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Penispidins A-C (1-3), new aromatic sesquiterpenoids with two classes of rare carbon skeletons, were isolated from the endophytic fungus Penicillium virgatum HL-110. 1 represents the first example of a dunniane-type aromatic sesquiterpenoid, possessing a novel 4/6/6 tricyclic system, while (±)-2 and 3 have a 7,12-cyclized bisabolene skeleton, featuring a 3,4-benzo-fused 2-oxabicyclo[3.3.1]nonane central framework. Their structures were elucidated on the basis of spectroscopic methods, single-crystal X-ray diffraction, and ECD calculations. 1 inhibited hepatic lipid accumulation in HepG2 cells.

Curcuma longa (Turmeric) is a tropical herbaceous perennial plant of the family Zingiberaceae and contains curcuminoids, sesquiterpenoids and monoterpenoids as its major components. Given the broad range of activities that Curcuma longa possesses and also its use as a traditional epilepsy remedy, this review attempts to systematically review the experimentally proven activities of Curcuma longa and its bioactive components, which are related to the management of epileptic seizures. Using the PRISMA model, five databases (Google Scholar, PubMed, ScienceDirect, SCOPUS and SpringerLink) were searched using the keywords [“Curcuma longa” AND “Epilepsy”] and [“Curcuma longa” AND “Seizures”], leaving 34 articles that met the inclusion criteria. The present systematic review elaborated on the experimentally proven potential of Curcuma longa components, such as an aqueous extract of Curcuma longa itself, Curcuma longa oil and active constituents like curcuminoids and bisabolene sesquiterpenoids found in Curcuma longa with anti-seizure potential. Using human equivalent dose calculations, human treatment parameters were suggested for each component by analysing the various studies in this review. This review also determined that the principal components possibly exert their anti-seizure effect via the reduction of corticosterone, modulation of neurotransmitters signalling, modulation of sodium ion channels, reduction of oxidative DNA damage, reduction of lipid peroxidation, upgregulation of brain-derived neurotrophic factor (BDNF) and γ-aminobutyric acid (GABA) mediated inhibition. It is anticipated that this review will help pave the way for future research into the development of Curcuma longa and its neuroactive constituents as potential drug candidates for the management of epilepsy.

A new rearranged nitrogenous bisabolone-type sesquiterpene, halichonic acid B (1), was isolated from a marine sponge Axinyssa sp. together with halichonic acid (2) and (6R,7S)-7-amino-7,8-dihydro-α-bisabolene (3). The structure of 1 was determined by extensive NMR and MS analyses, revealing an unprecedented carbon framework, and its absolute configuration was elucidated by time-dependent density-functional theory (TDDFT)-based electronic circular dichroism (ECD) spectrum calculation. We propose that 1 and 2 may be biosynthesized in the same pathway, involving the reaction between farnesyl pyrophosphate and glycine, followed by cyclization.

Sesquiterpenes are a large variety of terpene natural products, widely existing in plants, fungi, marine organisms, insects, and microbes. Value-added sesquiterpenes are extensively used in industries such as: food, drugs, fragrances, and fuels. With an increase in market demands and the price of sesquiterpenes, the biosynthesis of sesquiterpenes by microbial fermentation methods from renewable feedstocks is acquiring increasing attention. Synthetic biology provides robust tools of sesquiterpene production in microorganisms. This review presents a summary of metabolic engineering strategies on the hosts and pathway engineering for sesquiterpene production. Advances in synthetic biology provide new strategies on the creation of desired hosts for sesquiterpene production. Especially, metabolic engineering strategies for the production of sesquiterpenes such as: amorphadiene, farnesene, bisabolene, and caryophyllene are emphasized in: Escherichia coli, Saccharomyces cerevisiae, and other microorganisms. Challenges and future perspectives of the bioprocess for translating sesquiterpene production into practical industrial work are also discussed.

Energy crisis and global climate change have driven an increased effort toward biofuel synthesis from renewable feedstocks. Herein, purple nonsulfur photosynthetic bacterium (PNSB) of Rhodobacter capsulatus was explored as a platform for high-titer production of a terpene-based advanced biofuel-bisabolene. A multilevel engineering strategy such as promoter screening, improving the NADPH availability, strengthening the precursor supply, suppressing the side pathways, and introducing a heterologous mevalonate pathway, was used to improve the bisabolene titer in R. capsulatus. The above strategies enabled a 35-fold higher titer of bisabolene than that of the starting strain, reaching 1089.7 mg/L from glucose in a shake flask. The engineered strain produced 9.8 g/L bisabolene with a yield of >0.196 g/g-glucose under the two-phase fed-batch fermentation, which corresponds to >78% of theoretical maximum. Taken together, our work represents one of the pioneering studies to demonstrate PNSB as a promising platform for terpene-based advanced biofuel production.

(+)-β-Sesquiphellandrene-12-oic acid (1) and 4-oxo-lanceolic acid (3), two new bisabolane derivatives; (+)-2-methyl-6[4-oxo-2-cyclohexen-1-yl]-2-(E)-heptenoic acid (4), a new nor-bisabolane derivative; (+)-(E)-exo-α-bergamoten-12-oic acid (5), a new bergamotene-type sesquiterpene; (2E,6E)-2,6-dimethyl-10-methylene-dodecatrienoic acid (6), a farnesene-type sesquiterpene reported for the first time as a natural product; and (–)-curcumen-12-oic acid (7), an aromatic bisabolene derivative that is new in Lauraceae, were obtained, together with lanceolic acid (2) and the terpenic lactone loliolide (8), from the leaves of Ocotea minarum. The chemical structures of these compounds were determined by 1D- and 2D-NMR and HRESIMS. Evaluated in vitro against standard strains of Candida species and Cryptococcus neoformans, compounds 1, 2, and 4-8 exhibited MIC values in the 50–100 μg mL–1 range.

Summary The natural plant product bisabolene serves as a precursor for the production of a wide range of industrially relevant chemicals. However, the low abundance of bisabolene in plants renders its isolation from plant sources non‐economically viable. Therefore, creation of microbial cell factories for bisabolene production supported by synthetic biology and metabolic engineering strategies presents a more competitive and environmentally sustainable method for industrial production of bisabolene. In this proof‐of‐principle study, for the first time, we engineered the oleaginous yeast Yarrowia lipolytica to produce α‐bisabolene, β‐bisabolene and γ‐bisabolene through heterologous expression of the α‐bisabolene synthase from Abies grandis, the β‐bisabolene synthase gene from Zingiber officinale and the γ‐bisabolene synthase gene from Helianthus annuus respectively. Subsequently, two metabolic engineering approaches, including overexpression of the endogenous mevalonate pathway genes and introduction of heterologous multidrug efflux transporters, were employed in order to improve bisabolene production. Furthermore, the fermentation conditions were optimized to maximize bisabolene production by the engineered Y. lipolytica strains from glucose. Finally, we explored the potential of the engineered Y. lipolytica strains for bisabolene production from the waste cooking oil. To our knowledge, this is the first report of bisabolene production in Y. lipolytica using metabolic engineering strategies. These findings provide valuable insights into the engineering of Y. lipolytica for a higher‐level production of bisabolene and its utilization in converting waste cooking oil into various industrially valuable products., To our knowledge, we have unprecedentedly verified the function of α‐bisabolene synthase, β‐bisabolene synthase and γ‐bisabolene synthase in Y. lipolytica and successfully employed the enzyme to synthesize bisabolene in Y. lipolytica for the first time. Our study reports for the first time the utilization of terpenoid efflux pumps to improve the secretion, and thus the production, of bisabolene in microbial cell factories. We demonstrated for the first time the conversion of waste cooking oil to bisabolene, which has important potential applications in waste management as well as economical and sustainable production of valuable bisabolene from waste feedstocks.

A new aromatic bisabolene-type sesquiterpenoid (7S, 8S)-8-hydroxysydowic acid (1), together with a pair of new bisabolene derivative (±)-(7R*, 10R*)-10-hydroxysydowic acid (2), as well as two known analogues hydroxysydonic acid (3) and sydowic acid (4) were isolated and identified from the endophytic fungal strain Aspergillus sydowii EN-434 that was obtained from the marine red alga Symphyocladia latiuscula. Their structures were elucidated by nuclear magnetic resonance (NMR), high resolution electrospray ionization mass spectroscopy (HRESIMS), and quantum chemical electronic circular dichroism (ECD) calculations as well as comparison of the data with literature reports. Compound 1 exhibited potent DPPH free radical scavenging activity with IC50 value of 113.5 μmol/L.

Commercial fragrances consist of several thousand natural and synthetic substances formulated in complex combinations. These ingredients are frequently blended at very low concentrations but they are typically lipophilic and a few of them (e.g., synthetic musks) have been detected in aquatic systems, albeit at low concentrations. Few fragrances have guideline in vivo data on bioaccumulation, so in silico modeling has been widely used to estimate bioconcentration factors (BCFs). This study used in vitro metabolism assays with trout S9 cell fractions and cryopreserved hepatocytes to improve estimates of fish BCFs and to test published methods for extrapolating in vitro metabolic rate data to whole fish and corresponding BCFs. These estimates for several chemicals were compared with new in vivo bioconcentration measurements and previously published data on fragrances and the insect repellent, DEET. In total, 17 (20 including isomers) fragrance chemicals (abalyn, amberwood, amboryl acetate, bisabolene, cedroxide, coniferan, elemol, givescone, maritima, precyclemone B, polysantol, sandela, sanjinol, santalex, timberol and vernaldehyde) and DEET were metabolized at various rates. Only three materials tested did not appear to undergo enzymatic degradation (caryophyllene oxide, galaxolide and ketone patchouli). Even relatively slow rates of metabolism had a large influence on bioconcentration estimates. This work adds valuable information to the evolving body of work supporting the use of in vitro determinations of hepatic clearance to improve modeled predictions of bioaccumulation. It can also be used directly to help prioritize testing of potential bioaccumulative chemicals or serve as a more economical method for screening these chemicals.

Four new chlorinated bisabolene-type sesquiterpenoids (1-4) were isolated during the phytochemical investigation of an acetone extract of the whole plant of Parasenecio rubescens. The structures of 1-4 were determined by analysis of their HRESIMS and NMR spectroscopic data, and the absolute configuration of 1 was established through single-crystal X-ray diffraction. All isolates were evaluated for their cytotoxicity against three cancer cell lines (B16 mouse melanoma, HepG2 human hepatocellular carcinoma, and MCF7 human breast adenocarcinoma), as well as their antimicrobial effects against Staphylococcus aureus, Escherichia coli, and Monilia albicans. As a result, compounds 1-4 displayed a certain degree of antimicrobial activities.

Boswellia sacra Flueck. oleoresin extract (frankincense) has traditionally been used in the treatment of different diseases, but there are no sufficient studies on its potential activity against periodontal pathogens. Therefore, antibacterial and antibiofilm activity of frankincense extract against Porphyromonas gingivalis clinical isolates were studied. The phytochemical composition of the volatile components of the extract was identified by GC-MS analysis revealing 49 compounds as trans-nerolidyl formate, cycloartenol acetate, ursenoic acid 3-oxomethyl ester, bisabolene epoxide, and kaur-16-ene. It decreased the growth and increased the leakage of nucleotides in 58.3% and 33.3% of isolates, respectively. Additionally, it reduced the extracellular polysaccharide production and the cell surface hydrophobicity in 41.67% and 50% of the isolates, respectively. Crystal violet assay revealed inhibition of biofilm formation by the tested isolates. Light microscope and scanning electron microscope were used to examine the biofilms and they confirmed the reduction of biofilm formation by frankincense extract. Downregulation of the genes linked to biofilm formation (fimA, hagA, and hagB) was observed using qRT-PCR after treatment with the frankincense extract. This study suggested that the frankincense extract could exhibit antibacterial and antibiofilm activity against P. gingivalis isolates. Thus, the frankincense extract could be used as a treatment approach for periodontitis.

The saturated hydrocarbon bisabolane is a diesel fuel substitute that can be derived from sesquiterpene precursors bisabolene or curcumene. These sesquiterpenes are generated from farnesyl diphosphate in reactions catalyzed by eponymous terpenoid cyclases, but they can also be generated by engineered terpenoid cyclases in which cyclization cascades have been reprogrammed by mutagenesis. Here, we describe the X-ray crystal structure determination of F95Q epi-isozizaene synthase (EIZS), in which the new activity of curcumene biosynthesis has been introduced and the native activity of epi-isozizaene biosynthesis has been suppressed. F95Q EIZS generates β- and γ-curcumene regioisomers with greater than 50% yield. Structural analysis of the closed active site conformation, stabilized by the binding of 3 Mg2+ ions, inorganic pyrophosphate, and the benzyltriethylammonium cation, reveals a product-like active site contour that serves as the cyclization template. Remolding the active site contour to resemble curcumene instead of epi-isozizaene is the principal determinant of the reprogrammed cyclization cascade. Intriguingly, an ordered water molecule comprises part of the active site contour. This water molecule may also serve as a final proton acceptor, along with inorganic pyrophosphate, in the generation of curcumene regioisomers; it may also contribute to the formation of sesquiterpene alcohols identified as minor side products. Thus, the substitution of polar side chains for nonpolar side chains in terpenoid cyclase active sites can result in the stabilization of bound water molecules that, in turn, can serve template functions in isoprenoid cyclization reactions.

The objective of this study was to determine the efficacy of supercritical carbon dioxide turmeric extract (STE) on the oxidative stability of perilla oil (PO), as measured by acid value (AV), peroxide value (PV) and oxidation induction time and compared to control and α‐tocopherol containing PO as a positive control. The antioxidant activity was determined by DPPH, ABTS and FRAPS assays and individual bioactive compounds identified by chromatography. STE demonstrated significantly higher antioxidant activity than the α‐tocopherol. The major components of the STE were identified as ar‐turmerone, turmerone, curlone and bisabolene by GC‐MS and curcumin and demethoxycurcumin by UPLC‐Q‐TOF MS. During thermal treatment at 65 °C for 24 h, the PO containing STE at different concentrations (100, 300, 500 and 1000 μg mL⁻¹) had significantly lower AV and PV, and at least 1.5 times higher induction period than those of the control and α‐tocopherol‐incorporated PO.

A newly identified bacterial (Z)-γ-bisabolene synthase was used for investigating the cyclisation mechanism of the sesquiterpene. Since the stereoinformation of both chiral putative intermediates, nerolidyl diphosphate (NPP) and the bisabolyl cation, is lost during formation of the achiral product, the intriguing question of their absolute configurations was addressed by incubating both enantiomers of NPP with the recombinant enzyme, which resolved in an exclusive cyclisation of (R)-NPP, while (S)-NPP that is non-natural to the (Z)-γ-bisabolene synthase was specifically converted into (E)-β-farnesene. A hypothetical enzyme mechanistic model that explains these observations is presented.

Background Mitigation of climate change requires that new routes for the production of fuels and chemicals be as oil-independent as possible. The microbial conversion of lignocellulosic feedstocks into terpene-based biofuels and bioproducts represents one such route. This work builds upon previous demonstrations that the single-celled carotenogenic basidiomycete, Rhodosporidium toruloides, is a promising host for the production of terpenes from lignocellulosic hydrolysates. Results This study focuses on the optimization of production of the monoterpene 1,8-cineole and the sesquiterpene α-bisabolene in R. toruloides. The α-bisabolene titer attained in R. toruloides was found to be proportional to the copy number of the bisabolene synthase (BIS) expression cassette, which in turn influenced the expression level of several native mevalonate pathway genes. The addition of more copies of BIS under a stronger promoter resulted in production of α-bisabolene at 2.2 g/L from lignocellulosic hydrolysate in a 2-L fermenter. Production of 1,8-cineole was found to be limited by availability of the precursor geranylgeranyl pyrophosphate (GPP) and expression of an appropriate GPP synthase increased the monoterpene titer fourfold to 143 mg/L at bench scale. Targeted mevalonate pathway metabolite analysis suggested that 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGR), mevalonate kinase (MK) and phosphomevalonate kinase (PMK) may be pathway bottlenecks are were therefore selected as targets for overexpression. Expression of HMGR, MK, and PMK orthologs and growth in an optimized lignocellulosic hydrolysate medium increased the 1,8-cineole titer an additional tenfold to 1.4 g/L. Expression of the same mevalonate pathway genes did not have as large an impact on α-bisabolene production, although the final titer was higher at 2.6 g/L. Furthermore, mevalonate pathway intermediates accumulated in the mevalonate-engineered strains, suggesting room for further improvement. Conclusions This work brings R. toruloides closer to being able to make industrially relevant quantities of terpene from lignocellulosic biomass.

Trachymene incisa subsp. incisa is an Australian endemic taxon that varies greatly in the abundance and length of the leaf trichomes. The essential oil composition of five populations of this subspecies, three corresponding to the typical glabrous form and two of the particularly hairy variant, has been analyzed in an attempt to determinate if that variability is also reflected in their composition. The oils have been extracted by hydrodistillation and analyzed by Gas Chromatography (GC) and Gas Chromatography coupled to Mass Spectrometry (GC–MS). The essential oils of T. incisa subsp. incisa were characterized by the high amount of sesquiterpenes that were the major fraction. The sesquiterepene hydrocarbons were significantly higher in the hairy variant in comparison to the glabrous one. According to the main compound, three different chemotypes were found: I.—β-selinene + bicyclogermacrene and II.—γ-bisabolene + α-pinene for the typical glabrous variant and III.—bicyclogermacrene + β-caryophyllene for the hairy variant.

The vapor pressures and vaporization enthalpies of the major components of opopanax oil, a medicinal that has been in use since biblical times, are evaluated by correlation gas chromatography. Two sets of hydrocarbon standards are used, n-alkanes and a mixture of cyclic and polycyclic hydrocarbons. Two of the oil’s sesquiterpene components, evaluated in a previous study, were used both as standards and also as targets. Their use as targets was in an effort to confirm both their identity in the oil and the reproducibility of their properties. All correlations produce reproducible vaporization enthalpies and vapor pressures at ambient temperatures. At elevated temperatures, the use of the two different sets of standards resulted in some divergence in vapor pressure. Experiments are described aimed at attenuating this divergence. The results are compared to existing data.

In spite of mature processing techniques and the large availability of raw materials having sufficient quality, there is no study about the relation between the chemical components and material pretreatment method of ginger oleoresin extracted by supercritical fluid CO2. In order to obtain high content of effectual compositions in ginger oleoresin, Ginger was dehydrated by vacuum freeze–drying method and extracted by Supercritical CO2. The components in the extracts were quantified and qualified using Gas Chromatography-Mass Spectrometry method. Same studies were also done with the sun-dried ginger. The results show that the main components are different in the two samples. In the freeze-dried sample, zingerone is the highest substance at 19.472%, pungency compounds are the main components at53.920 %, and the content of sesquiterpene is 20.041%. By comparison, in the sun-dried sample, the content of bisabolene is 26.328% that is the highest, the pungency components are 24.832%, and the main components are sesquiterpene at 56.627%.

Metabolic engineering of microorganisms to produce sustainable chemicals has emerged as an important part of the global bioeconomy. Unfortunately, efforts to design and engineer microbial cell factories are challenging because design-built-test cycles, iterations of re-engineering organisms to test and optimize new sets of enzymes, are slow. To alleviate this challenge, we demonstrate a cell-free approach termedin vitroPrototyping and Rapid Optimization of Biosynthetic Enzymes (or iPROBE). In iPROBE, a large number of pathway combinations can be rapidly built and optimized. The key idea is to use cell-free protein synthesis (CFPS) to manufacture pathway enzymes in separate reactions that are then mixed to modularly assemble multiple, distinct biosynthetic pathways. As a model, we apply our approach to the 9-step heterologous enzyme pathway to limonene in extracts fromEscherichia coli. In iterative cycles of design, we studied the impact of 54 enzyme homologs, multiple enzyme levels, and cofactor concentrations on pathway performance. In total, we screened over 150 unique sets of enzymes in 580 unique pathway conditions to increase limonene production in 24 hours from 0.2 to 4.5 mM (23 to 610 mg/L). Finally, to demonstrate the modularity of this pathway, we also synthesized the biofuel precursors pinene and bisabolene. We anticipate that iPROBE will accelerate design-build-test cycles for metabolic engineering, enabling data-driven multiplexed cell-free methods for testing large combinations of biosynthetic enzymes to inform cellular design.TOC FigureHighlightsApplied the iPROBE framework to build the nine-enzyme pathway to produce limoneneAssessed the impact of cofactors and 54 enzyme homologs on cell-free enzyme performanceIteratively optimized the cell-free production of limonene by exploring more than 580 unique reactionsExtended pathway to biofuel precursors pinene and bisabolene

Metabolic engineering of microorganisms to produce sustainable chemicals has emerged as an important part of the global bioeconomy. Unfortunately, efforts to design and engineer microbial cell factories are challenging because design-build-test cycles, iterations of re-engineering organisms to test and optimize new sets of enzymes, are slow. To alleviate this challenge, we demonstrate a cell-free approach termed in vitro Prototyping and Rapid Optimization of Biosynthetic Enzymes (or iPROBE). In iPROBE, a large number of pathway combinations can be rapidly built and optimized. The key idea is to use cell-free protein synthesis (CFPS) to manufacture pathway enzymes in separate reactions that are then mixed to modularly assemble multiple, distinct biosynthetic pathways. As a model, we apply our approach to the 9-step heterologous enzyme pathway to limonene in extracts from Escherichia coli. In iterative cycles of design, we studied the impact of 54 enzyme homologs, multiple enzyme levels, and cofactor concentrations on pathway performance. In total, we screened over 150 unique sets of enzymes in 580 unique pathway conditions to increase limonene production in 24 h from 0.2 to 4.5 mM (23–610 mg/L). Finally, to demonstrate the modularity of this pathway, we also synthesized the biofuel precursors pinene and bisabolene. We anticipate that iPROBE will accelerate design-build-test cycles for metabolic engineering, enabling data-driven multiplexed cell-free methods for testing large combinations of biosynthetic enzymes to inform cellular design.

Cyanobacteria and microalgae are attractive phototrophic host systems for climate-friendly production of fuels and other high-value chemicals. The biosynthesis of an increasing diversity of industrially relevant compounds such as terpenoids has been demonstrated in recent years. To develop economically feasible and sustainable process designs, major challenges still remain regarding intracellular carbon partitioning, specific metabolic pathway activities and efficient cultivation strategies. Here, we present a technical study on comparative characteristics of sesquiterpene and sesquiterpene alcohol accumulation in engineered strains ofSynechocystissp. PCC 6803 (substrain GT-U) under different growth conditions and cell densities. This study particularly focuses on the basic applicability of a commercial High Density Cultivation platform in the presence of a dodecane overlay, which serves as a standardin-situextractant and sink for various hydrophobic biochemicals. Significantly, the presented data demonstrate high volumetric productivities of (E)-α-bisabolene under high-density conditions that are more than two orders of magnitude higher than previously reported for cyanobacteria. Operating in a two-step semi-batch mode over a period of eight days, average final volumetric titers of 179.4 ± 20.7 mg * L−1were detected. Likewise, the sesquiterpene alcohols (-)-patchoulol and (-)-α-bisabolol accumulated to many times higher levels in high density cultivation than under standard batch conditions, with final titers of 17.3 ± 1.85 mg * L−1and 96.3 ± 2.2 mg * L−1, respectively. In contrast, specific product accumulation (mg * L−1* OD750−1) was compromised particularly for bisabolene in the high density system during phases of high biomass accumulation rates. Volumetric productivities were high during linear growth at high densities, distinctly outperforming standard batch systems. While the presented data highlight the benefits of high-density strategies for highly efficient phototrophic terpenoid production, they further point at the presence of major metabolic bottlenecks for engineered terpenoid biosynthesis and the requirement for systematic and/or targeted strategies to sustainably redirect inherent carbon fluxes in cyanobacteria. Together, our data provide additional insights into growth- and density-related effects on the efficiency of product accumulation, introducing low-scale High Density Cultivation as a rapid and efficient platform for screening of heterologous terpenoid production in cyanobacteria.

Bisabolene-type sesquiterpenoids, which have multiple bioactivities, including anticancer activity, are one of the main groups of compounds in the essential oil extracted from Santalum album L. and other Santalum species. Bisabolene synthetase (SaBS) is a key enzyme for the synthesis of bisabolene in S. album, but the regulation of the SaBS gene&rsquo, s expression is poorly understood. In this study, a 1390-bp promoter sequence of the SaBS gene was isolated from the leaves of six-year-old S. album. A bioinformatics analysis showed that certain environment stresses and phytohormone-activated cis-acting elements were distributed in different regions of the SaBS promoter (PSaBS). Transgenic Arabidopsis carrying full-length PSaBS had significantly higher &beta, glucuronidase (GUS）activity than the untreated control after treatment with salicylic acid (SA), suggesting that PSaBS is a SA-inducible promoter. Histochemical GUS staining and GUS fluorometric assays of transgenic Arabidopsis showed that the GUS activity directed by PSaBS was mainly expressed in stem tissue, followed by leaves and flowers. Moreover, different regions of PSaBS showed significantly different GUS activity. A 171-bp fragment upstream of the transcriptional initiation codon (ATG) is the core promoter region of PSaBS. Our results provide insight into and a greater understanding of the transcriptional regulation mechanism of the SaBS gene, which could serve as an alternative inducible promoter for transgenic plant breeding.

The Mediterranean flora is characterized by an abundance of aromatic plants. These plants make a significant contribution to the Mediterranean ecological communities both in terms of species numbers and biomass. In this area, Teucrium is a polymorphic and cosmopolitan genus of perennial plants, the largest of the Lamiaceae family, which comprises more than 300 species, of which almost 50 are native to Europe. The members of this genus are commonly known as germanders: they are bitter, astringent and antirheumatic herbs, utilized as antispasmodic agents for gastric ulcer and intestinal inflammation, as diuretic, antiseptic, antipyretic and antihelmintic agents. Moreover, they were also used in food preparation. Teucrium species are rich in essential oils and the presence of various biologically active monoterpenoid and sesquiterpenoid with several biological activities has been reported. This review summarizes the chemical features of the essential oils of Teucrium species reported in literature: it provides an overview on their compositions with particular reference to the more abundant components and to the reported biological activities. Teucrium polium, T. chamaedrys, T. flavum and T. capitatum are the most studied taxa concerning the volatile oil composition. Moreover, it is possible to distinguish different types of essential oils, characterized by the prevalence of a specific class of compounds: in particular, we recognized volatile oils rich in caryophyllene and caryophyllene derivatives, α- and β-pinene, germacrene B and D, α-, γ-, δ-cadinene and/or cadinol, limonene, and bisabolol/bisabolene. This richness in chemodiversity confers to the essential oils from Teucrium species distinctive biological properties and applications, often confirming the traditional and folk uses of this genus. This review testifies the high great variability of Teucrium essential oils and shows that the possible factors responsible for the chemical polymorphism of essential oils and consequently for their bioactivity need further investigation.

Ginger (Zingiber officinale Rosc.) is a major rhizome spice and medicinal crop and belongs to the family Zingiberaceae. The spicy gingerols usually occur in oil cells of the rhizome as pungent yellow oil but can also form a crystalline solid with low melting point. Further important aroma compounds are the sesquiterpenes bisabolene, zingiberene, zingiberol, sesquiphellandrene and curcurmene, which contribute in moderate quantity to the flavour of ginger. Ginger plays an important role in Indian Ayurvedic medicine as a folk remedy to promote cleaning of the body through perspiration, to calm nausea and to stimulate treatments of colds. Ginger is a clonally propagated crop. Therefore, this species is bred through introduction, selection, mutation and polyploidy breeding. But also biotechnological approaches are being used for improving the ginger cultivars in India and other parts of world.

The inducible accumulation of desired products via in vitro cultures provides an experimental system for researching secondary metabolism in woody plants. This system is convenient, because environmental conditions can be strictly controlled. This is particularly important for East Indian sandalwood (Santalum album L.), a tree with desired sandal oil products that are restricted to the heartwood. In this study, we established a method to induce and proliferate callus from sandalwood shoot explants. Thidiazuron (TDZ) (0.2–1.5 mg/l) could induce the formation of callus, which proliferated rapidly within a month following three successive subcultures in liquid shake culture on Murashige and Skoog (MS) basal medium supplemented with 0.8–1.0-mg/l TDZ. Callus cultured in this liquid medium for 7 days was co-cultured with fungi, either Colletotrichum gloeosporioides or Penidiella kurandae. Gas chromatography–mass spectrometry (GC–MS) analysis of the solvent extract by chemical anhydrous diethyl ether of callus co-cultured with C. gloeosporioides showed the presence of santalenes and bisabolene, which are the precursors of santalol. However, another fungus, P. kurandae, could not induce santalenes or bisabolene. This study provides an opportunity to further studies on the santalene and bisabolene biosynthetic signaling pathway and the fungal endophyte–plant interaction in sandalwood.

Currently, algal fuel research has commenced to shift toward genetically engineered mutants able to express and excrete desired products directly into the culture. In this study, a mutant strain of Chlamydomonas reinhardtii, engineered for bisabolene (alternative biodiesel) excretion, was cultivated at different illumination and temperatures to investigate their effects on cell growth and bisabolene production. Moreover, a kinetic model was constructed to identify the desirable conditions for biofuel synthesis. Three original contributions were concluded. First, this work confirmed that bisabolene was partially synthesized independently of biomass growth, indicating its feasibility for continuous production. Second, it was found that while bisabolene synthesis was independent of light intensity, it was strongly affected by temperature, resulting in conflicting desirable conditions for cell growth and product synthesis. Finally, through model prediction, optimal operating conditions were identified for mutant growth and bisabolene synthesis. This study therefore paves the way toward chemostat production and process scale-up.

The eco-friendly management of mosquitoes with novel and effective larvicides and oviposition deterrents is a crucial challenge to prevent outbreaks of mosquito-borne diseases. However, most of the herbal formulations tested in these years showed LC50 values higher of 40 ppm, and significant oviposition deterrent activity only when tested at relatively higher doses (> 50 μg/ml). Herein, we studied the chemical composition of the Galinsoga parviflora essential oil (EO). This plant is an annual herb native to South America naturalized all over the world. We tested the EO larvicidal and oviposition deterrent action on 6 mosquito species. Totally 37 compounds were identified in the EO of G. parviflora by GC and GC-MS analyses. The major constituent was (Z)-γ-bisabolene (38.9%). The G. parviflora EO and (Z)-γ-bisabolene showed acute toxicity on An. stephensi (LC50 = 31.04 and 2.04 μg/ml, respectively), Ae. aegypti (LC50 = 34.22 and 2.26 μg/ml, respectively), Cx. quinquefasciatus (LC50 = 37.10 and 2.47 μg/ml, respectively), An. subpictus (LC50 = 40.97 and 4.09 μg/ml, respectively), Ae. albopictus (LC50 = 45.55 and 4.50 μg/ml, respectively) and Cx. tritaeniorhynchus (LC50 = 49.56 and 4.87 μg/ml, respectively) larvae. Furthermore, the oviposition deterrent potential of the G. parviflora EO and (Z)-γ-bisabolene was studied on six mosquito vectors, showing that 25 μg/ml of (Z)-γ-bisabolene led to an Oviposition Activity Index lower of − 0.79 in all tested mosquito vectors. Overall, all larvicidal LC50 values estimated for (Z)-γ-bisabolene were lower than 5 μg/ml. This result far encompasses current evidences of toxicity reported for the large majority of botanical products currently tested against mosquito young instars, allowing us to propose this compound as an highly effective mosquito larvicide and oviposition deterrent.

A new compound, halichonic acid (1), was isolated from a marine sponge Halichondria sp., together with (6R,7S)-7-amino-7,8-dihydro-α-bisabolene (2). The structure of 1 was elucidated by spectroscopic analysis and ECD spectrum calculation to be a rearranged bisabolene-type sesquiterpene having a 3-azabicyclo[3.3.1]nonene moiety. Compound 2 was cytotoxic against HeLa cells with an IC50 value of 50 μM, whereas 1 did not show cytotoxicity even at 50 μM. It is possible that 1 is biosynthesized from farnesyl pyrophosphate and glycine, with rearrangement.

Boswellia sacra resin (BS) is well known for its antimicrobial, antioxidant, and anti-inflammatory properties and hence could be used as an efficient dietary feed additive for ruminants during the transition period. This study was conducted to determine the effects of BS by characterizing the ruminal fermentation, nutrient digestibility, milk production, and energy status of early lactating goats fed different levels of BS. A total of 21 late pregnant Nubian goats (initial body weight 26.3 ± 3.9 kg) were stratified using a completely randomized design to the following three dietary treatments (n = 7 does/treatment): control (without supplementation of BS) and BS treatments; low BS and high BS received 2 and 4 g BS/day/animal, respectively. The treatments were started 2 weeks before parturition and continued till 7 weeks postpartum. Constituents with high biological activity (e.g., bisabolene, limonene, α-pinene, and α- and β-amyrins) were the most abundant components found in experimental BS. Linear reductions (P

Essential oils are considered of great efficacity regarding the various beneficial effects on human health. The aim of this study was to analyze the composition and to investigate the antiviral, antioxidant and antimicrobial effects of wild Daucus virgatus (Poir.) Maire from Tunisia. The essential oil obtained by hydrodistillation air-dried D. virgatus in a Clevenger type apparatus was analyzed using gas chromatography and mass spectrometry (GC-FID and GC–MS). Fifty-two components representing 92.4% of the total constituents were identified. Methyl eugenol (33.0%) and β -bisabolene (13.2%) were the most predominant components. The antibacterial and antifungal activities against the pathogens: Pseudomonas aeruginosa, Bacillus subtilis, Bacillus anthracis, Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis and Candida albicans were investigated using the agar diffusion and broth microdilution methods. The lowest minimum inhibitory concentration was 625 μg mL −1 against both B. subtilis and C. albicans . Investigation of the in vitro antiviral activity of the essential oil, ethylacetate and methanolic extracts was tested against Coxsackievirus B (CV-B) , a small nonenveloped single-stranded and positive-sense RNA virus of the Picornaviridae family that produce central nervous system disease frequently with acute syndromes. The reduction of viral infectivity of the extracts was determined by the plaque reduction assay. The cytotoxicity test demonstrated moderate cytotoxic effects of the crude extracts on the HEp-2 cell line. Fifty percent cytotoxic concentrations (CC 50 ) are 543.35 and 372.7 μg mL −1 for ethylacetate and methanol respectively. Furthermore, ethylacetate and methanol extracts exhibited significant inhibitory effects against CV-B4 virus with IC 50 vales of 98.16 and 60.08 μg mL −1 respectively.

The constituents of essential oils isolated by hydro-distillation of the aerial parts of six Ocimum species were examined by Gas Chromatography-Mass Spectrometry (GC-MS). Highest percentage of essential oils (dry weight basis) was found in O. kilimandscharium (2.47 %) and lowest in O. tenuiflorum green type (0.65 %). On fresh weight basis, the percentage of essential oils was highest in O. b. var. glabratum (0.76 %) and lowest in O. viride (0.001 %). A total of eight major components were identified. The oil of O. b. var. basilicum contained, as main components, methyl chavicol and linalool. Major compound in O. b. var. pilosum was methyl chavicol; in O.b. var. glabratum, O. b. var. thyrsiflora and O. b. var. difforme was linalool; β cedrene and linalool in O. b. var. purpurascens; methyl chavicol in O. citriodorum; camphor and 1,8-cineole in O. kilimandascharium; β bisabolene and 1,8-cineole in O. viride; eugenol in O. tenuiflorum, and eugenol and methyl eugenol in O. gratissimum.

Origanum vulgare L., recognized throughout the world as a popular medicinal and flavoring herb, contains a wide array of medicinally active components, including phenolic glucosides, flavonoids, tannins, sterols and high amounts of terpenoids. Especially the latter are often extracted by hydrodistillation resulting in the so-called essential oil that is rich in monoterpenes (e.g. carvacrol, thymol, linalyl acetate) and/or sesquiterpenes (e.g. ( E ) -β- caryophyllene, germacrene D, bicyclogermacrene, β -caryophyllene oxide). Water stresses in the arid and semiarid regions of the world severely affect growth and productivity of oregano. To determine the variation in essential oil and gene expression pathway of Iranian oregano under prolonged water stress, two native subspecies of O. vulgare (subsp. virens and subsp. gracile ) were studied. The plants, grown in pots, were subjected to three water stress conditions, i.e. no stress, mild stress (60± 5% FMC) and moderate stress (40± 5% FMC). The studied subspecies exhibited significant differences in essential oil content, compositions, and patterns of gene expression under water stress conditions. The essential oil of O. vulgare subsp. gracile was rich in the phenolic monoterpene carvacrol (46.86–52.07%), whereas the sesquiterpene hydrocarbon ( Z )- α -bisabolene (39.17–42.64%) was the major constituent in the oil of O. vulgare subsp. virens . Both the mild and moderate water stresses significantly increased the essential oil content of O. vulgare subsp. gracile , but did not significantly change the essential oil content of O. vulgare subsp. virens nor the level of carvacrol and ( Z )- α -bisabolene in the investigated subspecies. Interestingly, the amount of ( E )- β -caryophyllene in O. vulgare subsp. virens was significantly increased under water stress conditions. Gene expression studies supported the above findings and demonstrated that there are two different pathways affecting the biosynthesis of the terpenoid precursors geranyl pyrophosphate (GPP) and farnesyl pyrophosphate (FPP). In O. vulgare subsp. gracile , HMGR, Ovtps 2 and CYP71D180 transcript were up-regulated under mild and moderate water stress conditions. Transcription of FPPS was apparently down-regulated in water-stressed O. vulgare subsp. gracile . Investigation of terpene synthases expression levels in oregano subspecies demonstrated that Ovtps 2 and Ovtps 6 controlled the concentration of carvacrol and ( E )- β -caryophyllene in oregano essential oils, respectively.

A pair of novel sesquiterpenoids, (8R)-alismanoid A (1a) and (8S)-alismanoid A (1b), possessing an unprecedented 1,2-seco bisabolene carbon skeleton, were isolated from rhizomes of Alisma orientale together with two new sesquiterpenoids: a 15-nor guaiane alismanoid B (2) and alismanoid C (3). Their structures were elucidated by 1D and 2D NMR, HRESIMS, electronic circular dichroism (ECD), and theoretical calculations, and a plausible biosynthetic pathway for compound 1 is discussed. Meanwhile, compounds 1–3 were investigated for their protective effects on H2O2-induced damage in human dopaminergic neuroblastoma cells (SH-SY5Y), and compounds 1b, 2, and 3 showed significantly protective activities at a certain concentration. Further, the action mechanism of compound 3 was proved to be through inhibition of H2O2-induced apoptosis in SH-SY5Y cells. Herein, these results suggest that sesquiterpenoids are potential candidate drugs for treating Parkinson's disease induced by reactive oxygen species.

Mekhadmi NE, Ramdani M, Lograda T, Chalard P, Figueredo G. 2019. Chemotypes and antibacterial activity of Matricaria pubescens of Algeria. Biodiversitas 20: 3170-3179. The objectives of this study were to determine the chemical composition and to evaluate the antibacterial activity of Matricaria pubescens L. essential oils from 14 sampling locations in southern Algeria. The extraction of essential oils was carried out by the hydro-distillation; the analysis of chemical composition of essential oil was carried out by GC-MS. Antibacterial activity was performed by disc diffusion method at the undiluted and diluted essential oil (1/1, 1:2, 1:4 and 1:8 v:v of DMSO) against eight species of bacteria (Bacillus cereus, Escherichia coli, Klebsiella pneumoniae, Listeria innocua, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella enterica and Staphylococcus aureus). The results showed that the average yields of essential oils were 0.4 ± 0.27%. A total of 34 components were identified, averaging 99.22 ± 0.8% of the total oils. The main components were β-ocimene (Z) (47.41 ± 13.67%), α-pinene (19 ± 7.36%), β-bulnesene (4.96 ± 10.6%), ocimene-allo (4.46 ± 1.3%), 1-phenyl-penta-2,4-diyne (4.06 ± 5.7%), limonene (3.84 ± 1.8%), caryophyllene oxide (2.36 ± 3.6%) and γ-bisabolene-E (2.08 ± 4.3%). Based on the UPGMA cluster analysis, there were two distinct clades of M. pubescens. This species has a chemical polymorphism with different chemotypes marked in nature. There were four chemotypes identified in the essential oil of M. pubescens in the region studied. The essential oil of M. pubescens has antibacterial activity against eight bacteria tested in a concentration-dependent manner.

The original version of this article unfortunately contained a mistake. Under the heading "Insects" in "Methods and Materials" the sentence "A colony of N. viridula originated with field collections near Tifton, Georgia, USA" is incorrect.

Chemical investigation of the aerial parts of Mikania micrantha led to the isolation of eight sesquiterpenoids and ten diterpenoids, including five cadinane sesquiterpenoids (1−5), three bisabolene sesquiterpenoids (6 − 8), nine ent-kaurane diterpenoids (9–17), and an abietane diterpenoid (18). Among them, 1 − 3 are new and feature a rare lactone or furan ring derived from C-6 isopropyl group side chain. Compound 18 was isolated from genus Mikania for the first time, and was also the first example of abietane-type diterpenoids from this plant. Their structures were elucidated on the basis of extensive spectroscopic analyses (1D and 2D NMR, HRESIMS, and ECD). All compounds were examined for their inhibitory effects on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7 macrophage cells, and compound 18 exhibited pronounced inhibition on NO production (IC50 = 11.04 µM), being comparable to the positive control, quercetin (IC50 = 11.15 µM).

Carrot seed essential oil is a common fragrance component in cosmetics and perfumes and a flavor ingredient in different categories of food products. It is also a major source of sesquiterpene alcohol carotol. In this work we aimed to compare carotol content in commercially available (Moroccan and French) and hydrodistilled (Polish) wild carrot seed essential oils ( Daucus carota L. ssp. carota ) and to evaluate cytotoxicity of these essential oils, as well as isolated carotol. The chemical composition of studied essential oils was analysed by gas chromatography- mass spectrometry. Cytotoxicity tests were performed on green monkey kidney (VERO) and human pharynx squamous cell carcinoma (FaDu) cell lines. Carotol and other terpenoids were isolated using high performance counter current chromatography technique in the reversed phase mode, with a mixtures of n -hexane/acetonitrile/ tetr -butyl methyl ether (1:1:0.1 and 2:1:0.1 v/v). Carotol was the main constituent in three carrot seed essential oils of different origin amounting 19–33% of the sum of compounds. α-Pinene, sabinene, myrcene, limonene, geranyl acetate, bisabolene, cayophyllene oxide and daucol were identified as other main compounds. Separation of 280 mg of carrot seed essential oil yielded carotol (20 mg) and other terpenoids: α-pinene, sabinene, limonene, geranyl acetate, caryophyllene oxide, and daucol with the purity in the range of 80–99%. The applied method enabled isolation of all main constituents of essential oil in the single run. The comparison of cytotoxicity of tested essential oils on VERO and FaDu cell lines indicated that Moroccan and French essential oils have similar cytotoxicity (35.3–46.1 μg/mL), while Polish essential oil showed lower cytotoxic effect (70.9–96.3 μg/mL). Carotol being the main constituent of all tested essential oils showed moderate cytotoxicity on both tested cell lines without any selectivity.

One new perhydrobenzannulated 5,5-spiroketal sesquiterpene, pleurospiroketal F (1), as well as six new modified bisabolene sesquiterpenes pleurotins A–F (2–7) were isolated from solid-state fermentation of Pleurotus citrinopileatus. The structures of compounds 1–7 were determined by NMR and MS spectroscopic analysis. The absolute configuration of 1 was determined by X-ray diffraction analysis, while the absolute configurations of 3–7 were assigned using the in situ dimolybdenum circular dichroism method and circular dichroism data comparison. Protein tyrosine phosphatase 1B plays a crucial role as a negative regulator of the insulin-dependent signal cascades. Therefore, the protein tyrosine phosphatase 1B inhibitor can be used for treating type 2 diabetes mellitus and obesity. Compounds 2 and 6 showed moderate inhibitory effects on protein tyrosine phosphatase 1B with IC50 s of 32.1 µM and 30.5 µM, respectively. The kinetic study confirmed compound 2 to be a noncompetitive inhibitor. Compounds 1–7 did not show cytotoxic activity against cancer cell lines (IC50 > 50 µM).

The vaporization enthalpies and vapor pressures of (−) β-elemene and (−) β-bisabolene are evaluated by correlation gas chromatography using two sets of standards, a mixture of various cyclic hydrocarbons and a series of n-alkanes. Both compounds are sesquiterpenes found in a variety of plants. β-Elemene has been used in China for the treatment of a variety of cancers. β-Bisabolene is used as a flavoring agent and it has also been shown to exhibit cytotoxicity in breast cancer cell lines, both in vitro and in vivo. Vaporization enthalpies of [(66.8 ± 3.0) and (73.9 ± 3.0)] kJ·mol−1 and vapor pressures of [(3.1 ± 1.0) and (0.72 ± 0.2)] Pa at T = 298.15 K have been evaluated for (−) β-elemene and (−) β-bisabolene, respectively. Vapor pressures were also evaluated as a function of temperature from T = (298.15–450) K and fit to a second order polynomial.

Cyanobacterial biofuels have the potential to reduce the cost and climate impacts of biofuel production because primary carbon fixation and conversion to fuel are completed together in the cultivation of the cyanobacteria. Cyanobacterial biofuels, therefore, do not rely on costly organic carbon feedstocks that heterotrophs require, which reduces competition for agricultural resources such as arable land and freshwater. However, the published product titer achieved for most molecules of interest using cyanobacteria lag behind what has been achieved using yeast and Escherichia coli (E. coli) cultures. In Synechocystis sp. PCC 6803 (S. 6803), we attempted to increase the product titer of the sesquiterpene, bisabolene, which may be converted to bisabolane, a possible diesel replacement. We tested 19 strains of genetically modified S. 6803 with five different codon usage sequences of the bisabolene synthase from the grand fir tree (Abies grandis). At least three ribosome binding sites (most designed using the RBS Calculator) were tested for each codon usage sequence. We also tested strains with and without the farnesyl pyrophosphate synthase gene from E. coli. Bisabolene titers after five days of growth in continuous light ranged from un-detected to 7.8 ​mg/L. Bisabolene synthase abundance was measured and found to be well correlated with titer. Select strains were also tested in 12:12 light:dark cycles, where similar titers were reached after the same amount of light exposure time. One engineered strain was also tested in photobioreactors exposed to a simulated outdoor light pattern with maximum light intensity of 1600 ​μmol photons m−2 s−1. Here, the bisabolene titer reached 22.2 ​mg/L after 36 days of growth. Dramatic improvements in our ability to control gene expression in cyanobacteria such as S. 6803, and the co-utilization of additional metabolic engineering methods, are needed in order for these titers to improve to the levels reported for engineered E. coli., Highlights • Measured bisabolene synthase expression and bisabolene titer for 19 strains of engineered Synechocystis sp. PCC6803. • Varied ribosome binding sites and codon usage of bisabolene synthase to achieve 7.8 ​mg/L after five days. • When grown in photobioreactors with simulated outdoor light:dark cycles, the bisabolene titer reached 22.2 mg/L after 36 days.

Terpenoids constitute a class of compounds with remarkable potential for pharmaceutical, fragrance, specialty chemical, and biofuel applications. However, their industrial production is limited by their rarity within their native plant hosts, creating considerable interest in microbial hosts capable of manufacturing terpenoids. To reduce production costs, nondestructive product recovery from these microbial hosts is preferred, and is achievable using a hydrophobic organic overlay. Our prior research has indicated that oxidized fatty acyl products may permeate faster through host membranes, increasing overall biorefinery productivity. To test this hypothesis for terpenoids, we computed membrane permeabilities of conventional terpenoid target products (e.g., limonene, bisabolene, farnesene) and related oxidized compounds through molecular dynamics simulations. These simulations indicate that terpenoid product permeabilities from cytosol to overlay are oxidation independent, as increases in membrane extraction efficiency due to product oxidation are proportionally offset by decreases in the membrane crossing rate if the membrane and organic phase are in close contact. However, if aqueous extraction is required, oxidation will accelerate the slow product extraction from the membrane. Experimental toxicity assays performed indicated that most terpenoids tested were tolerated by microbial hosts, although exposure to oxidized terpenes often retarded microbial growth compared with conventional terpenes. Thus, terpenoid oxidation is not expected to significantly increase or decrease the extraction productivity in an industrial setting where cells are in close contact, unlike the previously studied fatty acyl products.

Background Aviation fuels are an important target of biofuels research due to their high market demand and competitive price. Isoprenoids have been demonstrated as good feedstocks for advanced renewable jet fuels with high energy density, high heat of combustion, and excellent cold-weather performance. In particular, sesquiterpene compounds (C15), such as farnesene and bisabolene, have been identified as promising jet fuel candidates. Results In this study, we explored three sesquiterpenes—epi-isozizaene, pentalenene and α-isocomene—as novel jet fuel precursors. We performed a computational analysis to calculate the energy of combustion of these sesquiterpenes and found that their specific energies are comparable to commercial jet fuel A-1. Through heterologous MVA pathway expression and promoter engineering, we produced 727.9 mg/L epi-isozizaene, 780.3 mg/L pentalenene and 77.5 mg/L α-isocomene in Escherichia coli and 344 mg/L pentalenene in Saccharomyces cerevisiae. We also introduced a dynamic autoinduction system using previously identified FPP-responsive promoters for inducer-free production and managed to achieve comparable amounts of each compound. Conclusion We produced tricyclic sesquiterpenes epi-isozizaene, pentalenene and α-isocomene, promising jet fuel feedstocks at high production titers, providing novel, sustainable alternatives to petroleum-based jet fuels. Electronic supplementary material The online version of this article (10.1186/s13068-018-1272-z) contains supplementary material, which is available to authorized users.

Insects use a diverse array of specialized terpene metabolites as pheromones in intraspecific interactions. In contrast to plants and microbes, which employ enzymes called terpene synthases (TPSs) to synthesize terpene metabolites, limited information from few species is available about the enzymatic mechanisms underlying terpene pheromone biosynthesis in insects. Several stink bugs (Hemiptera: Pentatomidae), among them severe agricultural pests, release 15-carbon sesquiterpenes with a bisabolene skeleton as sex or aggregation pheromones. The harlequin bug, Murgantia histrionica, a specialist pest of crucifers, uses two stereoisomers of 10,11-epoxy-1-bisabolen-3-ol as a male-released aggregation pheromone called murgantiol. We show that MhTPS (MhIDS-1), an enzyme unrelated to plant and microbial TPSs but with similarity to trans-isoprenyl diphosphate synthases (IDS) of the core terpene biosynthetic pathway, catalyzes the formation of (15,6S,7R)-1,10-bisaboladien-1-ol (sesquipiperitol) as a terpene intermediate in murgantiol biosynthesis. Sesquipiperitol, a so-far-unknown compound in animals, also occurs in plants, indicating convergent evolution in the biosynthesis of this sesquiterpene. RNAi-mediated knockdown of MhTPS mRNA confirmed the role of MhTPS in murgantiol biosynthesis. MhTPS expression is highly specific to tissues lining the cuticle of the abdominal sternites of mature males. Phylogenetic analysis suggests that MhTPS is derived from a trans-IDS progenitor and diverged from bona fide trans-IDS proteins including MhIDS-2, which functions as an (E,E)-farnesyl diphosphate (FPP) synthase. Structure-guided mutagenesis revealed several residues critical to MhTPS and MhFPPS activity. The emergence of an IDS-like protein with TPS activity in M. histrionica demonstrates that de novo terpene biosynthesis evolved in the Hemiptera in an adaptation for intraspecific communication. US Department of Agriculture National Institute of Food and Agriculture [2016-67013-24759]; Virginia Polytechnic Institute; State University Department of Biochemistry; State University Department of Biological Sciences; Translational Plant Sciences Program We thank Megan V. Herlihy and Jeremy K. Turner for assisting with harlequin bug rearing and volatile collection and Filadelfo Guzman for assisting with GC analyses. This work was supported by US Department of Agriculture National Institute of Food and Agriculture Grant 2016-67013-24759 (to D.T., D.E.G.-R., A.K., T.P.K., C.T., and D.C.W.) and the Virginia Polytechnic Institute and State University Departments of Biochemistry and Biological Sciences and the Translational Plant Sciences Program.

Insects use a wide range of structurally diverse pheromones for intra-specific communication. Compounds in the class of terpenes are emitted as sex, aggregation, alarm, or trail pheromones. Despite the common occurrence of terpene pheromones in different insect lineages, their origin from dietary host plant precursors or de novo biosynthetic pathways often remains unknown. Several stink bugs (Hemiptera: Pentatomidae) release bisabolene-type sesquiterpenes for aggregation and mating. Here we provide evidence for de novo biosynthesis of the sex pheromone trans−/cis-(Z)-α-bisabolene epoxide of the Southern green stink bug, Nezara viridula. We show that an enzyme (NvTPS) related to isoprenyl diphosphate synthases (IDSs) of the core terpene metabolic pathway functions as a terpene synthase (TPS), which converts the general intermediate (E,E)-farnesyl diphosphate (FPP) to the putative pheromone precursor (+)-(S,Z)-α-bisabolene in vitro and in protein lysates. A second identified IDS-type protein (NvFPPS) makes the TPS substrate (E,E)-FPP and functions as a bona fide FPP synthase. NvTPS is highly expressed in male epidermal tissue associated with the cuticle of ventral sternites, which is in agreement with the male specific release of the pheromone from glandular cells in this tissue. Our study supports findings of the function of similar TPS enzymes in the biosynthesis of aggregation pheromones from the pine engraver beetle Ips pini, the striped flea beetle Phyllotreta striolata, and the harlequin bug Murgantia histrionica, and hence provides growing evidence for the evolution of terpene de novo biosynthesis by IDS-type TPS families in insects.

This is a quick guide for the preparation of (E)-α-bisabolene samples and external standards in dodecane for GC analysis. This protocol has been established in the Lindberg lab at Ångström laboratory (Uppsala University) for direct analysis of dodecane-based ex-situ extracts from cyanobacterial strains producing (E)-α-bisabolene. The protocol includes the preparation of stock solutions for the internal standard β-caryophyllene (BCP) and the external standard. The preparation of a fresh external standard series is suggested for each sample analysis. The calibration curve is required for quantification of (E)-α-bisabolene in the samples of interest. This protocol is used in combination with the following GC protocol: dx.doi.org/10.17504/protocols.io.kj2cuqe

The essential oils from Commiphora species have for centuries been recognized to possess medicinal properties. Here, we performed gas chromatography-mass spectrometry on the essential oil from opoponax (Commiphora guidotti) and identified bisabolene isomers as the main constituents of this essential oil. Opoponax essential oil, a chemical component; β-bisabolene and an alcoholic analogue, α-bisabolol, were tested for their ability to selectively kill breast cancer cells. Only β-bisabolene, a sesquiterpene constituting 5% of the essential oil, exhibited selective cytotoxic activity for mouse cells (IC50 in normal Eph4: >200 µg/ml, MG1361: 65.49 µg/ml, 4T1: 48.99 µg/ml) and human breast cancer cells (IC50 in normal MCF-10A: 114.3 µg/ml, MCF-7: 66.91 µg/ml, MDA-MB-231: 98.39 µg/ml, SKBR3: 70.62 µg/ml and BT474: 74.3 µg/ml). This loss of viability was because of the induction of apoptosis as shown by Annexin V-propidium iodide and caspase-3/7 activity assay. β-bisabolene was also effective in reducing the growth of transplanted 4T1 mammary tumours in vivo (37.5% reduction in volume by endpoint). In summary, we have identified an anti-cancer agent from the essential oil of opoponax that exhibits specific cytotoxicity to both human and murine mammary tumour cells in vitro and in vivo, and this warrants further investigation into the use of β-bisabolene in the treatment of breast cancers.

The chemical composition of the essential oils obtained by hydrodistillation from the aerial parts of Thymus serpyllum and Thymus serpyllum‘Aureus’ has been investigated by gas chromatography-mass spectrometry (GC-MS). Forty-seven compounds (99.67% of the total oil) wereidentified in the essential oil of T. serpyllum. The main components found in the oil were carvacrol (37.49%), -terpinene (10.79%), -caryophyllene (6.51%), p-cymene (6.06%), (E)--ocimene (4.63%) and -bisabolene (4.51%). Similarly, carvacrol (44.93%), -terpinene(10.08%), p-cymene (7.39%) and -caryophyllene (6.77%) dominated in the oil of T. serpyllum ‘Aureus’. A total of forty three compounds wereidentified in this oil, representing 99.49% of the total oil content. On the basis of the obtained data it was proved that the content of 1-octen-3-ol,eucalyptol, (Z)--ocimene, (E)--ocimene, -terpinene, carvacrol methyl ether, germacrene D and -bisabolene was significantly higher for T.serpyllum while T. serpyllum ‘Aureus’ was characterized by a significantly higher content of 3-octanone, 3-octanol, p-cymene, borneol andcarvacrol. The isolated essential oils were evaluated for their antimicrobial activity against nine reference strains (Escherichia coli, Staphylococcusaureus, Staphylococcus epidermidis, Streptococcus agalactiae, Enterococcus faecalis, Bacillus cereus, Micrococcus luteus, Proteus vulgaris and Candidaalbicans) by the microdilution technique. Based on this test, the minimum inhibitory concentrations (MIC) of essential oil were calculated. Thevolatile oil obtained from T. serpyllum showed the highest antimicrobial activity relative to the strain of E. coli (MIC=0.025 μL/mL) and to theyeast C. albicans (MIC=0.05 μL/mL). Similarly, a significant antimicrobial activity exhibited T. serpyllum ‘Aureus’ essential oil, although the MICvalues obtained in that case for E. coli and C. albicans strains were twice as high and were respectively 0.05 μL/mL and 0.1 μL/mL.

The hydro-distillation of the dried aerial parts of Meconopsis simplicifolia yielded essential oil (0.4 % v/w). The GC-FID coupled with GC-MS analysis detected at least 41 constituents of the chromatographable fraction of the total injected oil with bisabolene (29.2 %) as the major constituents of the oil. Strong antimalarial, moderate anti-inflammatory and weak antibacterial activities reported for the crude extracts and the alkaloids of this plant are also discussed.