may provide useful structures in developing new classes of natural product-based pesticides, often via novel mechanisms of action. The latter is particularly important since new pesticides are also needed to combat the evolution of resistance to synthetic pesticides. The 2nd aim of WP2 in TriForC is to screen for triterpenoids that can be leads for future development of biopesticides with focus on insects, fungi and bacteria.
Bioengineering in plant and microalgal production systems
TriForC has built a unique toolbox (WP3) to enable the synthesis and regio-selective modification of triterpenes through metabolic engineering (WP4) and provide novel enzyme catalysts for chemistries derived from the biochemical versatility of plant triterpene biosynthesis. There are four main objectives in the toolbox: 1) Mining genomes and transcriptomes for candidates for new triterpene biosynthetic enzymes and pathways; 2) Functional analysis of the candidate triterpene enzymes to build the TriForC toolbox; 3) Identiy a set of regulators involved in regulating when a selected set genes in triterpenoid biosynthesis is turned on or off; 4) construction of a public database that allows for searches in triterpenoid structures, genes and pathways.
As plants and microalgae naturally accumulate the triterpene precursor 2,3-oxidosqualene for sterole biosynthesis they are directly amenable to triterpene metabolic engineering. Using the TriForC toolbox (WP3), plant bioreactor yeast cultures, tobacco leaves, cucumber and legume hairy roots and microalgae systems have been used to metabolically engineer enzymatic steps in triterpene biosynthesis to produce novel and new-to-nature triterpenes by stacking triterpenoid pathway genes in this expression platforms (WP4). This offers a potent complementary approach to our exploitation of 3rd generation temporary immersion bioreactors that will secure a stable and sustainable production of rare or endangered triterpene-producing plant tissue.
From ‘Triterpene bioactives platform’ to upscaling and commercialisation.
TriForC has selected a few cases for upscaled supply of simple and novel target triterpenes. This will be achieved through the development of small- and large-scale bioreactors for production in differentiated plant biomass from source species, and in engineered biomass as a proof of concept (WP5).
Finally, TriForC has as objective to address the techno-economic potential of the different production platforms, in terms of production, exploitation and regulation. This includes i) benchmarking (economic analysis) of production systems; ii) the process of (pre)-clinical trials (Pharma) and field trials (Agro); iii) biorefinery/cascade approaches; and iv) toxicity and safety testing of formulated products, including identification and consideration of any regulatory issues associated with the intended use of products and co-products within specific markets. This completed the TriForC pipeline and established the ‘Triterpene commercialisation platform’.
The interactions between the TriForC Work Packages are shown in Figure 2 below. The RTD Work Packages 1-5 were initiated in month 1 and WP6 in month 18.
The objectives of WP7 was to implement the TriForC dissemination and engagement strategy and to ensure a productive and fertile training environment for the participating researchers. WP7 involves:
• Engagement with other consortia and research groups working in complementary areas to ensure optimal delivery of the programme.
• Dissemination of TriForC research and outputs to stakeholders, the international scientific community and society.
• Training of researchers in TriForC science and technologies, and in engagement and dissemination activities that will enhance awareness and appreciation of EU-funded science.
Potential Impact:
The major scientific impact of TriForC will lie in an improved understanding of plant secondary metabolism in general- and in the synthesis and the diversity therein for triterpenes. TriForC has enhanced our understanding of the structure-activity-relationships (SAR) of triterpenes. TriForC has also set new new standards for use of plant bioreactors and use of microalgae for bioproduction of triterpenoids. TriForC has performed basic research that has upgraded our current views of metabolic pathways in plants and how these pathways are structured, activated, controlled and how they might have evolved in time. This basic knowledge has provided novel tools that have facilitated (and will continue to do so in the future) rational metabolic engineering of plant metabolic pathways in general towards enhanced and diversified synthesis of novel, new-to-nature or existing bioactive molecules in plant, microalgal or yeast-based systems. Until now, more than 22 peer reviewed papers, more than 100 scientific talks at international meetings and a public TriForC database (http://bioinformatics.psb.ugent.be/triforc/#/home) has been disseminated to the wider scientific community, demonstrating the impact TriForC has had scientifically. Extensive training of early career scientists has been achieved through workshops and mentoring, to shape future scientists within academia and industry. TriForC has not only had wide-reaching socio-economic impact through delivery and dissemination of basic science, filing of a patent, training of participating researchers, but has also reached out further to broader audiences, in particular through the SAW Trust initiative, capturing the interest in science for the public. The SAW Trust practice has been disseminated by TriForC partners in different countries, including the UK, Greece, Israel, France, China and the USA. A reflective article reporting on the successes and challenges of TriForC project “Triterpene messages from the EU-FP7 project TriForC” has recently been submitted to the leading international science journal Trends in Plant Science. TriForC has also been disseminated to stakeholders thought journal ”Impact” (https://impact.pub/January2017digitaledition/ page 38-39) to promote the TriForC project and in particular the SMEs. Finally, gender issues were particularly addressed and special events were organized accordingly (i.e. Committee for Gender issues, Round table meeting, information on website etc)
Third grade elementary school pupils exploring plant and microbial natural products in Greece
Commercial impact: the complex biochemistry of triterpenoids makes their direct chemical synthesis extremely difficult, and hence extremely expensive, or impossible. TriForC has developed a fractionation protocol that allows for generation of extracts from plant material for systematic screening for bioactivities. TriForC has used this protocol for producing ~750 extracts for screening for activities related to triterpenoids. As the protocol is generic it can be used for other classes of compounds outside of TriForC. To help disseminate the protocol a video has been produced (https://drive.google.com/file/d/1WvWyuMJS5vOWCFEv7Ar68leIOjNbyheB/view). TriForC has also demonstrated ways to increase bioactivity by taking a semi-synthetic approach creating ~100 new-to-nature triterpenoid. This strategy has been patented (EP16139684), and can be used for future studies within triterpenoid research, but also other compound classes, and therefore has a potential impact on the ongoing exploitation in the pharmaceutical and agrochemical markets. The substantial IP generated throughout the project has been handled by our exploitation board headed by the Exploitation Manager, Andrew Spicer, in agreement with the Consortium Agreement. The Exploitation board has pro-actively screened the foreground coming out of TriForC for possible IP protection (summarized below).
The exploitable foreground from the TriForC project is varied and wide-ranging and expected to have an impact in the short term to support and catalyse world-class scientific research & development; in the mid to long-term to 1) drive commercial development opportunities around the exploitation of specific triterpenes and terpene/sterol/sapogenin related chemicals, 2) create job opportunities and 3) feed into educational and social policies; in the long-term to 1) drive substantial economic development, 2) improve agricultural outputs, 3) reduce our over-reliance on oil-based agricultural chemicals, 4) feed the world of the future and 5) care for our environment through the implementation of lower carbon footprint and sustainable technologies.
Within human health and medicine, new hits have been identified that may eventually find their way into the clinic in the form of new medicines to treat various medical conditions. New sustainable supply chains for existing pharmaceutical bioactives could be realised – indeed GSK has already been in active discussions with Algenuity around access to specific triterpenes produced through a synthetic biology supply chain as opposed to from biomass harvested from nature. These type of ongoing discussions highlight the growing realisation that we are and will be faced with limited natural resources from which we can continue to draw the raw materials we need to maintain or improve our standard of living. This problem is only expected to get worse with time and increasing global population.
Research & development findings range from furthering our understanding of structure function activity relationships (SAR) between enzymes and their ultimate biosynthetic product, an understanding of how combinations of heterologous enzymes can be used together to create novel or even new-to-nature compounds and a discovery of the enzymes responsible for the production of chemicals where no known biosynthetic pathway had been previously defined. Exploitable resources range from libraries of chemicals and plant extracts (~750 extracts/ ~100 semi synthetic analogs/~80 pure compounds), a diverse TriForC genetic toolbox encoding for enzymes in involved in triterpenoid biosynthesis, a library of gene cassettes encoding biosynthetic and/or regulatory enzymes important for combinatorial production of triterpenes in any one of three synthetic biology platforms that were the subject of this project, and an online database relating the project outputs in such a way that structure activity relationships can be identified and used as the basis to drive further cutting edge research on a wider, more global research canvas.
The exploitation routes for the key identified foreground that falls within a commercial sphere are quite specific and will be considered individually:
HIF-1alpha agonists for pharma
It has been reported in WP2 that several NCEs (TFC10-152, TFC10-153, TFC10-156 and TFC10-157) derived from oleanolic and betulinic acid presented as new biological activities as activators and stabilizers of the hypoxia inducible factor 1 alpha (HIF1-α). A European patent (EP16193684.4) describing these novel pentacyclic triterpenoid derivatives (triterpenoid derivatives) that show capacity to bind prolyl hydroxylase domain-containing protein (PHD2), stabilize HIF-1 and HIF-2 proteins, and activate the HIF pathway in different cell types, induce angiogenesis in human endothelial vascular cells, show neuroprotective activity in vivo, and increase the plasma levels of erythropoietin in vivo.
In March 2017 a consideration of the examiner’s response to the patent application led the inventors to devise an alternate approach whereby a new PCT application would be filed. The ongoing development of the target compounds is moving at a significant pace. Two lead compounds were identified as key considerations. Both were shown to have similarly potent inhibitory activity against PHD2. The compound derived from oleanolic acid was considered to be the more attractive based on raw material availability of the triterpene scaffold versus potential supply chain inconsistencies for betulinic acid.
Additional structure activity relationship studies have defined further the basis for the bioactivity towards PHD2. Plans are moving forward now to include manufacturing, non-clinical development (animal testing) and to enter into regulatory phase prior to any clinical trials. This includes a 4 year R&D and preclinical/nonclinical timeline; this has been described at length within the WP6 Final report.
In 2016-2017 controlling shareholding in VivaCell Technologies was acquired by Emerald Health Research Inc (Canada) – the continued development of the hits described above will be supported by an ongoing commitment from Emerald Health to pursue these leads at this time. It should be pointed out that the majority of hits or leads that enter into this process will not end up in the clinical. The average cost of taking a chemical through to the clinic represents is about $1 billion and the process can take as long as 10-15 years. The potential impact of successful launch of one of these chemicals into the clinic would be considerable. Advanced treatment of conditions ranging from stroke and wound healing to various other more specific diseases and conditions. Economic impact would be substantial and not simply due to sales or the final drug but the revenues and taxes drawn from all associated industries and disciplines that would need to be engaged along the drug development process. Social impact would be significant from a health perspective as well as a job creation perspective.
Triterpenes with pesticidal activity for agriculture
Twenty two TriForC compounds and extracts with potential pesticidal activity for potential use as biopesticides in agriculture were discovered in the WP2 screening activity. These compounds were considered “Hits” which are candidates for scaling up and further work in WP5 and WP6.
• Seven Hits were further produced by P6 in WP5 in small quantities (2- 15 gr), enabling small scale experiments in planta in growth chambers.
• One Hit, (euphol- euphorbol mixture) was obtained from P10 in 50 & 500 gr batches, which was sufficient for formulation and testing it in field and greenhouses trials.
• In parallel, four Hits were also obtained from vendors outside TrifoC in 1 kg batches, and formulated for further testing in field and greenhouse trials.
• The formulated Hits were evaluated for biosafety and regulatory potential (by data screening), and tested for phytotoxicity, pesticidal efficacy and positioning in field and greenhouse trials on a variety of crops and insect pests.
Exploitable foreground:
All Hits showed biological activity but the Euphol-euphorbol mixture, protopanaxatriol and betulinic acid ranked highest and will be further tested for development as new commercial biopesticide products at the end of TriForC.
The need for biopesticide solutions in agriculture is increasing due to restrictions on use of chemical pesticides as well as increasing customer awareness.
Biopesticides development is a 10-year process that requires investments of 5-7 million Euros per candidate. The process includes scaling up production to tonne scale and establishing industrial biorefineries. Bio- production and biorefineries are our next major challenges and solving them may require additional research. However, it is reasonable to assume that P5 (STK) will develop at least one TriforC Hit as a biopesticide product. IP will be generated where appropriate.
Extraction process for vegan cholesterol (from microalgae)
In the course of developing microalgal strains for triterpene production, Algenuity screened a large panel of commercially produced microalgae including freshwater and marine strains, to assess the baseline sterol productivity and profiles. One microalgal strain, a marine microalgal strain currently cultivated in the high 10’s to low 100’s of tons globally primarily to support aquaculture markets, emerged as a strain of significant interest based upon an extremely high cholesterol content and extremely simple sterol profile.
Within WP6, Extrasynthese and Algenuity worked together to develop this opportunity further both in technical and commercial terms. At present, negotiations are quite mature with a commercial client identified in the USA within the nutritional supplements sector; a pilot scale production process has been proposed to deliver a first 100g pilot batch to this client followed by a commitment to deliver 10kg in year 1 and 10’s of kilos up to a projected 100 kg in subsequent years. IP Protection is not possible with regard to the process or the target. Trade secret and stable supply chain for the biomass plus access and knowhow around new strains that could yield higher levels of the target compound as well as an exclusive agreement to supply and purchase all create barriers to entry for competitors.
Algenuity and Extrasynthese are negotiating a commercial agreement to exploit this opportunity. In the event that the client agrees with the terms of the pilot scale process development and commitment to supply product in the 10’s to 100’s of kilos, the pilot production phase will start immediately through a projected 3 month timescale. Subject to successful transition through the stage gate at 3 months, 10 kg of purified product would be supplied in the 1st year. This has the potential to bring in revenues in the low millions of Euros per year with profit sharing agreed between Algenuity and Extrasynthese. The impact of the successful commercial exploitation of this vegan cholesterol supply chain will be economic and environmental. At present, natural sources of vegan cholesterol as well as its desirable higher value derivatives are harvested from nature through a capped (limited) annual harvest. Removal of this plant material from the environment does have an impact on the relevant ecosystems.
The commercial opportunity could be substantially increased by identification of customers for other chemicals (biorefinery model) produced by the microalgal biomass in question including long-chain polyunsaturated fatty acids (omega-3’s). The commercial value of this fraction will largely be impacted by the extraction process being used and the purity of any resultant product – particularly with regard to the presence of any solvent traces in the final product.
Summary
It is difficult to quantify the long-term impacts of the foreground developed by this project. With only the three commercial opportunities outlined above as examples, these alone could bring 100’s of millions to billions of Euros in sales revenues globally over the next 25 years. There is little doubt that the fundamental research advances that have been made will provide a strong springboard from which further commercial opportunities will arise. The three commercial opportunities also highlight social and environmental impacts – job creation, development of linked businesses within supply chains, and reduced impact on the environment through development of new sustainable supply chains. Lastly, the potential impact of the strong and enthusiastic commitment within the consortium to participate in public-facing educational activities particularly spearheaded by the SAW Trust link provided by P8 cannot be overestimated. By taking time to invest in younger potential scientists, engineers, business developers, artists, writers, policymakers, and end-users – to pass on ideas and plant seeds of knowledge and inspiration – the return on investment of the TriForC project is expected to be staggering and longstanding.
List of Websites:
www.triforc.eu
Søren Bak Bak@plen.ku.dk
