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  • Periodic Report Summary 2 - TRIFORC (A pipeline for the discovery, sustainable production and commercial utilisation of known and novel high-value triterpenes with new or superior biological activities.)

Periodic Report Summary 2 - TRIFORC (A pipeline for the discovery, sustainable production and commercial utilisation of known and novel high-value triterpenes with new or superior biological activities.)

Project Context and Objectives:
The TriForC goal is 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.
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 tackles this issue by establishing an integrative and innovative pipeline for the exploitation of plant triterpenes, which is one of the largest classes of plant bioactive compounds with an astonishing array of structural diversity and spectrum of biological activities.
Triterpenes’ known roles in nature is in defence against pathogens, pests and predators. As triterpenes are structurally diverse and highly 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. To unleash the potential of triterpenes, we are in the TriForC project screening extracts and purified triterpenes from a selected range of plants known to be rich in triterpenoids. We focus on screening for biologically active triterpenoids for agrochemical applications within insecticidal and fungicidal potential and/or as drug medicinal targets within cancer, inflammation, metabolism, and HIV-1 infection and at the same time establishing a unique collection of genes encoding for enzymes catalysing this vast structural diversity. This TriForC genetic toolbox will be used in our metabolic engineering platform for bio-based production of known triterpenes and new-to-nature structures by combining genes between species and even across Kingdoms of Life in a combinatorial manor. Extracts and purified compounds from these production platforms will feed back into our screening platform. A range of biobased production platforms are being developed in TriForC, including yeast cells, hairy roots, shoot cultures, tobacco plants and various salt and fresh water algae which will allow us to scale-up a biobased sustainable production of triterpenes with superior bioactivities for commercial utilisation.
WP1 is providing TriForC with a selection of bioactive triterpenoids suitable for further development by capitalizing on three methodologically complementary strategies: a) identification of lead structures from a collection of triterpenoid-rich extracts of plant origin, b) the semi-synthetic modification of triterpenoid scaffolds available in multi-gram amounts from easily available plant biomasses to increase of the structural diversity of the library of natural plant triterpenoids, and c) identification of natural or “new-to-nature” triterpenes generated in yeast, tobacco or algal expression systems from synthetic biology/ combinatorial biochemistry approaches.
The aim of WP2 is to identify biologically active triterpenes for agrochemical applications and/or as drug medicinal targets. These novel compounds are isolated from plant extracts, produced in bioreactors or obtained by semi synthesis starting from known templates. Hence, WP2 has two prime objectives:
i) To screen material sourced from the TriForC activities to identify natural and new-to-nature triterpenes with novel or superior bioactivities.
ii) To identify lead and hit compounds (novel chemical entities) with potential agrochemical and pharmacological applications.
The aim of WP3 is to provide the TriForC consortium with a validated gene resource that can be used for the biosynthesis of natural and “new-to-nature” triterpenes in yeast, algae, and plant production systems.
WP4 is designing a range of platforms for producing natural or new-to-nature bioactive triterpenes in plant or microbial production systems that are amenable to upscaling.
The main goal of WP5 is to supply purified and formulated triterpenoids identified in TriForC at an industrial scale through developing 3 technological platforms. WP6 assesses parameters that influence the economics of production at larger scale. For that each partner is specialized in several field involving production, exploitation and regulation.
WP7 aims to implement the TriForC dissemination and engagement strategy and to ensure a productive and fertile training environment for participating researchers. Finally, WP8 is ensuring that all the scientific and contractual obligations are kept on track and that the deadlines are kept.

Project Results:
A library of over 700 extracts from triterpenoid-rich plants has been assembled under WP1 capitalizing on plant biomasses of European origin or commercially available in Europe. Crude extracts were pre-fractionated in sub-fractions of different polarity by solid-phase extraction in order to increase the concentration of the compounds in the material to be bio-assayed and to remove polymeric compounds that could interfere with the assays.
Semi-synthetic modification of triterpenoid scaffold has been focused on both pentacyclic and tetracyclic scaffolds. Espcially the semi-synthetic modified triterpenoids has allowed WP2 to identify a series of promising lead structures whose optimization is currently pursued. Some interesting and novel chemistry has also been discovered, paving the way to the exploration of novel areas of the biological space associated to the scaffolds.

Furthermore, a collection of 514 plant extracts and 125 pure compounds have been tested in screening platforms that include selected targets for pharmaceutical and agronomical applications. On the basis of selectivity and potency against selected targets of pharmacological and agronomical interest a selection of 34 plant extracts have been selected for fractionation and bio-guide based isolation of pure compounds.
Known pure compounds were used for testing in novel targets to identify novel activities and also to serve as template of novel chemical entities (NCEs) with promising bioactivity on specific targets.
The consortia has taken advantage of the increasing amount of sequence data publically available on the internet as well as sequenced selective species. This has enabled that a large number of genes encoding enzymes producing the more traditional as well as more unusual triterpene scaffolds have been isolated and the substrate or product specificities determined. Genes encoding downstream tailoring enzymes that further modify the scaffolds have been isolated and characterized, providing a TriForC toolbox for metabolic engineering in WP4. To complete the tool box a number of regulators of steroidal triterpenoids have been isolated and characterized.
The upscaling of production systems is being achieved by stacking genes already provided as background by the consortia partners as well as genes coming out of WP3 in heterologous expression systems such as yeast cultures, tobacco leaves, cucumber and legume hairy roots and microalgae. Within yeast and tobacco we have been very successful in producing a large number of triterpenoids, with different backbones and degrees of decoration, and corresponding to natural triterpenoid building blocks as well as new-to-nature molecules, which provides proof of concept for our synthetic biology platform. In a marine diatom species we managed to engineer basic triterpenoid structures, in particular. Furthermore, triterpenoid synthesis gene cassettes have been assembled to assess the combinatorial biochemistry capacity of legume hairy roots.
TriForC’s three technological platforms can be described as follow; 1) organ cultures of medicinal plants in specialised bioreactors. The upscaling is in progress, with already a gram-scale production of purified celastrol achieved in this system. 2) The transient expression of triterpenoid pathways in Nicotiana benthamiana. Successful production and purification of β-amyrin, an important triterpenoid backbone, was also reached at gram-scale level, 3) the third platform based on transgenic specific algae strains, provided the first transgenic lines with significant production of the triterpenoid scaffold lupeol.
Finally, different formulations have been prepared to establish the understanding that will be required for rapid development of robust and effective formulations to be made. The findings are based temporarily on celastrol. We also started the process of scale-up for sterols as well as β-amyrin including first cost analysis.
The work within TriForC has until now resulted in 12 publications, 20 oral presentations and 3 poster presentations. In addition to this we have organised 5 workshops of which two are part of a career development course for the ESR in TriForC.

Potential Impact:
Plant tissue cultures have been successfully developed for the production of triterpenoids of problematic availability from isolation, making them available to the project in multigram amounts. We expect to further build on these results to identify a portfolio of optimized candidates amenable to further development for pharmaceutical, and/or agrochemical use.
Several known compounds and novel chemical entities (NCEs) were found to be active in agronomical and pharmaceutical targets and we are exploring the mechanism of action and assessing its potential for developing novel products for the agronomical and pharmaceutical markets.
All SMEs involved in WP5 are highly committed and therefore a decision was made to move quickly in the pipeline for a specific triterpenoid based on the first screening results. This compound was produced at gram-scale by Alkion, purified by Extrasynthese and formulated by Stockton for a bio-pesticide application. At the end of M30, field trials are already on going, providing TriForC with a global overview of a complete industrial development of a triterpenoid from a lead compound in screening until field trials as an innovative bio-pesticide.
On top of these successful activities, WP5 will now increase its focus on i) producing new-to-nature compounds provided by WP4, ii) developing the biorefinery strategies and parameters as well as iii) benchmarking all production platforms to demonstrate economic feasibility of such green chemistry industries.
Regarding the findings and output from WP6, we expect have increased focus on obtaining IP formulation technique, on the active ingredients itself if will be novel. We will also continue to publish high ranking basic science papers within evolution, diversity, semi-synthetic synthesis and biochemistry of triterpenoids.

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