Induction of sesquiterpenoid biosynthesis in tobacco cell suspension cultures by fungal elicitor

Large amounts of the sesquiterpenoid capsidiol accumulated in the media of tobacco (Nicotiana tabacum L. cv KY14) cell suspension cultures upon addition of fungal elicitor. Capsidiol accumulation was proportional to the amount of elicitor added. The accumulation of capsidiol was preceded by a transient increase in the capsidiol de novo synthesis rate as measured by the incorporation of exogenous [¹⁴C]acetate. Changes in 3-hydroxy-3-methylglutaryl-CoA reductase activity (HMGR; EC 1.1.1.34), an enzyme of general isoprenoid metabolism, paralleled the changes in [¹⁴C]acetate incorporation into capsidiol. Incubation of the cell cultures with mevinolin, a potent in vitro inhibitor of the tobacco HMGR enzyme activity, inhibited the elicitor-induced capsidiol accumulation in a concentration dependent manner. [¹⁴C]Acetate incorporation into capsidiol was likewise inhibited by mevinolin treatment. Unexpectedly, [³H] mevalonate incorporation into capsidiol was also partially inhibited by mevinolin, suggesting that mevinolin may effect secondary sites of sesquiterpenoid biosynthesis in vivo beyond HMGR. The data indicated the importance of the induced HMGR activity for capsidiol production in elicitor-treated tobacco cell suspension cultures.     

Effect of salicylic acid and yeast extract on the accumulation of jasmonic acid and sesquiterpenoids in Panax ginseng adventitious roots

In different plant species, secondary metabolite biosynthesis is regulated by the phytohormone jasmonic acid (JA), which is derived by the action of lipoxygenase. In this study, we examined mono- and sesquiterpenoid accumulation and the related signal transduction pathways and biosynthetic genes in adventitious root cultures of Panax ginseng C.A. Meyer as induced by yeast extract (YE, 3 g/L), a biotic elicitor, and salicylic acid (SA, 200 μM), a signaling elicitor. The lipoxygenase (LOX) gene was highly expressed in 24 and 12 h after treatment with SA and YE. JA content was significantly increased in 24 h after SA treatment. The H₂O₂ content was the highest in 24 and 72 h after the onset of SA and YE treatment, respectively. RNA blot analysis showed that farnesyl diphosphate synthase (FPS) and isopentenyl pyrophosphate isomerase (IPPI) genes encoding enzymes of the biosynthesis of mono- and sesquiterpenoids were up-regulated by both elicitors. Farensol, isochiapin B sesquiterpenoids, champhor, and cineole monoterpenoids were highly accumulated after 24 h of SA treatment, while YE treatment induced bacchotricuneatin C, guaiazulene, isochiapin B, and p-benzoquinone sesquiterpenoid production. These results suggest that mono- and sesquiterpenoid accumulation induced by SA and YE occurs due to the IPPI and FPS expression and may be mediated by reactive oxygen species signaling and jasmonic acid signal transduction.     

Induction of sesquiterpene cyclase and suppression of squalene synthetase activities in plant cell cultures treated with fungal elicitor

Addition of elicitor, cell wall fragments of the fungus Phytophthora parasitica, to tobacco cell suspension cultures (Nicotiana tabacum) resulted in the rapid synthesis and secretion of large amounts of antibiotic sesquiterpenoids. Pulse-labeling experiments with [¹⁴C]acetate and [³H] mevalonate demonstrated that the induction of sesquiterpenoid biosynthesis, maximal by 6 to 9 hours after elicitor addition to the cell cultures, was paralleled by a rapid and large decline in the incorporation rate of radioactivity into sterols. Consequently, sterol accumulation was also inhibited upon addition of elicitor to the cell cultures. Sesquiterpene cyclase activity was absent from control cell cultures but induced to a maximum within 10 hours of elicitor addition to the cell cultures. The cyclase activity remained elevated for an additional 30 hours before declining. In contrast, squalene synthetase activity was suppressed to less than 15% of that found in control cells within 7 hours of elicitor addition. Our results suggest that the channeling of isoprenoid intermediates, and especially farnesyl diphosphate, into sesquiterpenoids occurred by a coordinated increase in the sesquiterpene cyclase and a decrease in the squalene synthetase enzyme activities. A reexamination of the data pertaining to the transient induction of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity (EC 1.1.1.34) in elicitor-treated cells suggested that, while the reductase activity was necessary for sesquiterpenoid biosynthesis, it functioned more to maintain a sufficient level of intermediates between mevalonate and farnesyl diphosphate rather than as a rate limiting step controlling the synthesis rate of any one class of isoprenoids.     

Anti-inflammatory spiroaxane and drimane sesquiterpenoids from Talaromyces minioluteus (Penicillium minioluteum)

A new spiroaxane sesquiterpenoid talaminoid A (1) and two drimane sesquiterpenoid talaminoids B and C (2 and 3), together with four known compounds (4–7), were isolated from the solid culture broth of fungus Talaromyces minioluteum. The structures were determined by extensive 1D and 2D NMR and HRESIMS spectroscopic data analyses, and the absolute configuration of these new compounds were undoubtedly confirmed by X-ray crystal diffrations. Compound 1 is a rare spiroaxane sesquiterpenoid and the absolute configuration of spiroaxane sesquiterpenoid was determined for the first time. Compound 2 is the first drimane-type sesquiterpenoid containing both amino acid residue and butanediol group. Compounds 1, 4, and 5 showed significant suppressive effect on the production of NO on LPS induced BV-2 cells, with IC₅₀ values ranging from 4.97 to 7.81 μM. In addition, 1, 4, and 5 exhibited significant anti-inflammatory activities against the production of TNF-α and IL-6. Further immunofluorescence experiments revealed the mechanism of action to be inhibitory the NF- κB-activated pathway.     

Elicitor-inducible 3-hydroxy-3-methylglutaryl coenzyme a reductase activity is required for sesquiterpene accumulation in tobacco cell suspension cultures

Addition of cell wall fragments from Phytophthora species or cellulase from Trichoderma viride, but not pectolyase from Aspergillus japonicus, to tobacco (Nicotiana tabacum) cell suspension cultures induced the accumulation of the extracellular sesquiterpenoid capsidiol. Pulse-labeling experiments with [¹⁴C]acetate and [³H]mevalonate suggested that enzymatic steps preceding mevalonate were limiting capsidiol biosynthesis in the pectolyase-treated cell cultures. Treatment of the cell cultures with either Phytophthora cell wall fragments or cellulase induced 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) and sesquiterpene cyclase activities, enzymes of the sesquiterpene biosynthetic pathway, and phenylalanine ammonia lyase activity, an enzyme of the general phenylpropanoid pathway. Pectolyase treatment induced sesquiterpene cyclase and phenylalanine ammonia lyase activities, but not HMGR activity. These results corroborate the importance of inducible HMGR enzyme activity for sesquiterpene accumulation.     

Endophytic Bacterium-Triggered Reactive Oxygen Species Directly Increase Oxygenous Sesquiterpenoid Content and Diversity in Atractylodes lancea

Oxygenous terpenoids are active components of many medicinal plants. However, current studies that have focused on enzymatic oxidation reactions cannot comprehensively clarify the mechanisms of oxygenous terpenoid synthesis and diversity. This study shows that an endophytic bacterium can trigger the generation of reactive oxygen species (ROS) that directly increase oxygenous sesquiterpenoid content and diversity in Atractylodes lancea. A. lancea is a famous but endangered Chinese medicinal plant that contains abundant oxygenous sesquiterpenoids. Geo-authentic A. lancea produces a wider range and a greater abundance of oxygenous sesquiterpenoids than the cultivated herb. Our previous studies have shown the mechanisms behind endophytic promotion of the production of sesquiterpenoid hydrocarbon scaffolds; however, how endophytes promote the formation of oxygenous sesquiterpenoids and their diversity is unclear. After colonization by Pseudomonas fluorescens ALEB7B, oxidative burst and oxygenous sesquiterpenoid accumulation in A. lancea occur synchronously. Treatment with exogenous hydrogen peroxide (H₂O₂) or singlet oxygen induces oxidative burst and promotes oxygenous sesquiterpenoid accumulation in planta. Conversely, pretreatment of plantlets with the ROS scavenger ascorbic acid significantly inhibits the oxidative burst and oxygenous sesquiterpenoid accumulation induced by P. fluorescens ALEB7B. Further in vitro oxidation experiments show that several oxygenous sesquiterpenoids can be obtained from direct oxidation caused by H₂O₂ or singlet oxygen. In summary, this study demonstrates that endophytic bacterium-triggered ROS can directly oxidize oxygen-free sesquiterpenoids and increase the oxygenous sesquiterpenoid content and diversity in A. lancea, providing a novel explanation of the mechanisms of oxygenous terpenoid synthesis in planta and an essential complementarity to enzymatic oxidation reactions.     

Stress compounds from Nicotiana rustica inoculated with TMV

Three new sesquiterpenoid stress compounds, occidenol, occidentalol and trans-occidentalol, have been isolated from the leaves of Nicotiana rustica inoculated with TMV.     

Foetidin,a sesquiterpenoid coumarin from Ferula assa-foetida

A new sesquiterpenoid coumarin, foetidin, has been isolated from the roots of Ferula assa-foetida.     

A new bisabolane-type sesquiterpenoid from Curcuma domestica

Biochemical Systematics and Ecology, Volume x, Issue x, Pages x–x (dd mm yyyy). A new bisabolane-type sesquiterpenoid from Curcuma domestica. Takahiro Ishii, Hiroshi Matsuura, Kunimitsu Kaya, Charles Santhanaraju Vairappan.

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Highlights? Chemical constituents of lowland and highland Curcuma domestica of Borneo are reported. ? Three sesquiterpenes and three curcuminoids secondary metabolites were found in common. ? Highland populations contained two additional bisabolane-type sesquiterpenoid metabolites. ? Curcuma domestica is reported as a new source for novel compound, bisacurol B.     

(−)-Ent-12β-acetoxylongipin-2(10)-en-3-one,an Ent-longipinane derivative from the liverwort Marsupella aquatica

The structure of a new ent-longipinane type of sesquiterpenoid, (?)-ent-12β-acetoxylongipin- 2(10)-en-3-one from the liverwort Marsupella aquatica, has been elucidated by spectroscopic and chemical methods.     

Microbial transformation of antimalarial terpenoids

The fungal and bacterial transformation of terpenoids derived from plant essential oils, especially the sesquiterpenoid artemisinin from Artemisia annua, has produced several new candidate drugs for the treatment of malaria. Obtaining new derivatives of terpenoids, including artemisinin derivatives with increased antimalarial activity, is an important goal of research in microbial biotechnology and medicinal chemistry.     

Structures of melleolides B-D,three antibacterial sesquiterpenoids from Armillaria mellea

The structures of melleolides B-D, three new protoilludene sesquiterpenoid O-methylorsellinates isolated from a culture of Armillaria mellea, have been elucidated on the basis of chemical and spectral data.     

Eudesmane derivatives from cyperus corymbosus

From the rhizomes of Cyperus corymbosus a new eudesmane sesquiterpenoid, isocorymbolone, was isolated besides the known compounds corymbolone and (+)-α-cyperone. The structure of the new compound was established by means of spectroscopic methods.     

4-Hydroxydehydromyoporone from infected Ipomoea batatas root tissue

A new sesquiterpenoid, 4-hydroxydehydromyoporone, was isolated from Ceratocystis fimbriata— infected root tissue of Ipomoea batatas. We showed that it was a derivative of myoporane with one hydroxyl group at C-8 and one double bond at C-12 by spectroscopic comparison with known compounds.     

Stereostructure of curlone,a sesquiterpenoid of Curcuma longa rhizomes

From the crude drug ‘ukon’ (turmeric) (obtained from the rhizomes of Curcuma longa) a new sesquiterpenoid, curlone, has been isolated and shown as (6S)-2-methyl-6-[(1S)-4-methylene-2-cyclohexen-1-yl]-2-hepten-4-one on the basis of its spectral properties and its chemical conversion to the known (+)-ar-turmerone.     

Molecular cloning and expression of Hedychium coronarium farnesyl pyrophosphate synthase gene and its possible involvement in the biosynthesis of floral and wounding/herbivory induced leaf volatile sesquiterpenoids

Farnesyl pyrophosphate synthase (FPPS EC 2.5.1.10) catalyzes the production of farnesyl pyrophosphate (FPP), which is a key precursor for many sesquiterpenoids such as floral scent and defense volatiles against herbivore attack. Here we report a new full-length cDNA encoding farnesyl diphosphate synthase from Hedychium coronarium. The open reading frame for full-length HcFPPS encodes a protein of 356 amino acids, which is 1068 nucleotides long with calculated molecular mass of 40.7 kDa. Phylogenetic tree analysis indicates that HcFPPS belongs to the plant FPPS super-family and has strong relationship with FPPS from Musa acuminata. Expression of the HcFPPS gene in Escherichia coli yielded FPPS activity. Tissue-specific and developmental analyses of the HcFPPS mRNA and corresponding volatile sesquiterpenoid levels in H. coronarium flowers revealed that the HcFPPS might play a regulatory role in floral volatile sesquiterpenoid biosynthesis. The emission of the FPP-derived volatile terpenoid correlates with strong expression of HcFPPS induced by mechanical wounding and Udaspes folus-damage in leaves, which suggests that HcFPPS may have an important ecological function in H. coronarium vegetative organ.     

Apotrisporin: A major metabolite ofBlakeslea trispora

A major metabolite, an apotrisporoid and a sesquiterpenoid, has been identified in cultures ofBlakeslea trispora. It is apotrisporin: 2,4,4,-trimethyl-3-(5′-hydroxyl-3′-methylpenta-1′,3′-dienyl)-cyclohex-2-enone. Its inability to stimulate the development of zygophores is consistent with its putative origin as an inactivation product of trisporin.     

Sesquiterpene lactone from Salmalia malbarica

A new cadalane type sesquiterpenoid, 1, 6-dihydroxy-3-methyl-5-(1-methylethyl)-7-methoxy-8-carboxylic acid (8 → 1 lactone) has been isolated from a 50% aqueous methanolic extract of 6–9 months old cultivated roots of Salmalia malbarica (Bombax malbaricum).     

Raimondal,a new sesquiterpenoid from pigment glands of Gossypium raimondii

A new sesquiterpenoid isolated from pigment glands of leaves and immature bolls of Gossypium raimondii has been named raimondal and identified as 5-iso-propyl-2-methoxy-3-methyl-1,6,7-trihydroxy-8-naphthaldehyde. Raimondal was oxidized by ferric chloride to o-hemigossypolone (8-formyl-5-iso-propyl-3-methyl-6,7-dihydroxy-1,2-naphthoquinone) which was readily reduced to its hydroquinone, 2-hydroxyhemigossypol. Neither of the two oxidation products were detected in extracts from G. raimondii.     

Efficient phototrophic production of a high-value sesquiterpenoid from the eukaryotic microalga Chlamydomonas reinhardtii

The heterologous expression of terpene synthases in microbial hosts has opened numerous possibilities for bioproduction of desirable metabolites. Photosynthetic microbial hosts present a sustainable alternative to traditional fermentative systems, using freely available (sun)light and carbon dioxide as inputs for bio-production. Here, we report the expression of a patchoulol synthase from Pogostemon cablin Benth in the model green microalga Chlamydomonas reinhardtii. The sesquiterpenoid patchoulol was produced from the alga and was used as a marker of sesquiterpenoid production capacity. A novel strategy for gene loading was employed and patchoulol was produced up to 922±242 µg g⁻¹ CDW in six days. We additionally investigated the effect of carbon source on sesquiterpenoid productivity from C. reinhardtii in scale-up batch cultivations. It was determined that up to 1.03 mg L⁻¹ sesquiterpenoid products could be produced in completely photoautotrophic conditions and that the alga exhibited altered sesquiterpenoid production metabolism related to carbon source.