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 will tackle this issue by establishing an integrative and innovative pipeline 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 will deploy state-of-the-art technologies, developed by the partners, to achieve its aims.

The ambitious goal of TriForC can be divided into five scientific and technical objectives:

• Identify new bioactive triterpenes for commercial development as exemplified for pharmaceuticals and agrochemicals.
• Elucidate Structure-Activity-Relationships (SAR) of triterpenes through biological activity screenings.
• Constitute a genetic toolbox that will allow mimicking ‘rainforest’-like structural triterpene 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 each bring to the consortium the necessary tools, resources, methods and production systems required to assemble the pipeline (Figure 1) and reach the scientific and technical objectives of our ambitious goal. Although the TriForC pipeline is sequential in structure, all individual TriForC activities will be operational from the beginning of the project, when the consortium partners deliver the initial parts of the pipeline components.

The TriForC pipeline will specifically address 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, creating a genetic toolbox and establishing 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.

Publications

1. Moses T., Papadopoulou K. K., Osbourn A. (2014) Metabolic and functional diversity of saponins, biosynthetic intermediates and semi-synthetic derivatives. Critical Reviews in Biochemistry and Molecular Biology 49(6) 439–46, doi: 10.3109/10409238.2014.953628 (P8)http://www.plantcell.org/content/27/1/286.long
2. Thimmappa R., Geisler K., Louveau T., O’Maille P., Osbourn A. (2014) Triterpene biosynthesis in plants.  Annual Reviews Plant Biology 65: 225-257 (P8)
3. Cárdenas PD, Sonawane PD, Bocobza SE, Aharoni A (2014).  The bitter side of the nightshades: genomics drives discovery in Solanaceae steroidal alkaloid metabolism. Phytochemistry 2015 May: 113:24-32. doi: 10.1016/j.phytochem.2014.12.010. Epub 2014 Dec 31. (P9)
4. Moses T,  Pollier  J,  Shen Q,  Soetaert S,  Reed J,  Erffelinck  ML , Van Nieuwerburgh FcW, Vanden Bossche R, Osbourn, A, Thevelein  JM, Deforce D, Tang K, Goossens A (2015). OSC2 and CYP716A14v2 catalyze the biosynthesis of triterpenoids for the cuticle of aerial organs of Artemisia annua. Plant Cell 27, 286-301. doi: 10.1105/tpc.114.134486. Epub 2015 Jan 9. (P8, P7)
5. Goossens A (2015).  It is easy to get huge candidate gene lists for plant metabolism now, but how to get beyond? Molecular Plant 8, 2-5.  doi: 10.1016/jmolp.2014.08.001. (P7)
6. Patron NJ, Orzaez D, Marillonnet S, Warzecha H, Matthewman C, Youles M, Raitskin O, Leveau A, Farré G, Rogers C, Smith A, Hibberd J, Webb AA, Locke J, Schornack S, Ajioka J, Baulcombe DC, Zipfel C, Kamoun S, Jones J D, Kuhn H, Robatzek S, Van Esse HP, Sanders D, Oldroyd G, Martin C, Field R, O'Connor S, Fox S, Wulff  B,  Miller B, Breakspear A, Radhakrishnan G, Delaux  PM, Loqué D, Granell A, Tissier A, Shih P, Brutnell T P, Quick WP, Rischer H, Fraser PD, Aharoni A, Raines C, South PF, Ané JM, Hamberger B R, Langdale J, Stougaard J, Bouwmeester H, Udvardi M, Murray JA, Ntoukakis V, Schäfer P, Denby K, Edwards K J, Osbourn A, Haseloff J. (2015). Standards for plant synthetic biology: a common syntax for exchange of DNA parts.  New Phytologist 14 July 2015, doi: 10.1111/nph.13532. (P8)
7. Hao Y, Hu G, Breitel D, Liu M, Mila I, Frasse P, Aharoni A, Bouzayen M, Zouine M. Auxin response factor S1ARF2 is an essential component of the regulatory mechanism controlling fruit ripening in tomato. PloS Genet (in press). (P9)
8. Khakimov B, Kuzina V, Erthmann PØ, Fukushima EO, Augustin JM, Olsen CE, Scholtalbers J, Volpin H, Andersen SB, Hauser TP, Muranaka T, Bak S. Identification and genome organization of saponin pathway genes from a wild crucifer, and their use for transient production of saponins in Nicotiana benthamiana.   Plant Journal Nov;84(3):478-90. doi: 10.1111/tpj.13012. (P1)
9. Cárdenas PD, Sonawane PD, Pollier J, Vanden Bossche R, Dewangan V, Weithorn E, Tal L, Meir S, Rogachev I, Malitsky S, Giri AP, Goossens A, Burdman S, Aharoni A (2015). GAME9 Regulates steroidal alkaloid and its upstream isopremoid biosynthesis in the plant mevalonate pathway.  Nature Communications.  Feb 15:7: 10654. doi: 10.1038/ncomms10654. (P9, P7)
10. Arendt P, Pollier J, Callewaert N, Goossens A (2015).  Synthetic biology for production of natural and new-to-nature terpenoids in photosynthetic organisms.  The Plant Journal. Feb 12. doi: 10.1111/tpj.13138 (P7)
11. Salmon M, Thimmappa R, Minto R, Melton R, O’Maille P, Hemmings A, Osbourn A (2016). Functional analysis of a triterpene synthase required for the synthesis of antimicrobial defence compounds in oat. PNAS. (P8)
12. Salmon M., Thimmappa R., Minto R., Melton R., Hughes R., O'Maille P., Hemmings A. M., Osbourn A.(2016) A conserved amino acid residue critical for product and substrate specificity in plant triterpene synthases. Proceedings of the National Academy of Sciences of the United States of America 113(30) E4407-E4414 Project(s): CA556W08B C0614W08A CA533W08E CA521W08B
13. A pipeline for triterpene production. A pipeline of developing the value of plants. www.Impactpub. Jan 2017 p 37-39.

 Submitted/in press: 

1. Miettinen K, Pollier J, Buyst D, Arendt P, Csuk R, Sommerwerk S, Moses T, Mertens J, Sonawane PD, Aharoni A, Martins J, Nelson DR, Goossens A (2016) CYP716 enzymes form the cradle of triterpenoid diversity in eudicots.  Nature Communications submitted (P7)

• Evonik Nutrition and Care GmbH
• Algenuity (a division of Spicer Consulting Ltd)
• Vivacell Biotechnology España SL
• Stockton Israel Ltd.
• Extrasyntheses SAS
• VIB
• John Innes Centre
• Weizmann Institute of Science
• Universita Degli Studi del Piedmonte Orientale Amedeo Avogradro
• University of Thessaly