Evaluation of chemical constituents and antinociceptive properties of Myricaria elegans Royle

The phytochemical screening of Myricaria elegans Royle (Tamaricaceae) gave strongly positive results for terpenes. A total of six triterpenes were isolated from the CHCl₃ fraction, including eleganene‐A, eleganene‐B, corsolic acid, betulin, ursolic acid, and erythrodiol. The in vivo antinociceptive investigation of the plant showed a significant increase in the tail‐flick latency, accompanied by mild sedation and severe ataxia. Considering the known activities of some of the compounds isolated from the plant, it may be hypothesized that the increase in the tail‐flick latency may be the combined effect of analgesia, ataxia, and sedation, rather than analgesia alone. These findings suggest M. elegans to be a potential source for activity‐guided isolation of important natural compounds with a variety of effects.     

Interactions of oxidosqualene cyclase (Erg7p) with 3-keto reductase (Erg27p) and other enzymes of sterol biosynthesis in yeast

In Saccharomyces cerevisiae and Candida albicans, two enzymes of the ergosterol biosynthetic pathway, oxidosqualene cyclase (Erg7p) and 3-keto reductase (Erg27p) interact such that loss of the 3-keto reductase also results in a concomitant loss of activity of the upstream oxidosqualene cyclase. This interaction wherein Erg27p has a stabilizing effect on Erg7p was examined to determine whether Erg7p reciprocally has a protective effect on Erg27p. To this aim, three yeast strains each lacking the ERG7 gene were tested for 3-ketoreductase activity by incubating either cells or cell homogenates with unlabeled and radiolabeled 3-ketosteroids. In these experiments, the ketone substrates were effectively reduced to the corresponding alcohols, providing definitive evidence that oxidosqualene cyclase is not required for the 3-ketoreductase activity. This suggests that, in S. cerevisiae, the protective relationship between the 3-keto reductase (Erg27p) and oxidosqualene cyclase (Erg7p) is not reciprocal. However, the absence of the Erg7p, appears to affect other enzymes of sterol biosynthesis downstream of lanosterol formation. Following incubation with radiolabeled and non-radiolabeled 3-ketosteroids we detected differences in hydroxysteroid accumulation and ergosterol production between wild-type and ERG7 mutant strains. We suggest that oxidosqualene cyclase affects Erg25p (C-4 sterol oxidase) and/or Erg26p (C-3 sterol dehydrogenase/C-4 decarboxylase), two enzymes that, in conjunction with Erg27p, are involved in C-4 sterol demethylation.     

Squalene cyclase and oxidosqualene cyclase from a fern

Ferns are the most primitive vascular plants. The phytosterols of ferns are the same as those of higher plants, but they produce characteristic triterpenes. The most distinct feature is the lack of oxygen functionality at C-3, suggesting that the triterpenes of ferns may be biosynthesized by direct cyclization of squalene. To obtain some insights into the molecular bases for the biosynthesis of triterpenes in ferns, we cloned ACX, an oxidosqualene cyclase homologue, encoding a cycloartenol synthase (CAS) and ACH, a squalene cyclase homologue, encoding a 22-hydroxyhopane synthase from Adiantum capillus-veneris. Phylogenetic analysis revealed that ACH is located in the cluster of bacterial SCs, while ACX is in the cluster of higher plant CASs.     

Identification of marneral synthase,which is critical for growth and development in Arabidopsis

Plants produce structurally diverse triterpenoids, which are important for their life and survival. Most triterpenoids and sterols share a common biosynthetic intermediate, 2,3‐oxidosqualene (OS), which is cyclized by 2,3‐oxidosqualene cyclase (OSC). To investigate the role of an OSC, marneral synthase 1 (MRN1), in planta, we characterized a Arabidopsis mrn1 knock‐out mutant displaying round‐shaped leaves, late flowering, and delayed embryogenesis. Reduced growth of mrn1 was caused by inhibition of cell expansion and elongation. Marnerol, a reduced form of marneral, was detected in Arabidopsis overexpressing MRN1, but not in the wild type or mrn1. Alterations in the levels of sterols and triterpenols and defects in membrane integrity and permeability were observed in the mrn1. In addition, GUS expression, under the control of the MRN1 gene promoter, was specifically detected in shoot and root apical meristems, which are responsible for primary growth, and the mRNA expression of Arabidopsis clade II OSCs was preferentially observed in roots and siliques containing developing seeds. The eGFP:MRN1 was localized to the endoplasmic reticulum in tobacco protoplasts. Taken together, this report provides evidence that the unusual triterpenoid pathway via marneral synthase is important for the growth and development of Arabidopsis.     

Identification of triterpene biosynthetic genes from Momordica charantia using RNA-seq analysis

Cucurbitaceae plants contain characteristic triterpenoids. Momordica charantia, known as a bitter melon, contains cucurbitacins and multiflorane type triterpenes, which confer bitter tasting and exhibit pharmacological activities. Their carbon skeletons are biosynthesized from 2,3-oxidosqualene by responsible oxidosqualene cyclase (OSC). In order to identify OSCs in M. charantia, RNA-seq analysis was carried out from ten different tissues. The functional analysis of the resulting four OSC genes revealed that they were cucurbitadienol synthase (McCBS), isomultiflorenol synthase (McIMS), β-amyrin synthase (McBAS) and cycloartenol synthase (McCAS), respectively. Their distinct expression patterns based on RPKM values and quantitative RT-PCR suggested how the characteristic triterpenoids were biosynthesized in each tissue. Although cucurbitacins were finally accumulated in fruits, McCBS showed highest expression in leaves indicating that the early step of cucurbitacins biosynthesis takes place in leaves, but not in fruits.

Abbreviations: OSC: oxidosqualene cyclase; RPKM: reads perkilobase of exon per million mapped reads     

High throughput screening of mutants of oat that are defective in triterpene synthesis

The triterpenes are a large and diverse group of plant natural products that have important functions in plant protection and food quality, and a range of pharmaceutical and other applications. Like sterols, they are synthesised from mevalonate via the isoprenoid pathway, the two pathways diverging after 2,3-oxidosqualene. During triterpene synthesis 2,3-oxidosqualene is cyclised to one of a number of potential products, the most common of these being the pentacyclic triterpene β-amyrin. Plants often produce complex mixtures of conjugated triterpene glycosides which may be derived from a single triterpene skeleton. The delineation, functional analysis and exploitation of triterpene pathways in plants therefore represent a substantial challenge. Here we have carried out high throughput screening to identify mutants of diploid oat (Avena strigosa) that are blocked in the early steps of triterpene synthesis. We also show that mutants that are affected in the first committed step in synthesis of β-amyrin-derived triterpenes, and so are unable to cyclise 2,3-oxidosqualene to β-amyrin (sad1 mutants), accumulate elevated levels of primary sterols. The major differences were in Δ-7-campesterol and Δ-7-avenasterol, which both increased several fold relative to wild-type levels. This is presumably due to accumulation of squalene and 2,3-oxidosqualene and consequent feedback into the sterol pathway, and is consistent with previous reports in which specific oxidosqualene cyclase inhibitors and elicitors of triterpene biosynthesis were shown to have inverse effects on the flux through the sterol and triterpene pathways.     

Activation-independent cyclization of monoterpenoids

The biosynthesis of cyclic monoterpenes (C(10)) generally requires the cyclization of an activated linear precursor (geranyldiphosphate) by specific terpene cyclases. Cyclic triterpenes (C(30)), on the other hand, originate from the linear precursor squalene by the action of squalene-hopene cyclases (SHCs) or oxidosqualene cyclases (OSCs). Here, we report a novel terpene cyclase from Zymomonas mobilis (ZMO1548-Shc) with the unique capability to cyclize citronellal to isopulegol. To our knowledge, ZMO1548-Shc is the first biocatalyst with diphosphate-independent monoterpenoid cyclase activity. A combinatorial approach using site-directed mutagenesis and modeling of the active site with a bound substrate revealed that the cyclization of citronellal proceeds via a different mechanism than that of the cyclization of squalene.     

Evaluation of pyruvate decarboxylase‐negative Saccharomyces cerevisiae strains for the production of succinic acid

Dicarboxylic acids are important bio‐based building blocks, and Saccharomyces cerevisiae is postulated to be an advantageous host for their fermentative production. Here, we engineered a pyruvate decarboxylase‐negative S. cerevisiae strain for succinic acid production to exploit its promising properties, that is, lack of ethanol production and accumulation of the precursor pyruvate. The metabolic engineering steps included genomic integration of a biosynthesis pathway based on the reductive branch of the tricarboxylic acid cycle and a dicarboxylic acid transporter. Further modifications were the combined deletion of GPD1 and FUM1 and multi‐copy integration of the native PYC2 gene, encoding a pyruvate carboxylase required to drain pyruvate into the synthesis pathway. The effect of increased redox cofactor supply was tested by modulating oxygen limitation and supplementing formate. The physiologic analysis of the differently engineered strains focused on elucidating metabolic bottlenecks. The data not only highlight the importance of a balanced activity of pathway enzymes and selective export systems but also shows the importance to find an optimal trade‐off between redox cofactor supply and energy availability in the form of ATP.     

Glucose‐6‐Phosphate Dehydrogenase from the Oleaginous Microalga Nannochloropsis Uncovers Its Potential Role in Promoting Lipogenesis

Microalgae have long been considered as potential biological feedstock for the production of wide array of bioproducts, such as biofuel feedstock because of their lipid accumulating capability. However, lipid productivity of microalgae is still far below commercial viability. Here, a glucose‐6‐phosphate dehydrogenase from the oleaginous microalga Nannochloropsis oceanica is identified and heterologously expressed in the green microalga Chlorella pyrenoidosa to characterize its function in the pentose phosphate pathway. It is found that the G6PD enzyme activity toward NADPH production is increased by 2.19‐fold in engineered microalgal strains. Lipidomic analysis reveals up to 3.09‐fold increase of neutral lipid content in the engineered strains, and lipid yield is gradually increased throughout the cultivation phase and saturated at the stationary phase. Moreover, cellular physiological characteristics including photosynthesis and growth rate are not impaired. Collectively, these results reveal the pivotal role of glucose‐6‐phosphate dehydrogenase from N. oceanica in NADPH supply, demonstrating that provision of reducing power is crucial for microalgal lipogenesis and can be a potential target for metabolic engineering.     

Cloning and Functional Expression of Cycloartenol Synthases from Mangrove Species Rhizophora stylosa Griff. and Kandelia candel (L.) Druce

To obtain cDNAs encoding oxidosqualene cyclase (OSC), we cloned two cDNAs, KcCAS and RsCAS, from roots of Kandelia candel (L.) Druce and leaves of Rhizophora stylosa Griff. by homology based PCR method respectively. The deduced amino acid sequences of both OSCs showed 82% homology to cycloartenol synthases from Lotus japonicus (OSC5) and Ricinus cummunis (RcCAS), suggesting that these are cycloartenol synthases of K. candel and R. stylosa. The genes obtained were expressed in a lanosterol synthase deficient Saccharomyces cerevisiae (ERG7) strain, GIL77. GC–MS analysis identified the accumulated reaction product in the yeast transformant to be cycloartenol, indicating that both KcCAS and RsCAS encode cycloartenol synthase.     

Production of Plant Bioactive Triterpenoid Saponins: Elicitation Strategies and Target Genes to Improve Yields

Triterpenoid saponins are a class of plant secondary metabolites with structure derived from the precursor oxidosqualene in which one or more sugar residues are added. They have a wide range of pharmacological applications, such as antiplatelet, hypocholesterolemic, antitumoral, anti-HIV, immunoadjuvant, anti-inflammatory, antibacterial, insecticide, fungicide and anti-leishmanial agents. Their accumulation in plant cells is stimulated in response to changes mediated by biotic and abiotic elicitors. The enhancement of saponin yields by methyl jasmonate in plants and cell cultures in several species indicates the involvement of these metabolites in plant defence mechanisms. The elucidation of their biosynthesis at the molecular level has advanced recently. Most studies to date have focused on the participation of early enzymes in the pathway, including oxidosqualene cyclase, squalene synthase and dammarenediol synthase, as well as in isolating and characterizing genes that encode β-amyrin synthase. Yields of bioactive saponins in various plant species and experimental systems have been successfully increased by treating cells and tissues with jasmonate or by exposing these to oxidative stress. These elicitation and molecular studies are consolidating a robust knowledge platform from which to launch the development of improved sources for commercial supply of bioactive saponins.     

New antibacterial pentacyclic triterpenes from Myricaria elegans Royle. (tamariscineae)

Two new pentacyclic triterpenes eleganene-A (1) and eleganene-B (2), along with four known pentacyclic triterpenes betulin (3), ursolic acid (4), erythrodiol (5) and corosolic acid (6) were isolated from the aerial parts of Myricaria elegans. These compounds exhibited significant antibacterial activity. The structure of compounds 1 and 2 were deduced on the basis of their spectral analysis.     

Cytotoxic and Apoptosis‐Inducing Activities,and Anti‐Tumor‐Promoting Effects of Cyanogenated and Oxygenated Triterpenes

Two of each semisynthetic lanostane‐ and cycloartane‐type triterpenes with a cyano‐enone functionality, i.e., 13 and 18 , and 23 and 28 , respectively, sixteen of their synthetic intermediates, 9 – 12, 14 – 17, 19 – 22 , and 24 – 27 , along with seven semisynthetic oxygenated triterpene acetates, 29 – 35 , and eight natural hydroxy triterpenes, 1 – 8 , were evaluated for their cytotoxic activities against leukemia (HL60), lung (A549), stomach (AZ521), and breast (SK‐BR‐3) cancer cell lines. One natural triterpene, 8 , and ten semisynthetic triterpenes, 9, 13, 15, 18, 23, 25, 28, 29, 32 , and 33 , exhibited potent cytotoxicities against one or more cell lines with IC₅₀ values in the range of 1.4–9.9 μM . Two lanostane‐type triterpenes with a cyano‐enone functionality, 3‐oxolanosta‐1,8,24‐triene‐2‐carbonitrile ( 13 ) and 3‐oxolanosta‐1,8‐diene‐2‐carbonitrile ( 18 ), induced apoptosis in HL60 cells, as observed by membrane phospholipid exposure in flow cytometry. Western blot analysis showed that 13 and 18 significantly reduced procaspases‐3, ‐8, and ‐9, and increased cleaved caspases‐3, ‐8, and ‐9. These findings indicated that compounds 13 and 18 induced apoptosis in HL60 cells via both the mitochondrial and the death receptor‐mediated pathways. In addition, upon evaluation of the inhibitory effects on Epstein? Barr virus early antigen (EBV‐EA) activation induced with 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) in Raji cells, seven natural triterpenes, 1 – 6 and 8 , and ten semisynthetic triterpenes, 9, 10, 14, 15, 19, 20, 24, 25, 29 , and 30 , exhibited inhibitory effects which were higher than that of β‐carotene, a vitamin A precursor studied widely in cancer‐chemoprevention animal models.     

Distribution and expression characteristics of triterpenoids and OSC genes in white birch (<Emphasis Type="Italic">Betula platyphylla</Emphasis> suk.)

Betulin and oleanolic acids (pentacyclic triterpenoid secondary metabolites) have broad pharmacological activities and can be potentially used for the development of anti-cancer and anti-AIDS drugs. In this study, we detected the accumulation and the distribution characteristics of betulin and oleanolic acid in various organs of white birch at different ages. We also determined the expression of 4 OSC genes (LUS, β-AS, CAS1 and CAS2) involved in the triterpenoid synthesis pathways by real time RT-PCR. The result showed that the 1-year old birch can synthesize betulin and oleanolic acid. In addition, betulin and oleanolic acids were mainly distributed in the bark, while the content in the root skin and leaf was very low. The content of betulin and oleanolic acid in birch varied in different seasons. The content of betulin and oleanolic acid and their corresponding LUS and β-AS gene expression were very low in 1-year old birch. With increasing age of birch, betulin content was increased, while oleanolic acid was decreased. Similar changes were also observed for their corresponding synthesis genes LUS and β-AS. In the leaf of 1-year old plant, the highest expression of CAS1 and CAS2 occurred at end of September, while expression of LUS and the β-AS was low from June to October. In the stem skin,high expression of β-AS and the LUS genes occurred from the end of July to September. In the root, high expression of the β-AS gene was observed at the end of October. These results indicated that triterpenoid gene expression was similar to the triterpene accumulation. Expression of LUS gene and β-AS gene in birch with different ages were corresponding to the betulinic and oleanolic acid accumulation. Expression of CAS1 and CAS2 genes were elevated with increasing age of birch. This study provides molecular mechanisms of triterpenes synthesis in birch plants.     

Pentacyclic triterpenes in birch bark extract inhibit early step of herpes simplex virus type 1 replication

Antiviral agents frequently applied for treatment of herpesvirus infections include acyclovir and its derivatives. The antiviral effect of a triterpene extract of birch bark and its major pentacyclic triterpenes, i.e. betulin, lupeol and betulinic acid against acyclovir-sensitive and acyclovir-resistant HSV type 1 strains was examined. The cytotoxic effect of a phytochemically defined birch bark triterpene extract (TE) as well as different pentacyclic triterpenes was analyzed in cell culture, and revealed a moderate cytotoxicity on RC-37 cells. TE, betulin, lupeol and betulinic acid exhibited high levels of antiviral activity against HSV-1 in viral suspension tests with IC₅₀ values ranging between 0.2 and 0.5 μg/ml. Infectivity of acyclovir-sensitive and clinical isolates of acyclovir-resistant HSV-1 strains was significantly reduced by all tested compounds and a direct concentration- and time-dependent antiherpetic activity could be demonstrated. In order to determine the mode of antiviral action, TE and the compounds were added at different times during the viral infection cycle. Addition of these drugs to uninfected cells prior to infection or to herpesvirus-infected cells during intracellular replication had low effect on virus multiplication. Minor virucidal activity of triterpenes was observed, however both TE and tested compounds exhibited high anti-herpetic activity when viruses were pretreated with these drugs prior to infection. Pentacyclic triterpenes inhibit acyclovir-sensitive and acyclovir-resistant clinical isolates of HSV-1 in the early phase of infection.     

构建酿酒酵母细胞工厂高效合成摩尔酸

[目的] 摩尔酸作为齐墩果烷型三萜化合物具有抗HIV、抗炎等多种生物学活性,其前体物质是计曼尼醇,本研究基于合成生物学策略构建酿酒酵母细胞工厂高效合成摩尔酸。[方法] 运用CRISPR/Cas9技术,首先分别整合不同来源的氧化鲨烯环化酶（OSCs）,筛选高产计曼尼醇底盘细胞;进一步异源表达长春花来源的细胞色素P450氧化酶（CYP716AL1）和麻风树来源的细胞色素P450还原酶（JcCPR）,构建摩尔酸生物合成途径;并通过CYP716AL1和不同来源的CPR适配研究以及过表达甲羟戊酸（MVA）代谢途径中关键酶的方式提高摩尔酸的产量。[结果] 整合苹果来源的氧化鲨烯环化酶MdOSC获得的重组菌株计曼尼醇产量最高,达68.3 mg/L;以此为底盘细胞进一步整合CYP716AL1和JcCPR实现了摩尔酸的生物合成,产量为15.0 mg/L;共表达CYP716AL1和拟南芥来源的CPR获得的重组菌株摩尔酸产量最高,达到24.3 mg/L;最后过表达MVA代谢途径中的关键酶法呢基焦磷酸合酶（ERG20）和鲨烯环氧酶（ERG1）,获得的重组菌株摩尔酸产量高达34.1 mg/L。[结论] 本研究实现了摩尔酸的高效生物合成,为构建高产齐墩果烷型三萜酿酒酵母细胞工厂提供了理论和技术依据。     

Biosynthesis of friedelane and quinonemethide triterpenoids is compartmentalized in Maytenus aquifolium and Salacia campestris

Maytenus aquifolium (Celastraceae) and Salacia campestris (Hippocrateaceae) species accumulate friedelane and quinonemethide triterpenoids in their leaves and root bark, respectively. Enzymatic extracts obtained from leaves displayed cyclase activity with conversion of the substrate oxidosqualene to the triterpenes, 3beta-friedelanol and friedelin. In addition, administration of (+/-)5-(3)H mevalonolactone in leaves of M. aquifolium seedlings produced radio labelled friedelin in the leaves, twigs and stems, while the root bark accumulated labelled maytenin and pristimerin. These experiments indicated that the triterpenes once biosynthesized in the leaves are translocated to the root bark and further transformed to the antitumoral quinonemethide triterpenoids.