Search

Pentacyclic triterpenoids, recognized for their natural bioactivity, display complex spatiotemporal accumulation patterns within the ecological model plant Nicotiana attenuata. Despite their ecological importance, the underlying biosynthetic enzymes and functional attributes of triterpenoid synthesis in N. attenuata remain unexplored. Here, we show that 3 cytochrome P450 monooxygenases (NaCYP716A419, NaCYP716C87, and NaCYP716E107) from N. attenuata oxidize the pentacyclic triterpene skeleton, as evidenced by heterologous expression in Nicotiana benthamiana. NaCYP716A419 catalyzed a consecutive 3-step oxidation reaction at the C28 position of β-amyrin/lupeol/lupanediol, yielding the corresponding alcohol, aldehyde, and carboxylic acid. NaCYP716C87 hydroxylated the C2α position of β-amyrin/lupeol/lupanediol/erythrodiol/oleanolic acid/betulinic acid, while NaCYP716E107 hydroxylated the C6β position of β-amyrin/oleanolic acid. The genes encoding these 3 CYP716 enzymes are highly expressed in flowers and respond to induction by ABA, MeJA, SA, GA3, and abiotic stress treatments. Using VIGS technology, we revealed that silencing of NaCYP716A419 affects the growth and reproduction of N. attenuata, suggesting the ecological significance of these specialized metabolite biosynthetic steps., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Artemisia argyi is a traditional medicinal and edible plant, generating various triterpenoids with pharmacological activities, such as anti-virus, anti-cancer, and anti-oxidant. The 2,3-oxidosqualene cyclase family of A. argyi offers novel insights into the triterpenoid pathway, which might contribute to the medicinal value of its tissue extracts. Nevertheless, the biosynthesis of active triterpenoids in Artemisia argyi is still uncertain. In this study, four putative OSC (2,3-oxidosqualene cyclase) genes (AaOSC1-4) were first isolated and identified from A. argyi. Through the yeast heterologous expression system, three AaOSCs were characterized for the biosynthesis of diverse triterpenoids including cycloartenol, β-amyrin, (3S,13R)-malabarica-14(27),17,21-trien-3β-ol, and dammara-20,24-dien-3β-ol. AaOSC1 was a multifunctional dammara-20,24-dien-3β-ol synthase, which yielded 8 different triterpenoids, including tricyclic, and tetracyclic products. AaOSC2 and AaOSC3 were cycloartenol, and β-amyrin synthases, respectively. As a result, these findings provide a deeper understanding of the biosynthesis pathway of triterpenes in A. argyi., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Platycodon grandiflorus is a medicinal plant whose main component is platycodins, which have a variety of pharmacological effects and nutritional values. The farnesyl pyrophosphate synthase (FPS) is a key enzyme in the isoprenoid biosynthesis pathway, which catalyzes the synthesis of farnesyl diphosphate (FPP). In this study, we cloned the FPS gene from P. grandiflorus ( PgFPS ) with an ORF of 1260 bp, encoding 419 amino acids with a deduced molecular weight and theoretical pI of 46,200.98 Da and 6.52, respectively. The squalene content of overexpressed PgFPS in tobacco leaves and yeast cells extract was 1.88-fold and 1.21-fold higher than that of the control group, respectively, and the total saponin content was also increased by 1.15 times in yeast cells extract, which verified the biological function of PgFPS in terpenoid synthesis. After 48 h of MeJA treatment and 6 h of ethephon treatment, the expression of the PgFPS gene in roots and stems reached its peak, showing a 3.125-fold and 3.236-fold increase compared to the untreated group, respectively. Interestingly, the expression of the PgFPS gene in leaves showed a decreasing trend after exogenous elicitors treatment. The discovery of this enzyme will provide a novel perspective for enhancing the efficient synthesis of platycodins.

Triterpenoids from Camellia species comprise a diverse class of bioactive compounds with great therapeutic potential. However, triterpene biosynthesis in tea plants ( Camellia sinensis ) remains elusive. Here, we identified eight putative 2,3-oxidosqualene cyclase (OSC) genes ( CsOSC1-8 ) from the tea genome and characterized the functions of five through heterologous expression in yeast and tobacco and transient overexpression in tea plants. CsOSC1 was found to be a β-amyrin synthase, whereas CsOSC4, 5, and 6 exhibited multifunctional α-amyrin synthase activity. Molecular docking and site-directed mutagenesis showed that the CsOSC6M259T/W260L double mutant yielded >40% lupeol, while the CsOSC1 W259L single mutant alone was sufficient for lupeol production. The V732F mutation in CsOSC5 altered product formation from friedelin to taraxasterol and ψ-taraxasterol. The L254 M mutation in the cycloartenol synthase CsOSC8 enhanced the catalytic activity. Our findings shed light on the molecular basis governing triterpene diversity in tea plants and offer potential avenues for OSC engineering.

Triterpenoids are important, pharmacologically active substances in jujube ( Ziziphus jujuba Mill.), and play an important role in the plant's resistance to abiotic stress. However, regulation of their biosynthesis, and the underlying mechanism of their balance with stress resistance, remain poorly understood. In this study, we screened and functionally characterized the ZjWRKY18 transcription factor, which is associated with triterpenoid accumulation. The transcription factor is induced by methyl jasmonate and salicylic acid, and its activity was observed by gene overexpression and silencing experiments, combined with analyses of transcripts and metabolites. ZjWRKY18 gene silencing decreased the transcription of triterpenoid synthesis pathway genes and the corresponding triterpenoid content. Overexpression of the gene promoted the biosynthesis of jujube triterpenoids, as well as triterpenoids in tobacco and Arabidopsis thaliana . In addition, ZjWRKY18 binds to W-box sequences to activate promoters of 3-hydroxy-3-methyl glutaryl coenzyme A reductase and farnesyl pyrophosphate synthase, suggesting that ZjWRKY18 positively regulates the triterpenoid synthesis pathway. Overexpression of ZjWRKY18 also increased tolerance to salt stress in tobacco and Arabidopsis thaliana . These results highlight the potential use of ZjWRKY18 to improve triterpenoid biosynthesis and salt stress tolerance in plants, and provide a strong basis for metabolic engineering to improve the content of triterpenoids and breeding of jujube varieties that are resistant to stress.

Background: Helicteres angustifolia has long been used in Chinese traditional medicine. It has multiple pharmacological benefits, including anti-inflammatory, anti-viral and anti-tumor effects. Its main active chemicals include betulinic acid, oleanolic acid, helicteric acid, helicterilic acid, and other triterpenoid saponins. It is worth noting that some acylated triterpenoids, such as helicteric acid and helicterilic acid, are characteristic components of Helicteres and are relatively rare among other plants. However, reliance on natural plants as the only sources of these is not enough to meet the market requirement. Therefore, the engineering of its metabolic pathway is of high research value for enhancing the production of secondary metabolites. Unfortunately, there are few studies on the biosynthetic pathways of triterpenoids in H. angustifolia, hindering its further investigation., Results: Here, the RNAs of different groups treated by metabolic stimulation were sequenced with an Illumina high-throughput sequencing platform, resulting in 121 gigabases of data. A total of 424,824 unigenes were obtained after the trimming and assembly of the raw data, and 22,430 unigenes were determined to be differentially expressed. In addition, three oxidosqualene cyclases (OSCs) and four Cytochrome P450 (CYP450s) were screened, of which one OSC (HaOSC1) and one CYP450 (HaCYPi3) achieved functional verification, suggesting that they could catalyze the production of lupeol and oleanolic acid, respectively., Conclusion: In general, the transcriptomic data of H. angustifolia was first reported and analyzed to study functional genes. Three OSCs, four CYP450s and three acyltransferases were screened out as candidate genes to perform further functional verification, which demonstrated that HaOSC1 and HaCYPi3 encode for lupeol synthase and β-amyrin oxidase, which produce corresponding products of lupeol and oleanolic acid, respectively. Their successful identification revealed pivotal steps in the biosynthesis of acylated triterpenoids precursors, which laid a foundation for further study on acylated triterpenoids. Overall, these results shed light on the regulation of acylated triterpenoids biosynthesis., (© 2022. The Author(s).)

Friedelin, a pentacyclic triterpene found in the leaves of the Celastraceae species, demonstrates numerous biological activities and is a precursor of quinonemethide triterpenes, which are promising antitumoral agents. Friedelin is biosynthesized from the cyclization of 2,3-oxidosqualene, involving a series of rearrangements to form a ketone by deprotonation of the hydroxylated intermediate, without the aid of an oxidoreductase enzyme. Mutagenesis studies among oxidosqualene cyclases (OSCs) have demonstrated the influence of amino acid residues on rearrangements during substrate cyclization: loss of catalytic activity, stabilization, rearrangement control or specificity changing. In the present study, friedelin synthase from Maytenus ilicifolia (Celastraceae) was expressed heterologously in Saccharomyces cerevisiae . Site-directed mutagenesis studies were performed by replacing phenylalanine with tryptophan at position 473 (Phe473Trp), methionine with serine at position 549 (Met549Ser) and leucine with phenylalanine at position 552 (Leu552Phe). Mutation Phe473Trp led to a total loss of function; mutants Met549Ser and Leu552Phe interfered with the enzyme specificity leading to enhanced friedelin production, in addition to α-amyrin and β-amyrin. Hence, these data showed that methionine 549 and leucine 552 are important residues for the function of this synthase.

Key Message: De novo transcriptome analysis from callus, leaf, and fruit of Trichosanthes cucumerina L. for the identification of genes associated with triterpenoid biosynthesis, especially bryonolic acid and cucurbitacin B. Trichosanthes cucumerina L. (TC) has been used as a medicinal plant in Thailand with various potential functions. Two major triterpenoids found in this plant, bryonolic acid and cucurbitacin B, are receiving increased attention for their activities. Here, we provide TC transcriptome data to identify genes involved in the triterpenoid biosynthetic pathway through callus, where was previously suggested as a novel source for bryonolic acid production as opposed to leaf and fruit. A de novo assembly of approximately 290-thousand transcripts generated from these tissues led to two putative oxidosqualene cyclases: isomultiflorenol synthase (IMS) and cucurbitadienol synthase (CBS). TcIMS and TcCBS, genes that encode substrates for two characteristic triterpenoids in cucurbitaceous plants, were identified as isomultiflorenol synthase and cucurbitadienol synthase, respectively. These two genes were functionally characterised in mutant yeast Gil77 systems, which led to the productions of isomultiflorenol and cucurbitadienol. Moreover, the callus-specific gene expression profiles were also presented. These obtained information showed candidate cytochrome P450s with predicted full-length sequences, which were most likely associated with triterpenoid biosynthesis, especially bryonolic acid. Our study provides useful information and a valuable reference for the further studies on cucurbitaceous triterpenoids., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Pentacyclic triterpenoids have wide applications in the pharmaceutical industry. The precise glucosylation at C-3 OH of pentacyclic triterpenoids mediated by uridine 5'-diphospho-glucosyltransferase (UDP-glucosyltransferase [UGT]) is an important way to produce valuable derivatives with various improved functions. However, most reported UGTs suffer from low regiospecificity toward the OH and COOH groups of pentacyclic triterpenoids, which significantly decreases the reaction efficiency. Here, two new UGTs (UGT73C33 and UGT73F24) were discovered in Glycyrrhiza uralensis. UGT73C33 showed high activity but poor regioselectivity toward the C-3 OH and C-30 COOH of pentacyclic triterpenoid, producing three glucosides. UGT73F24 showed rigid regioselectivity toward C-3 OH of typical pentacyclic triterpenoids producing only C-3 O-glucosylated derivatives. In addition, UGT73C33 and UGT73F24 showed a broad substrate scope toward typical flavonoids with various sugar donors. Next, the substrate recognition mechanism of UGT73F24 toward glycyrrhetinic acid (GA) and UDP-glucose was investigated. Two key residues, I23 and L84, were identified to determine activity, and site-directed mutagenesis of UGT73F24-I23G/L84N increased the activity by 4.1-fold. Furthermore, three in vitro GA glycosylation systems with UDP-recycling were constructed, and high yields of GA-3-O-Glc (1.25 mM), GA-30-O-Glc (0.61 mM), and GA-di-Glc (0.26 mM) were obtained. The de novo biosynthesis of GA-3-O-glucose (26.31 mg/L) was also obtained in engineered yeast., (© 2020 Wiley Periodicals LLC.)

N-(2-Chloro-4-pyridyl)-N'-phenylurea (CPPU) is a cytokinin-like plant growth regulator, which application in melon fruit set often produced bitter fruit caused by cucurbitacin B (Cu B) accumulation. However, more evidence is required to uncover the role of CPPU in regulating Cu B synthesis. In this study, two oriental melon cultivars 'YMR' (easy to present bitter fruit in maturation) and 'HDB' (hardly produce bitter fruit at maturity) were used. Four concentrations of CPPU (2.5, 5, 10 and 20 mg L -1 ) were set and hand pollination was used as control. Cu B accumulated in roots and fruit of 7 days after flower (7 DAF), which in 'YMR' was higher than those in 'HDB', and consistent with Cu B biosynthetic genes expression patterns. Furthermore, Cu B content in fruit significantly increased with CPPU concentrations and reached the highest level at 7 DAF, then decreased after 14 DAF, and which treated by 20 mg L -1 CPPU was always higher than that of controls and other low CPPU concentrations. Meanwhile, fruit bitterness evaluation suggested 20 mg L -1 CPPU increased the occurrence of bitterness during melon maturation. Transcription analysis suggested that the expression of Cu B biosynthetic genes (CmBi, Cm710, CmACT) and CmBt, the fruit-specific transcription factor, were significantly induced by 20 mg L -1 CPPU. Transient over-expression of CmBt in young fruit of 'YMR' increased Cu B biosynthetic genes (CmBi, CmACT, Cm710 and Cm890) expression and promoted Cu B accumulation. Taken together, this study demonstrates that 20 mg L -1 CPPU promotes Cu B accumulation in melon fruit by inducing CmBt and its biosynthetic genes., (Copyright © 2020. Published by Elsevier Masson SAS.)

Plants produce a vast array of defense compounds to protect themselves from pathogen attack or herbivore predation. Saponins are a specific class of defense compounds comprising bioactive glycosides with a steroidal or triterpenoid aglycone backbone. The model legume Medicago truncatula synthesizes two types of saponins, hemolytic saponins and nonhemolytic soyasaponins, which accumulate as specific blends in different plant organs. Here, we report the identification of the seed-specific transcription factor TRITERPENE SAPONIN ACTIVATION REGULATOR3 (TSAR3), which controls hemolytic saponin biosynthesis in developing M. truncatula seeds. Analysis of genes that are coexpressed with TSAR3 in transcriptome data sets from developing M. truncatula seeds led to the identification of CYP88A13, a cytochrome P450 that catalyzes the C-16α hydroxylation of medicagenic acid toward zanhic acid, the final oxidation step of the hemolytic saponin biosynthesis branch in M. truncatula In addition, two uridine diphosphate glycosyltransferases, UGT73F18 and UGT73F19, which glucosylate hemolytic sapogenins at the C-3 position, were identified. The genes encoding the identified biosynthetic enzymes are present in clusters of duplicated genes in the M. truncatula genome. This appears to be a common theme among saponin biosynthesis genes, especially glycosyltransferases, and may be the driving force of the metabolic evolution of saponins., (© 2020 American Society of Plant Biologists. All rights reserved.)

Platycosides, the saponins abundant in Platycodi radix (the root of Platycodon grandiflorum ), have diverse pharmacological activities and have been used as food supplements. Since deglycosylated saponins exhibit higher biological activity than glycosylated saponins, efforts are on to enzymatically convert glycosylated platycosides to deglycosylated platycosides; however, the lack of diversity and specificities of these enzymes has limited the kinds of platycosides that can be deglycosylated. In the present study, we examined the enzymatic conversion of platycosides and showed that Cytolase PCL5 completely converted platycoside E and polygalacin D3 into deapiose-xylosylated platycodin D and deapiose-xylosylated polygalacin D, respectively, which were identified by LC-MS analysis. The platycoside substrates were hydrolyzed through the following novel hydrolytic pathways: platycoside E → platycodin D3 → platycodin D → deapiosylated platycodin D → deapiose-xylosylated platycodin D; and polygalacin D3 → polygalacin D → deapiosylated polygalacin D → deapiose-xylosylated polygalacin D. Our results show that cytolast PCL5 may have a potential role in the development of biologically active platycosides that may be used for their diverse pharmacological activities., Competing Interests: The authors declare no conflict of interest.

Cucurbitacins are highly oxygenated triterpenoids characteristic of plants in the family Cucurbitaceae and responsible for the bitter taste of these plants. Fruits of bitter melon ( Momordica charantia ) contain various cucurbitacins possessing an unusual ether bridge between C5 and C19, not observed in other Cucurbitaceae members. Using a combination of next-generation sequencing and RNA-Seq analysis and gene-to-gene co-expression analysis with the ConfeitoGUIplus software, we identified three P450 genes, CYP81AQ19 , CYP88L7 , and CYP88L8 , expected to be involved in cucurbitacin biosynthesis. CYP81AQ19 co-expression with cucurbitadienol synthase in yeast resulted in the production of cucurbita-5,24-diene-3β,23α-diol. A mild acid treatment of this compound resulted in an isomerization of the C23-OH group to C25-OH with the concomitant migration of a double bond, suggesting that a nonenzymatic transformation may account for the observed C25-OH in the majority of cucurbitacins found in plants. The functional expression of CYP88L7 resulted in the production of hydroxylated C19 as well as C5-C19 ether-bridged products. A plausible mechanism for the formation of the C5-C19 ether bridge involves C7 and C19 hydroxylations, indicating a multifunctional nature of this P450. On the other hand, functional CYP88L8 expression gave a single product, a triterpene diol, indicating a monofunctional P450 catalyzing the C7 hydroxylation. Our findings of the roles of several plant P450s in cucurbitacin biosynthesis reveal that an allylic hydroxylation is a key enzymatic transformation that triggers subsequent processes to produce structurally diverse products., (© 2019 Takase et al.)

3-Hydroxy-3-methylglutaryl-CoA synthase (HMGS) is the first committed enzyme in the MVA pathway and involved in the biosynthesis of terpenes in Tripterygium wilfordii. The full-length cDNA and a 515 bp RNAi target fragment of TwHMGS were ligated into the pH7WG2D and pK7GWIWG2D vectors to respectively overexpress and silence, TwHMGS was overexpressed and silenced in T. wilfordii suspension cells using biolistic-gun mediated transformation, which resulted in 2-fold increase and a drop to 70% in the expression level compared to cells with empty vector controls. During TwHMGS overexpression, the expression of TwHMGR, TwDXR and TwTPS7v2 was significantly upregulated to the control. In the RNAi group, the expression of TwHMGR, TwDXS, TwDXR and TwMCT visibly displayed downregulation to the control. The cells with TwHMGS overexpressed produced twice higher than the control value. These results proved that differential expression of TwHMGS determined the production of triptolide in T. wilfordii and laterally caused different trends of relative gene expression in the terpene biosynthetic pathway. Finally, the substrate acetyl-CoA was docked into the active site of TwHMGS, suggesting the key residues including His247, Lys256 and Arg296 undergo electrostatic or H-bond interactions with acetyl-CoA., (Copyright © 2019 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.)

Functional manipulation of biosynthetic enzymes such as cytochrome P450s (or P450s) has attracted great interest in metabolic engineering of plant natural products. Cucurbitacins and mogrosides are plant triterpenoids that share the same backbone but display contrasting bioactivities. This structural and functional diversity of the two metabolites can be manipulated by engineering P450s. However, the functional redesign of P450s through directed evolution (DE) or structure-guided protein engineering is time consuming and challenging, often because of a lack of high-throughput screening methods and crystal structures of P450s. In this study, we used an integrated approach combining computational protein design, evolutionary information, and experimental data-driven optimization to alter the substrate specificity of a multifunctional P450 (CYP87D20) from cucumber. After three rounds of iterative design and evaluation of 96 protein variants, CYP87D20, which is involved in the cucurbitacin C biosynthetic pathway, was successfully transformed into a P450 mono-oxygenase that performs a single specific hydroxylation at C11 of cucurbitadienol. This integrated P450-engineering approach can be further applied to create a de novo pathway to produce mogrol, the precursor of the natural sweetener mogroside, or to alter the structural diversity of plant triterpenoids by functionally manipulating other P450s.

Triterpenes, consisting of six isoprene units, are one of the largest classes of natural compounds in plants. The genus Taraxacum is in the family Asteraceae and is widely distributed in the Northern Hemisphere. Various triterpenes, especially taraxerol and taraxasterol, are present in Taraxacum plants. Triterpene biosynthesis occurs through the action of oxidosqualene cyclase (OSC), which generates various types of triterpenes from 2,3-oxidosqualene after the rearrangement of the triterpene skeleton. However, no functional characterization of the OSC genes involved in triterpene biosynthesis, except for a lupeol synthase in Taraxacum officinale, has been performed. Taraxacum coreanum, or Korean dandelion, grows in Korea and China. Putative OSC genes in T. coreanum plants were isolated by transcriptome analysis, and four of these (TcOSC1, TcOSC2, TcOSC3 and TcOSC4) were functionally characterized by heterologous expression in yeast. Both TcOSC1 and TcOSC2 were closely related to dammarenediol-II synthases. TcOSC3 and TcOSC4 were strongly grouped with β-amyrin synthases. Functional analysis revealed that TcOSC1 produced several triterpenes, including taraxasterol; Ψ-taraxasterol; α-, β- and δ-amyrin; and dammarenediol-II. TcOSC2 catalyzed the production of bauerenol and another unknown triterpene, TcOSC3 catalyzed the production of β-amyrin. TcOSC4 catalyzed the production of taraxerol. Moreover, we identified taraxasterol, ψ-taraxasterol, taraxerol, lupeol, δ-amyrin, α-amyrin, β-amyrin and bauerenol in the roots and leaves of T. coreanum. Our results suggest that TcOSC1, TcOSC2, TcOSC3 and TcOSC4 are key triterpene biosynthetic enzymes in T. coreanum. These enzymes are novel triterpene synthases involved in the production of taraxasterol, bauerenol and taraxerol., (� The Author(s) 2019. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

To better understand the molecular mechanism of color formation in different varieties of the mulberry fruit, we investigated the functional genes related to anthocyanin and betulinic acid biosynthesis using high-throughput transcriptome sequencing and detected the primary and secondary metabolites in the white (Morus alba L. cv. 'Turkey') and red (Morus alba L. cv. 'Cheongil') mulberry cultivars. We obtained 171,702,058 high-quality reads with an average read length of 125 bp. These reads were assembled into 51,272 and 51,159 unigenes in Turkey and Cheongil, respectively. We also identified the genes related to anthocyanin and triterpene biosynthesis and investigated their expression and metabolite profiles. Overall, our transcriptome sequencing provides valuable information that could be used in gene discovery, marker-assisted selection, and investigation of metabolic pathways in mulberry. Additionally, gene expression and metabolite profiles provide new insights into the underlying mechanism of anthocyanin and betulinic acid biosynthesis and relationship between primary and secondary metabolites., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The potential of six ancient Tuscan sweet cherry ( Prunus avium L.) varieties as a source of health-promoting pentacyclic triterpenes is here evaluated by means of a targeted gene expression and metabolite analysis. By using a sequence homology criterion, we identify five oxidosqualene cyclase genes ( OSC s) and three cytochrome P450s ( CYP85 s) that are putatively involved in the triterpene production pathway in sweet cherries. We performed 3D structure prediction and induced-fit docking using cation intermediates and reaction products for some OSCs to predict their function. We show that the Tuscan varieties have different amounts of ursolic and oleanolic acids and that these variations are related to different gene expression profiles. This study stresses the interest of valorizing ancient fruits as alternative sources of functional molecules with nutraceutical value. It also provides information on sweet cherry triterpene biosynthetic genes, which could be the object of follow-up functional studies.

Only very little is known about the resin composition of natural rubber from the dandelion species Taraxacum koksaghyz, thus its full characterization could provide new insights into how the isoprenoid end-products influence the physical properties of natural rubber, and this resin might be a good source of highly diverse triterpenoids. Here, we present a comprehensive analysis of the triterpenoid composition in an acetone extract and identified 13 triterpenes and triterpenoids also including the so far unknown pentacyclic compounds lup-19(21)-en-3-ol (1) and its ketone lup-19(21)-en-3-one (2). We purified single triterpenes from the acetone extract by developing a two-step HPLC system that is adapted to the structural differences of the described triterpenoids. Furthermore, we isolated six different oxidosqualene cyclases (OSCs) and two P450 enzymes, and we functionally characterized TkOSC1 and CYP716A263 in Nicotiana benthamiana and Saccharomyces cerevisiae in detail. TkOSC1 is a multifunctional OSC that was capable of synthesizing at least four of the latex-predominant pentacyclic triterpenes (taraxasterol, α-, β-amyrin and lup-19(21)-en-3-ol) while CYP716A263 oxidized pentacyclic triterpenes at the C-3 position. The identified enzymes responsible for biosynthesis and modification of pentacyclic triterpenes in T. koksaghyz latex may represent excellent tools for bioengineering approaches to produce pentacyclic triterpenes heterologously.

Euphorbia lathyris was proposed about fifty years ago as a potential agroenergetic crop. The tremendous amounts of triterpenes present in its latex has driven investigations for transforming this particular biological fluid into an industrial hydrocarbon source. The huge accumulation of terpenes in the latex of many plant species represent a challenging question regarding cellular homeostasis. In fact, the enzymes, the mechanisms and the controllers that tune the amount of products accumulated in specialized compartments (to fulfill ecological roles) or deposited at important sites (as essential factors) are not known. Here, we have isolated oxidosqualene cyclases highly expressed in the latex of Euphorbia lathyris. This triterpene biosynthetic machinery is made of distinct paralogous enzymes responsible for the massive accumulation of steroidal and non-steroidal tetracyclic triterpenes. More than eighty years after the isolation of butyrospermol from shea butter (Heilbronn IM, Moffet GL, and Spring FS J. Chem. Soc. 1934, 1583), a butyrospermol synthase is characterized in this work using yeast and in folia heterologous expression assays.

Mogrosides, the main bioactive compounds isolated from the fruits of Siraitia grosvenorii , are a group of cucurbitane-type triterpenoid glycosides that exhibit a wide range of notable biological activities and are commercially available worldwide as natural sweeteners. However, the extraction cost is high due to their relatively low contents in plants. Therefore, molecular breeding needs to be achieved when conventional plant breeding can hardly improve the quality so far. In this study, the levels of 21 active mogrosides and two precursors in 15 S. grosvenorii varieties were determined by HPLC-MS/MS and GC-MS, respectively. The results showed that the variations in mogroside V content may be caused by the accumulation of cucurbitadienol. Furthermore, a total of four wild-type cucurbitadienol synthase protein variants (50R573L, 50C573L, 50R573Q, and 50C573Q) based on two missense mutation single nucleotide polymorphism (SNP) sites were discovered. An in vitro enzyme reaction analysis indicated that 50R573L had the highest activity, with a specific activity of 10.24 nmol min -1 mg -1 . In addition, a site-directed mutant, namely, 50K573L, showed a 33% enhancement of catalytic efficiency compared to wild-type 50R573L. Our findings identify a novel cucurbitadienol synthase allele correlates with high catalytic efficiency. These results are valuable for the molecular breeding of luohanguo.

Triterpenes are thirty-carbon compounds derived from the universal five-carbon prenyl precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Normally, triterpenes are synthesized via the mevalonate (MVA) pathway operating in the cytoplasm of eukaryotes where DMAPP is condensed with two IPPs to yield farnesyl diphosphate (FPP), catalyzed by FPP synthase (FPS). Squalene synthase (SQS) condenses two molecules of FPP to generate the symmetrical product squalene, the first committed precursor to sterols and most other triterpenes. In the green algae Botryococcus braunii, two FPP molecules can also be condensed in an asymmetric manner yielding the more highly branched triterpene, botryococcene. Botryococcene is an attractive molecule because of its potential as a biofuel and petrochemical feedstock. Because B. braunii, the only native host for botryococcene biosynthesis, is difficult to grow, there have been efforts to move botryococcene biosynthesis into organisms more amenable to large-scale production. Here, we report the genetic engineering of the model monocot, Brachypodium distachyon, for botryococcene biosynthesis and accumulation. A subcellular targeting strategy was used, directing the enzymes (botryococcene synthase [BS] and FPS) to either the cytosol or the plastid. High titres of botryococcene (>1 mg/g FW in T 0 mature plants) were obtained using the cytosolic-targeting strategy. Plastid-targeted BS + FPS lines accumulated botryococcene (albeit in lesser amounts than the cytosolic BS + FPS lines), but they showed a detrimental phenotype dependent on plastid-targeted FPS, and could not proliferate and survive to set seed under phototrophic conditions. These results highlight intriguing differences in isoprenoid metabolism between dicots and monocots., (© 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Main Conclusion: An oxidosqualene cyclase (PdFRS) from Populus davidiana was characterized as a monofunctional friedelin synthase by its heterologous expression in yeast and overexpression in plants. Triterpenes are one of the largest classes of plant chemical compounds composed of three terpene units, which form the basic skeleton of all sterols and saponins. Friedelin (friedelan-3-one), a pentacyclic triterpene, occurs in many plant families and is particularly present in rich amounts in cork tissues from trees. The biosynthesis of friedelin occurs through the oxidosqualene cyclase (OSC) enzyme that generates friedelin from 2,3-oxidosqualene after the maximum rearrangement of a triterpene skeleton. Populus davidiana is called Korean aspen and grows in northern East Asia. From 57,322 unique sequences generated from the P. davidiana transcriptome database, one complete coding sequence (PdFRS) was obtained from a contig, which showed 74% identity to Betula platyphylla β-amyrin synthase and 73% identity with friedelin synthase from Maytenus ilicifolia. The open reading frame (ORF) region of the PdFRS sequence was 2280 bp long and composed a 759 amino acid protein with a predicted molecular mass of 87.81 kDa. qPCR analysis revealed that methyl jasmonate treatments strongly upregulated PdFRS gene expression and resulted in enhanced friedelin accumulation in leaves. Heterologous expression of the PdFRS gene in yeast resulted in the production of friedelin triterpene as a single product, which was confirmed by comparison with the mass fragmentation pattern from an authentic friedelin standard by GC/MS analysis. Transgenic P. davidiana overexpressing the PdFRS gene was constructed via Agrobacterium-mediated transformation. Overexpression of PdFRS in transgenic P. davidiana lines resulted in enhanced friedelin production.

The current water scarcity forces farmers to adopt new irrigation strategies to save water without jeopardizing the fruit yield and quality. In this study, the influence of 3 regulated deficit irrigation (RDI) treatments and 3 rootstocks on the functional quality of pistachios were studied. The functional parameters studied included, polyphenols, triterpenoids, and inhibition of α-amylase. The results showed that P. terebinthus and P. atlantica rootstocks led to pistachio kernels with higher contents of polyphenols and triterpenoids (mainly betulinic acid with 111 and 102 µg g -1 , respectively) than pistachios obtained using P. integerrima rootstock (81 µg g -1 ). On the other hand, the use of moderate RDI (T1 treatment) increased the total content of polyphenols (∼10%), quercetin-O-galloyl-hexoside (∼15%), keampferol-3-O-glucoside (∼19%), and polymeric procyanidins (∼20%), as compared to the control trees, resulting in pistachios with a better functional profile, lower economic cost and with a lesser environmental impact., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Laticifers are specialized plant cells capable of indefinite elongation that ramify extensively and are responsible for latex biosynthesis and accumulation. However, the mechanisms underlying laticifer cell differentiation, growth and production of latex remain largely unknown. In a search for mutants showing enhanced accumulation of latex we identified two LOT OF LATEX (LOL) loci in Euphorbia lathyris. lol2 and lol5 mutants show enhanced production of latex contained within laticifer cells. The recessive lol2 mutant carries increased biosynthesis of the plant hormone jasmonoyl-isoleucine (JA-Ile) and therefore establishes a genetic link between jasmonic acid (JA) signaling and latex production in laticifers. Instead, heightened production of latex in lol5 plants obeys to enhanced proliferation of laticifer cells. Phylogenetic analysis of laticifer-expressed genes in E. lathyris and in two other latex-bearing species, Euphorbia corallioides and Euphorbia palustris, allowed the identification of canonical JA responsive elements present in the gene promoter regions of laticifer marker genes. Moreover, we identified that the hormone JA functions not as a morphogen for laticifer differentiation but as a trigger for the fill out of laticifers with latex and the associated triterpenoids. The identification of LOL loci represents a further step towards the understanding of mechanisms controlling latex production in laticifer cells., (No claim to original US Government works New Phytologist © 2018 New Phytologist Trust.)

Triterpene cyclases catalyze the first committed step in triterpene biosynthesis, by forming mono- to pentacyclic backbone structures from oxygenated C30 isoprenoid precursors. Squalene epoxidase precedes this cyclization by providing the oxygenated and activated substrate for triterpene biosynthesis. Three squalene epoxidases from Cucurbita pepo (CpSEs) were isolated and shown to have evolved under purifying selection with signs of sites under positive selection in their N- and C-termini. They all localize to the Endoplasmic Reticulum (ER) and produce 2,3-oxidosqualene and 2,3:22,23-dioxidosqualene when expressed in a yeast erg1 (squalene epoxidase) erg7 (lanosterol synthase) double mutant. Co-expression of the CpSEs with four different triterpene cyclases, either transiently in Nicotiana benthamiana or constitutively in yeast, showed that CpSEs boost triterpene production. CpSE2 was the best performing in this regard, which could reflect either increased substrate production or superior channeling of the substrate to the triterpene cyclases. Fluorescence Lifetime Imaging Microscopy (FLIM) analysis with C. pepo cucurbitadienol synthase (CpCPQ) revealed a specific interaction with CpSE2 but not with the other CpSEs. When CpSE2 was transformed into C. pepo hairy root lines, cucurbitacin E production was increased two folds compared to empty vector control lines. This study provides new insight into the importance of SEs in triterpene biosynthesis, suggesting that they may facilitate substrate channeling, and demonstrates that SE overexpression is a new tool for increasing triterpene production in plants and yeast., (Copyright © 2018. Published by Elsevier Inc.)

Ferns are the most primitive of all vascular plants. One of the characteristics distinguishing them from flowering plants is its triterpene metabolism. Most cyclic triterpenes in ferns are hydrocarbons derived from the direct cyclization of squalene by squalene cyclases (SCs). Both ferns and more complex plants share sterols and biosynthetic enzymes, such as cycloartenol synthases (CASs). Polystichum belongs to Dryopteridaceae, and is one of the most species-rich of all fern genera. Several Polystichum ferns in Japan are classified as one of three possible chemotypes, based on their triterpene profiles. In this study, we describe the molecular cloning and functional characterization of cDNAs encoding a SC (PPH) and a CAS (PPX) from the type species Polystichum polyblepharum. Heterologous expression in Pichia pastoris revealed that PPH and PPX are hydroxyhopane synthase and CAS, respectively. By using the PPH and PPX sequences, we successfully isolated SC- and CAS-encoding cDNAs from six Polystichum ferns. Phylogenetic analysis, based on SCs and oxidosqualene cyclase sequences, suggested that the Polystichum subclade in the fern SC and CAS clades reflects the chemotype-but not the molecular phylogeny constructed using plastid molecular markers. These results show a possible relation between triterpenes and their biosynthetic enzymes in Polystichum.

Centella asiatica is widely used as a medicinal plant due to accumulation of the ursane-type triterpene saponins asiaticoside and madecassoside. The molecular structure of both compounds suggests that they are biosynthesized from α-amyrin via three hydroxylations, and the respective Cyt P450-dependent monooxygenases (P450 enzymes) oxidizing the C-28 and C-2α positions have been reported. However, a third enzyme hydroxylating C-23 remained elusive. We previously identified 40,064 unique sequences in the transcriptome of C. asiatica elicited by methyl jasmonate, and among them we have now found 149 unigenes encoding putative P450 enzymes. In this set, 23 full-length cDNAs were recognized, 13 of which belonged to P450 subfamilies previously implicated in secondary metabolism. Four of these genes were highly expressed in response to jasmonate treatment, especially in leaves, in accordance with the accumulation patterns of asiaticoside. The functions of these candidate genes were tested using heterologous expression in yeast cells. Gas chromatography-mass spectrometry (GC-MS) analysis revealed that yeast expressing only the oxidosqualene synthase CaDDS produced the asiaticoside precursor α-amyrin (along with its isomer β-amyrin), while yeast co-expressing CaDDS and CYP716A83 also contained ursolic acid along with oleanolic acid. This P450 enzyme thus acts as a multifunctional triterpenoid C-28 oxidase converting amyrins into corresponding triterpenoid acids. Finally, yeast strains co-expressing CaDDS, CYP716A83 and CYP714E19 produced hederagenin and 23-hydroxyursolic acid, showing that CYP714E19 is a multifunctional triterpenoid oxidase catalyzing the C-23 hydroxylation of oleanolic acid and ursolic acid. Overall, our results demonstrate that CaDDS, CYP716A83 and CYP714E19 are C. asiatica enzymes catalyzing consecutive steps in asiaticoside biosynthesis.

Cynara cardunculus: L. represents a natural source of terpenic compounds, with the predominant molecule being cynaropicrin. Cynaropicrin is gaining interest since it has been correlated to anti-hyperlipidaemia, antispasmodic and cytotoxicity activity against leukocyte cancer cells. The objective of this work was to screen a collection of C. cardunculus, from different origins, for new allelic variants in germacrene A synthase (GAS) gene involved in the cynaropicrin biosynthesis and correlate them with improved cynaropicrin content and biological activities. Using high-resolution melting, nine haplotypes were identified. The putative impact of the identified allelic variants in GAS protein was evaluated by bioinformatic tools and polymorphisms that putatively lead to protein conformational changes were described. Additionally, cynaropicrin and main pentacyclic triterpenes contents, and antithrombin, antimicrobial and antiproliferative activities were also determined in C. cardunculus leaf lipophilic-derived extracts. In this work we identified allelic variants with putative impact on GAS protein, which are significantly associated with cynaropicrin content and antiproliferative activity. The results obtained suggest that the identified polymorphisms should be explored as putative genetic markers correlated with biological properties in Cynara cardunculus.

Centella asiatica (L.) Urban contains two ursane-type triterpene saponins, asiaticoside and madecassoside, as major secondary metabolites. In order to select candidate genes encoding UDP-glucosyltransferases (UGTs) involved in asiaticoside biosynthesis, we performed transcriptomic analysis of leaves elicited by methyl jasmonate (MeJA). Among the unigenes, 120 isotigs and 13 singletons of unique sequences were annotated as UGTs, including 37 putative full-length cDNAs, and 15 of the putative UGT genes were named according to the UGT committee nomenclature protocols. One of them, UGT73AH1, was characterized by heterologous expression in Escherichia coli BL21 (DE3) cells. After induction with IPTG, a total protein extract was assayed with UDP-glucose and asiatic acid. UPLC-QTOF/MS analysis showed that UGT73AH1 catalyzes the glycosylation of asiatic acid to its monoglucoside. It remains unclear whether glycosylation occurs on the triterpene C-2α, C-3β, C-23, or C-28 position. However, it is very likely that UGT73AH1 glucosylates the C-28 position, because only C-28 bears a glucose moiety in the final pathway product of asiatic acid, while C-2α, C-3β, and C-23 remain un-conjugated., Competing Interests: The authors declare no conflict of interest.

The alternative oxidase (AOX), which forms a branch of the mitochondrial respiratory electron transport pathway, functions to sustain electron flux and alleviate reactive oxygen species (ROS) production. In this article, a homologous AOX gene was identified in Ganoderma lucidum. The coding sequence of the AOX gene in G. lucidum contains 1038 nucleotides and encodes a protein of 39.48 kDa. RNA interference (RNAi) was used to study the function of AOX in G. lucidum, and two silenced strains (AOXi6 and AOXi21) were obtained, showing significant decreases of approximately 60 and 50 %, respectively, in alternative pathway respiratory efficiency compared to WT. The content of ganoderic acid (GA) in the mutant strains AOXi6 and AOXi21 showed significant increases of approximately 42 and 44 %, respectively, compared to WT. Elevated contents of intermediate metabolites in GA biosynthesis and elevated transcription levels of corresponding genes were also observed in the mutant strains AOXi6 and AOXi21. In addition, the intracellular ROS content in strains AOXi6 and AOXi21 was significantly increased, by approximately 1.75- and 1.93-fold, respectively, compared with WT. Furthermore, adding N-acetyl-l-cysteine (NAC), a ROS scavenger, significantly depressed the intracellular ROS content and GA accumulation in AOX-silenced strains. These results indicate that AOX affects GA biosynthesis by regulating intracellular ROS levels. Our research revealed the important role of AOX in the secondary metabolism of G. lucidum.

Triterpene saponins include bioactive compounds with structures consisting of triterpene aglycones (sapogenins) and one or more sugar moieties linked through acetal or ester glycosidic linkages at one or more sites. Centella asiatica (L.) Urban is a medicinal plant that contains bioactive ursane-type saponins, such as madecassoside and asiaticoside. In this work, glucosylation of triterpenoids in C. asiatica was investigated starting with plant extracts. An enzyme capable of glucosylating asiatic and madecassic acids was partially purified. Proteomics methods and cDNA sequence data were employed as tools to obtain a full-length cDNA clone encoding a glucosyltransferase. The recombinant gene product, UGT73AD1, was functionally expressed in Escherichia coli and purified by immobilized metal-affinity chromatography. Purified recombinant UGT73AD1 was found to have a narrow specificity, glucosylating asiatic and madecassic acids at the C28 carboxyl. mRNA accumulated in all tissues tested (leaves, stems, roots and flowers), with highest expression in leaves. Thus, UGT73AD1 was identified as a triterpenoid carboxylic acid: UDP-glucose 28-O-glucosyltransferase that appears to be involved in saponin biosynthesis in C. asiatica., (Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.)

Charantin, a natural cucurbitane type triterpenoid, has been reported to have beneficial pharmacological functions such as anticancer, antidiabetic, and antibacterial activities. However, accumulation of charantin in bitter melon has been little studied. Here, we performed a transcriptome analysis to identify genes involved in the triterpenoid biosynthesis pathway in bitter melon seedlings. A total of 88,703 transcripts with an average length of 898 bp were identified in bitter melon seedlings. On the basis of a functional annotation, we identified 15 candidate genes encoding enzymes related to triterpenoid biosynthesis and analyzed their expression in different organs of mature plants. Most genes were highly expressed in flowers and/or fruit from the ripening stages. An HPLC analysis confirmed that the accumulation of charantin was highest in fruits from the ripening stage, followed by male flowers. The accumulation patterns of charantin coincide with the expression pattern of McSE and McCAS1, indicating that these genes play important roles in charantin biosynthesis in bitter melon. We also investigated optimum light conditions for enhancing charantin biosynthesis in bitter melon and found that red light was the most effective wavelength.

The roots of Platycodon grandiflorus are widely used as a crude drug. The active components include a variety of triterpenoid saponins. Recent studies have revealed that Cyt P450 monooxygenases (P450s) function as triterpene oxidases in triterpenoid saponin biosynthesis in many plant species. However, there have been no reports regarding triterpene oxidases in P. grandiflorus. In this study, we performed transcriptome analysis of three different P. grandiflorus tissues (roots, leaves and petals) using RNA sequencing (RNA-Seq) technology. We cloned six P450 genes that were highly expressed in roots, and classified them as belonging to the CYP716A, CYP716D and CYP72A subfamilies. We heterologously expressed these P450s in an engineered yeast strain that produces β-amyrin, one of the most common triterpenes in plants. Two of the CYP716A subfamily P450s catalyzed oxidation reactions of the β-amyrin skeleton. One of these P450s, CYP716A140v2, catalyzed a three-step oxidation reaction at C-28 on β-amyrin to produce oleanolic acid, a reaction performed by CYP716A subfamily P450s in a variety of plant species. The other P450, CYP716A141, catalyzed the hydroxylation of β-amyrin at C-16β. This reaction is unique among triterpene oxidases isolated to date. These results enhance our knowledge of functional variation among CYP716A subfamily enzymes involved in triterpenoid biosynthesis, and provide novel molecular tools for use in synthetic biology to produce triterpenoid saponins with pre-defined structures., (© The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The medicinal plant sweet basil (Ocimum basilicum) accumulates bioactive ursane- and oleanane-type pentacyclic triterpenes (PCTs), ursolic acid and oleanolic acid, respectively, in a spatio-temporal manner; however, the biosynthetic enzymes and their contributions towards PCT biosynthesis remain to be elucidated. Two CYP716A subfamily cytochrome P450 monooxygenases (CYP716A252 and CYP716A253) are identified from a methyl jasmonate-responsive expression sequence tag collection and functionally characterized, employing yeast (Saccharomyces cerevisiae) expression platform and adapting virus-induced gene silencing (VIGS) in sweet basil. CYP716A252 and CYP716A253 catalyzed sequential three-step oxidation at the C-28 position of α-amyrin and β-amyrin to produce ursolic acid and oleanolic acid, respectively. Although CYP716A253 was more efficient than CYP716A252 for amyrin C-28 oxidation in yeast, VIGS revealed essential roles for both of these CYP716As in constitutive biosynthesis of ursolic acid and oleanolic acid in sweet basil leaves. However, CYP716A253 played a major role in elicitor-induced biosynthesis of ursolic acid and oleanolic acid. Overall, the results suggest similar as well as distinct roles of CYP716A252 and CYP716A253 for the spatio-temporal biosynthesis of PCTs. CYP716A252 and CYP716A253 might be useful for the alternative and sustainable production of PCTs in microbial host, besides increasing plant metabolite content through genetic modification., (© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.)

Key Message: CYP716A179, a cytochrome P450 monooxygenase expressed predominantly in tissue-cultured stolons of licorice ( Glycyrrhiza uralensis ), functions as a triterpene C-28 oxidase in the biosynthesis of oleanolic acid and betulinic acid. Cytochrome P450 monooxygenases (P450s) play key roles in the structural diversification of plant triterpenoids. Among these, the CYP716A subfamily, which functions mainly as a triterpene C-28 oxidase, is common in plants. Licorice (Glycyrrhiza uralensis) produces bioactive triterpenoids, such as glycyrrhizin and soyasaponins, and relevant P450s (CYP88D6, CYP72A154, and CYP93E3) have been identified; however, no CYP716A subfamily P450 has been isolated. Here, we identify CYP716A179, which functions as a triterpene C-28 oxidase, by RNA sequencing analysis of tissue-cultured stolons of G. uralensis. Heterologous expression of CYP716A179 in engineered yeast strains confirmed the production of oleanolic acid, ursolic acid, and betulinic acid from β-amyrin, α-amyrin, and lupeol, respectively. The transcript level of CYP716A179 was about 500 times higher in tissue-cultured stolons than in intact roots. Oleanolic acid and betulinic acid were consistently detected only in tissue-cultured stolons. The discovery of CYP716A179 helps increase our understanding of the mechanisms of tissue-type-dependent triterpenoid metabolism in licorice and provides an additional target gene for pathway engineering to increase the production of glycyrrhizin in licorice tissue cultures by disrupting competing pathways.

Triterpenoids are widespread bioactive plant defence compounds with potential use as pharmaceuticals, pesticides and other high-value products. Enzymes belonging to the cytochrome P450 family have an essential role in creating the immense structural diversity of triterpenoids across the plant kingdom. However, for many triterpenoid oxidation reactions, the corresponding enzyme remains unknown. Here we characterize CYP716 enzymes from different medicinal plant species by heterologous expression in engineered yeasts and report ten hitherto unreported triterpenoid oxidation activities, including a cyclization reaction, leading to a triterpenoid lactone. Kingdom-wide phylogenetic analysis of over 400 CYP716s from over 200 plant species reveals details of their evolution and suggests that in eudicots the CYP716s evolved specifically towards triterpenoid biosynthesis. Our findings underscore the great potential of CYP716s as a source for generating triterpenoid structural diversity and expand the toolbox available for synthetic biology programmes for sustainable production of bioactive plant triterpenoids., Competing Interests: The authors declare no competing financial interests.

Triterpenoid saponins are major components of secondary metabolites in soybean seeds and are divided into two groups: group A saponins, and 2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one (DDMP) saponins. The aglycone moiety of group A saponins consists of soyasapogenol A (SA), which is an oxidized β-amyrin product, and the aglycone moiety of the DDMP saponins consists of soyasapogenol B (SB). Group A saponins produce a bitter and astringent aftertaste in soy products, whereas DDMP saponins have known health benefits for humans. We completed map-based cloning and characterization of the gene Sg-5, which is responsible for SA biosynthesis. The naturally occurring sg-5 mutant lacks group A saponins and has a loss-of-function mutation (L164*) in Glyma15g39090, which encodes the cytochrome P450 enzyme, CYP72A69. An enzyme assay indicated the hydroxylase activity of recombinant CYP72A69 against SB, which also suggested the production of SA. Additionally, induced Glyma15g39090 mutants (R44* or S348P) lacked group A saponins similar to the sg-5 mutant, indicating that Glyma15g39090 corresponds to Sg-5. Endogenous levels of DDMP saponins were higher in the sg-5 mutant than in the wild-type lines due to the loss of the enzyme activity that converts SB to SA. Interestingly, the genomes of palaeopolyploid soybean and the closely related common bean carry multiple Sg-5 paralogs in a genomic region syntenic to the soybean Sg-5 region. However, SA did not accumulate in common bean samples, suggesting that Sg-5 activity evolved after gene duplication event(s). Our results demonstrate that metabolic switching of undesirable saponins with beneficial saponins can be achieved in soybean by disabling Sg-5., (© 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.)

Among the biologically active triterpenes, friedelin has the most-rearranged structure produced by the oxidosqualene cyclases and is the only one containing a cetonic group. In this study, we cloned and functionally characterized friedelin synthase and one cycloartenol synthase from Maytenus ilicifolia (Celastraceae). The complete coding sequences of these 2 genes were cloned from leaf mRNA, and their functions were characterized by heterologous expression in yeast. The cycloartenol synthase sequence is very similar to other known OSCs of this type (approximately 80% identity), although the M. ilicifolia friedelin synthase amino acid sequence is more related to β-amyrin synthases (65-74% identity), which is similar to the friedelin synthase cloned from Kalanchoe daigremontiana. Multiple sequence alignments demonstrated the presence of a leucine residue two positions upstream of the friedelin synthase Asp-Cys-Thr-Ala-Glu (DCTAE) active site motif, while the vast majority of OSCs identified so far have a valine or isoleucine residue at the same position. The substitution of the leucine residue with valine, threonine or isoleucine in M. ilicifolia friedelin synthase interfered with substrate recognition and lead to the production of different pentacyclic triterpenes. Hence, our data indicate a key role for the leucine residue in the structure and function of this oxidosqualene cyclase.

The consumption of sweeteners, natural as well as synthetic sugars, is implicated in an array of modern-day health problems. Therefore, natural nonsugar sweeteners are of increasing interest. We identify here the biosynthetic pathway of the sweet triterpenoid glycoside mogroside V, which has a sweetening strength of 250 times that of sucrose and is derived from mature fruit of luo-han-guo (Siraitia grosvenorii, monk fruit). A whole-genome sequencing of Siraitia, leading to a preliminary draft of the genome, was combined with an extensive transcriptomic analysis of developing fruit. A functional expression survey of nearly 200 candidate genes identified the members of the five enzyme families responsible for the synthesis of mogroside V: squalene epoxidases, triterpenoid synthases, epoxide hydrolases, cytochrome P450s, and UDP-glucosyltransferases. Protein modeling and docking studies corroborated the experimentally proven functional enzyme activities and indicated the order of the metabolic steps in the pathway. A comparison of the genomic organization and expression patterns of these Siraitia genes with the orthologs of other Cucurbitaceae implicates a strikingly coordinated expression of the pathway in the evolution of this species-specific and valuable metabolic pathway. The genomic organization of the pathway genes, syntenously preserved among the Cucurbitaceae, indicates, on the other hand, that gene clustering cannot account for this novel secondary metabolic pathway., Competing Interests: The authors declare no conflict of interest.

Triterpenes are structurally complex plant natural products with numerous medicinal applications. They are synthesized through an origami-like process that involves cyclization of the linear 30 carbon precursor 2,3-oxidosqualene into different triterpene scaffolds. Here, through a forward genetic screen in planta, we identify a conserved amino acid residue that determines product specificity in triterpene synthases from diverse plant species. Mutation of this residue results in a major change in triterpene cyclization, with production of tetracyclic rather than pentacyclic products. The mutated enzymes also use the more highly oxygenated substrate dioxidosqualene in preference to 2,3-oxidosqualene when expressed in yeast. Our discoveries provide new insights into triterpene cyclization, revealing hidden functional diversity within triterpene synthases. They further open up opportunities to engineer novel oxygenated triterpene scaffolds by manipulating the precursor supply.

Mogrosides, the principally bioactive compounds extracted from the fruits of Siraitia grosvenorii, are a group of glycosylated cucurbitane-type tetracyclic triterpenoid saponins that exhibit a wide range of notable biological activities and are commercially available worldwide as natural sweeteners. The biosynthesis of mogrosides involves initial cyclization of 2,3-oxidosqualene to the triterpenoid skeleton of cucurbitadienol, followed by a series of oxidation reactions catalyzed by Cyt P450s (P450s) and then glycosylation reactions catalyzed by UDP glycosyltransferases (UGTs). We previously reported the identification of a cucurbitadienol synthase (SgCbQ) and a mogrol C-3 hydroxyl glycosyltransferase (UGT74AC1). However, molecular characterization of further transformation of cucurbitadienol to mogrol by P450s remains unavailable. In this study, we report the successful identification of a multifunctional P450 (CYP87D18) as being involved in C-11 oxidation of cucurbitadienol. In vitro enzymatic activity assays showed that CYP87D18 catalyzed the oxidation of cucurbitadienol at C-11 to produce 11-oxo cucurbitadienol and 11-hydroxy cucurbitadienol. Furthermore, 11-oxo-24,25-epoxy cucurbitadienol as well as 11-oxo cucurbitadienol and 11-hydroxy cucurbitadienol were produced when CYP87D18 was co-expressed with SgCbQ in genetic yeast, and their structures were confirmed by liquid chromatography-solid-phase extraction-nuclear magnetic resonance-mass spectrometry coupling (LC-SPE-NMR-MS). Taken together, these results suggest a role for CYP87D18 as a multifunctional cucurbitadienol oxidase in the mogrosides pathway., (© The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Plant triterpenoids are a diverse group of secondary metabolites with wide distribution, high chemical diversity and interesting pharmacological and antimicrobial properties. The first step in the biosynthesis of all triterpenoids is the cyclization of the 2,3-oxidosqualene precursor, catalyzed by oxidosqualene cyclases (OSCs), which have characteristic product specificities. Biosynthesis and functions of pentacyclic triterpenes have been poorly studied in grapevine. In this study, we first investigated the profile of triterpenoids present in leaf cuticular waxes from eight Vitis vinifera cultivars cultivated in the Upper Rhine Valley. Further quantification of triterpenoids showed that these cultivars can be divided into two groups, characterized by high levels of lupeol (e.g., Pinot noir) or taraxerol (e.g., Gewurztraminer) respectively. We further analyzed the OSC family involved in the synthesis of pentacyclic triterpenes (called VvTTPSs) in the sequenced V. vinifera 40024 genome and found nine genes with similarity to previously characterized triterpene synthases. Phylogenetic analysis further showed that VvTTPS1-VvTTPS3 and VvTTPS5-VvTTPS9 belong to the β-amyrin synthase and multifunctional triterpene synthase clade, whereas VvTTPS10 belongs to the lupeol synthase clade. We studied the expression of several members of the VvTTPS family following biotic and abiotic stresses in V. vinifera 40024 as well as in the eight healthy cultivars. This study further revealed that one candidate gene, VvTTPS5, which does not belong to the lupeol synthase clade, is highly expressed in lupeol-rich cultivars. VvTTPS3, VvTTPS5, VvTTPS6, VvTTPS7 and VvTTPS10 were highly upregulated by UV stress, but only VvTTPS3, VvTTPS5, VvTTPS6 and VvTTPS10 were upregulated following downy mildew and gray mold infections respectively. These results suggest differential roles of VvTTPS against environmental stresses in grape leaves.

Linear, branch-chained triterpenes, including squalene (C30), botryococcene (C30), and their methylated derivatives (C31-C37), generated by the green alga Botryococcus braunii race B have received significant attention because of their utility as chemical and biofuel feedstocks. However, the slow growth habit of B. braunii makes it impractical as a production system. In this study, we evaluated the potential of generating high levels of botryococcene in tobacco (Nicotiana tabacum) plants by diverting carbon flux from the cytosolic mevalonate pathway or the plastidic methylerythritol phosphate pathway by the targeted overexpression of an avian farnesyl diphosphate synthase along with two versions of botryococcene synthases. Up to 544 µg g(-1) fresh weight of botryococcene was achieved when this metabolism was directed to the chloroplasts, which is approximately 90 times greater than that accumulating in plants engineered for cytosolic production. To test if methylated triterpenes could be produced in tobacco, we also engineered triterpene methyltransferases (TMTs) from B. braunii into wild-type plants and transgenic lines selected for high-level triterpene accumulation. Up to 91% of the total triterpene contents could be converted to methylated forms (C31 and C32) by cotargeting the TMTs and triterpene biosynthesis to the chloroplasts, whereas only 4% to 14% of total triterpenes were methylated when this metabolism was directed to the cytoplasm. When the TMTs were overexpressed in the cytoplasm of wild-type plants, up to 72% of the total squalene was methylated, and total triterpene (C30+C31+C32) content was elevated 7-fold. Altogether, these results point to innate mechanisms controlling metabolite fluxes, including a homeostatic role for squalene., (© 2016 American Society of Plant Biologists. All Rights Reserved.)

Plants respond to stresses by producing a broad spectrum of bioactive specialized metabolites. Hormonal elicitors, such as jasmonates, trigger a complex signaling circuit leading to the concerted activation of specific metabolic pathways. However, for many specialized metabolic pathways, the transcription factors involved remain unknown. Here, we report on two homologous jasmonate-inducible transcription factors of the basic helix-loop-helix family, TRITERPENE SAPONIN BIOSYNTHESIS ACTIVATING REGULATOR1 (TSAR1) and TSAR2, which direct triterpene saponin biosynthesis in Medicago truncatula. TSAR1 and TSAR2 are coregulated with and transactivate the genes encoding 3-HYDROXY-3-METHYLGLUTARYL-COENZYME A REDUCTASE1 (HMGR1) and MAKIBISHI1, the rate-limiting enzyme for triterpene biosynthesis and an E3 ubiquitin ligase that controls HMGR1 levels, respectively. Transactivation is mediated by direct binding of TSARs to the N-box in the promoter of HMGR1. In transient expression assays in tobacco (Nicotiana tabacum) protoplasts, TSAR1 and TSAR2 exhibit different patterns of transactivation of downstream triterpene saponin biosynthetic genes, hinting at distinct functionalities within the regulation of the pathway. Correspondingly, overexpression of TSAR1 or TSAR2 in M. truncatula hairy roots resulted in elevated transcript levels of known triterpene saponin biosynthetic genes and strongly increased the accumulation of triterpene saponins. TSAR2 overexpression specifically boosted hemolytic saponin biosynthesis, whereas TSAR1 overexpression primarily stimulated nonhemolytic soyasaponin biosynthesis. Both TSARs also activated all genes of the precursor mevalonate pathway but did not affect sterol biosynthetic genes, pointing to their specific role as regulators of specialized triterpene metabolism in M. truncatula., (© 2016 American Society of Plant Biologists. All Rights Reserved.)

Key Message: Overexpression of sterol glycosyltransferase (SGTL1) gene of Withania somnifera showing its involvement in glycosylation of withanolide that leads to enhanced growth and tolerance to biotic and abiotic stresses. Withania somnifera is widely used in Ayurvedic medicines for over 3000 years due to its therapeutic properties. It contains a variety of glycosylated steroids called withanosides that possess neuroregenerative, adaptogenic, anticonvulsant, immunomodulatory and antioxidant activities. The WsSGTL1 gene specific for 3β-hydroxy position has a catalytic specificity to glycosylate withanolide and sterols. Glycosylation not only stabilizes the products but also alters their physiological activities and governs intracellular distribution. To understand the functional significance and potential of WsSGTL1 gene, transgenics of W. somnifera were generated using Agrobacterium tumefaciens-mediated transformation. Stable integration and overexpression of WsSGTL1 gene were confirmed by Southern blot analysis followed by quantitative real-time PCR. The WsGTL1 transgenic plants displayed number of alterations at phenotypic and metabolic level in comparison to wild-type plants which include: (1) early and enhanced growth with leaf expansion and increase in number of stomata; (2) increased production of glycowithanolide (majorly withanoside V) and campesterol, stigmasterol and sitosterol in glycosylated forms with reduced accumulation of withanolides (withaferin A, withanolide A and withanone); (3) tolerance towards biotic stress (100 % mortality of Spodoptera litura), improved survival capacity under abiotic stress (cold stress) and; (4) enhanced recovery capacity after cold stress, as indicated by better photosynthesis performance, chlorophyll, anthocyanin content and better quenching regulation of PSI and PSII. Our data demonstrate overexpression of WsSGTL1 gene which is responsible for increase in glycosylated withanolide and sterols, and confers better growth and tolerance to both biotic and abiotic stresses.

Background: Neem tree (Azadirachta indica) is one of the richest sources of skeletally diverse triterpenoids and they are well-known for their broad-spectrum pharmacological and insecticidal properties. However, the abundance of Neem triterpenoids varies among the tissues. Here, we delineate quantitative profiling of fifteen major triterpenoids across various tissues including developmental stages of kernel and pericarp, flower, leaf, stem and bark using UPLC-ESI(+)-HRMS based profiling. Transcriptome analysis was used to identify the initial genes involved in isoprenoid biosynthesis. Based on transcriptome analysis, two short-chain prenyltransferases and squalene synthase (AiSQS) were cloned and functionally characterized., Results: Quantitative profiling revealed differential abundance of both total and individual triterpenoid content across various tissues. RNA from tissues with high triterpenoid content (fruit, flower and leaf) were pooled to generate 79.08 million paired-end reads using Illumina GA ΙΙ platform. 41,140 transcripts were generated by d e novo assembly. Transcriptome annotation led to the identification of the putative genes involved in isoprenoid biosynthesis. Two short-chain prenyltransferases, geranyl diphosphate synthase (AiGDS) and farnesyl diphosphate synthase (AiFDS) and squalene synthase (AiSQS) were cloned and functionally characterized using transcriptome data. RT-PCR studies indicated five-fold and ten-fold higher relative expression level of AiSQS in fruits as compared to leaves and flowers, respectively., Conclusions: Triterpenoid profiling indicated that there is tissue specific variation in their abundance. The mature seed kernel and initial stages of pericarp were found to contain the highest amount of limonoids. Furthermore, a wide diversity of triterpenoids, especially C-seco triterpenoids were observed in kernel as compared to the other tissues. Pericarp, flower and leaf contained mainly ring-intact triterpenoids. The initial genes such as AiGDS, AiFDS and AiSQS involved in the isoprenoids biosynthesis have been functionally characterized. The expression levels of AiFDS and AiSQS were found to be in correlation with the total triterpenoid content in individual tissues.

Key Message: Protopanaxadiol (PPD) is an aglycone of dammarene-type ginsenoside and has high medicinal values. In this work, we reported the PPD production in transgenic tobacco co-overexpressing PgDDS and CYP716A47. PPD is an aglycone of ginsenosides produced by Panax species and has a wide range of pharmacological activities. PPD is synthesized via the hydroxylation of dammarenediol-II (DD) by CYP716A47 enzyme. Here, we established a PPD production system via cell suspension culture of transgenic tobacco co-overexpressing the genes for PgDDS and CYP716A47. The concentration of PPD in transgenic tobacco leaves was 2.3-5.7 µg/g dry weight (DW), depending on the transgenic line. Leaf segments were cultured on medium with various types of hormones to induce callus. Auxin treatment, particularly 2,4-D, strongly enhanced the production of DD (783.8 µg g(-1) DW) and PPD (125.9 µg g(-1) DW). Treatment with 2,4-D enhanced the transcription of the HMG-Co reductase (HMGR) and squalene epoxidase genes. PPD production reached 166.9 and 980.9 µg g(-1) DW in a 250-ml shake flask culture and in 5-l airlift bioreactor culture, respectively.

Artemisia annua is widely studied for its ability to accumulate the antimalarial sesquiterpenoid artemisinin. In addition to producing a variety of sesquiterpenoids, A. annua also accumulates mono-, di-, and triterpenoids, the majority of which are produced in the glandular trichomes. A. annua also has filamentous trichomes on its aerial parts, but little is known of their biosynthesis potential. Here, through a comparative transcriptome analysis between glandular and filamentous trichomes, we identified two genes, OSC2 and CYP716A14v2, encoding enzymes involved in the biosynthesis of specialized triterpenoids in A. annua. By expressing these genes in Saccharomyces cerevisiae and Nicotiana benthamiana, we characterized the catalytic function of these proteins and could reconstitute the specialized triterpenoid spectrum of A. annua in these heterologous hosts. OSC2 is a multifunctional oxidosqualene cyclase that produces α-amyrin, β-amyrin, and δ-amyrin. CYP716A14v2 is a P450 belonging to the functionally diverse CYP716 family and catalyzes the oxidation of pentacyclic triterpenes, leading to triterpenes with a carbonyl group at position C-3, thereby providing an alternative biosynthesis pathway to 3-oxo triterpenes. Together, these enzymes produce specialized triterpenoids that are constituents of the wax layer of the cuticle covering the aerial parts of A. annua and likely function in the protection of the plant against biotic and abiotic stress., (© 2015 American Society of Plant Biologists. All rights reserved.)

Cucurbitacins are triterpenoids that confer a bitter taste in cucurbits such as cucumber, melon, watermelon, squash, and pumpkin. These compounds discourage most pests on the plant and have also been shown to have antitumor properties. With genomics and biochemistry, we identified nine cucumber genes in the pathway for biosynthesis of cucurbitacin C and elucidated four catalytic steps. We discovered transcription factors Bl (Bitter leaf) and Bt (Bitter fruit) that regulate this pathway in leaves and fruits, respectively. Traces in genomic signatures indicated that selection imposed on Bt during domestication led to derivation of nonbitter cucurbits from their bitter ancestors., (Copyright © 2014, American Association for the Advancement of Science.)