D586–D594 Nucleic Acids Research, 2018, Vol. 46, Database issue Published online 17 October 2017 doi: 10.1093/nar/gkx925 The TriForC database: a comprehensive up-to-date resource of plant triterpene biosynthesis Karel Miettinen1,2, Sabrina Inigo˜ 1,2, Lukasz Kreft3, Jacob Pollier1,2,ChristofDeBo3, Alexander Botzki3, Frederik Coppens1,2, Søren Bak4 and Alain Goossens1,2,* 1Ghent University, Department of Plant Biotechnology and Bioinformatics, 9052 Ghent, Belgium, 2VIB Center for Plant Systems Biology, 9052 Ghent, Belgium, 3VIB Bioinformatics Core, 9052 Ghent, Belgium and 4Plant Biochemistry Laboratory and Villum Research Center ‘Plant Plasticity’, Copenhagen Plant Science Center, Department of Plant and Environmental Sciences, University of Copenhagen, Copenhagen, Denmark Received August 14, 2017; Revised September 23, 2017; Editorial Decision September 26, 2017; Accepted October 03, 2017 ABSTRACT INTRODUCTION Triterpenes constitute a large and important class of Importance of triterpenes plant natural products with diverse structures and Triterpenes compose a diverse class of plant natural prod- functions. Their biological roles range from mem- ucts, both in structure and function. They comprise (i) pri- brane structural components over plant hormones mary metabolites such as the phytosterols, which are the to specialized plant defence compounds. Further- indispensable structural components of cell membranes, more, triterpenes have great potential for a vari- and hormones, such as brassinosteroids, and (ii) specialized ety of commercial applications such as vaccine ad- (also called secondary) metabolites with diverse biological juvants, anti-cancer drugs, food supplements and functions (1,2). The latter include among others defence agronomic agents. Their biosynthesis is carried out compounds with antimicrobial, antifungal, antiparasitic, through complicated, branched pathways by multiple insecticidal, anti-feedant and allelopathic activities (3)and leaf wax components (4). Primary metabolite triterpenes oc- enzyme types that include oxidosqualene cyclases, cur in all plant species. In contrast, specialized metabolite cytochrome P450s, and UDP-glycosyltransferases. triterpenes are often restricted to a specific species or taxa Given that the number of characterized triterpene although some of them can be quite widespread as a re- biosynthesis enzymes has been growing fast re- sult of convergent evolutionary events. For example, com- cently, the need for a database specifically focusing pounds of the most common type of specialized metabo- on triterpene enzymology became eminent. Here, we lite triterpenes, i.e. the oleananes and their derivatives, are present the TriForC database (http://bioinformatics. present in most flowering plant orders5 ( ). psb.ugent.be/triforc/), encompassing a comprehen- Specialized metabolite triterpenes show a striking diver- sive catalogue of triterpene biosynthesis enzymes. sity of structures from fairly simple unsubstituted triterpene This highly interlinked database serves as a user- backbones, such as the ones in leaf wax components, to friendly access point to versatile data sets of en- complex molecules carrying multiple oxidative decorations, heterocycles, elaborate sugar chains and/or other chemi- zyme and compound features, enabling the scanning cal groups (2). Because these structures represent a wide of a complete catalogue of experimentally validated range of biological activities, triterpenes have received con- triterpene enzymes, their substrates and products, siderable industrial interest as pharmaceuticals, cosmetics, as well as the pathways they constitute in various agronomic agents, etc. Renowned examples are the quinine plant species. The database can be accessed by di- methide celastrol from Tripterygium wilfordii (thunder god rect browsing or through convenient search tools vine), which has powerful antioxidant, anti-inflammatory, including keyword, BLAST, plant species and sub- and anti-cancer activities (6) and QS-21, a soluble triterpene structure options. This database will facilitate gene fraction from Quillaja saponaria, which is used as an adju- mining and creating genetic toolboxes for triterpene vant in vaccine formulations that are currently in clinical tri- synthetic biology. als for anti-HIV, HPV, malaria, melanoma, mycobacterium tuberculosis and varicella zoster virus activities (7). Triterpenes also have uses in the food industry, in which for instance glycyrrhizin from Glycyrrhiza species is used as *To whom correspondence should be addressed. Tel: +32 9 331 38 51; Fax: +32 9 331 38 09; Email: [email protected] C The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] Downloaded from https://academic.oup.com/nar/article-abstract/46/D1/D586/4555231 by Ghent University user on 18 January 2018 Nucleic Acids Research, 2018, Vol. 46, Database issue D587 a natural sweetener (8)andQ. saponaria extracts are being membranes and act as precursors for the biosynthesis used as foaming agents and emulsifiers (9,10). Furthermore, of the plant brassinosteroid hormones and of specialized plant sterols have been shown to reduce cholesterol serum taxa-specific steroidal triterpenes that include the steroidal levels. Plant sterols are frequently esterified with fatty acids (glyco)alkaloids. Phytosterols are synthesized from 2,3- to increase their lipid solubility when present in a food in- oxidosqualene via cycloartenol, generated by the OSC cy- gredient. Several studies have shown a significant reduction cloartenol synthase (CAS), followed by a 14–17 step enzy- in the LDL-cholesterol levels when people consumed food matic pathway (19,20). This pathway contains many differ- enriched with plant sterol or stanol esters (11). This fact ent types of enzymes and results in the production of sterols highly increased the interest of food manufacturers in using such as stigmasterol, ␤-sitosterol and campesterol, which phytosterols as supplements and food additives and thus in are the most commonly consumed plant sterols provided by phytosterol biosynthesis. vegetable oils (19,21). Using partly the same enzymes, but in a parallel pathway, plants also synthesize cholesterol (22), which in turn can serve as the starting point for the steroidal Biosynthesis of triterpenes glycoalkaloids in Solanaceae species (22,23). Whereas stig- Plants produce triterpenes in long and branched biosyn- masterol and ␤-sitosterol phytosterols are involved in the thetic pathways (Figure 1). Actual, known primary and spe- structure and function of cell membranes, campesterol is cialized triterpenes are all built around a triterpene back- the starting point of brassinosteroid biosynthesis, plant hor- bone based on six isopentenyl pyrophosphate (IPP) build- mones with main roles in plant growth, development and ing blocks originating from the mevalonate (MVA) path- stress responses (24–26). way (2). Part of the generated IPP pool is converted into Enzymes from the phytosterol and brassinosteroid the allylic isomer dimethylallyl pyrophosphate (DMAPP) biosynthesis pathways have been fairly well characterized by IPP isomerase. Two molecules of IPP and one molecule in the model plant Arabidopsis thaliana and to a lesser ex- of DMAPP are assembled to farnesyl pyrophosphate (FPP) tent in rice, pea and tomato (27,28). In contrast, only a by prenyltransferase farnesyl pyrophosphate synthase. FPP small fraction of the specialized metabolism pathway en- is the building block for various MVA-dependent terpene zymes has been characterized to date, mainly because of pathways, including the tri- and sesquiterpenes (12). The their immense variability. Indeed, most plant families have first committed step in triterpene biosynthesis is the conden- their own unique subset of triterpene biosynthesis enzymes sation of two molecules of FPP by squalene synthase (SQS) that contribute to a species-specific compendium of often to produce the first triterpene, squalene. This molecule unique structures with unique bio-activities. The diversity is then activated by epoxidation by a squalene epoxidase of specialized metabolism triterpenes arises from their mod- (SQE), resulting in 2,3-oxidosqualene (2,13,14). ular biosynthesis where a variety of species-, genus-, or The first branching point in triterpene biosynthesis is family-specific OSCs, P450s, UGTs and other decoration the cyclization of 2,3-oxidosqualene by oxidosqualene cy- enzymes act in rather web-like than linear pathways, pro- clase (OSC) enzymes into one of the more than hundred ducing arrays of bio-active compounds rather than single known triterpene backbones, such as cycloartenol for pri- effective compounds (1,2,13,14,17,18). mary sterol synthesis or ␤-amyrin for specialized triterpenes In recent years, the number of characterized triterpene (Figure 1). This cyclization is driven by an initial epoxy biosynthesis enzymes has increased extensively, mostly be- group protonation, followed by a cyclization and subse- cause of the tremendous increase in genome and transcrip- quent rearrangement of carbocation species and finally ter- tome sequencing data available. This has enabled large-scale