New abietane and tigliane diterpenoids from the roots of Euphorbia fischeriana and their cytotoxic activities.

Three new abietane and two new tigliane diterpenoids were isolated from the roots Euphorbia fischeriana. Their structures were elucidated by spectroscopic methods and quantum chemical calculation. Compounds 4 and 5 exhibited the inhibitory activities against human cancer cells HeLa and HepG2, with IC50 ranging from 3.54 to 11.45 µM.

Characterization of a Sesquiterpene Synthase Catalyzing Formation of Cedrol and Two Diastereoisomers of Tricho-Acorenol from Euphorbia fischeriana.

A sesquiterpene synthase gene was identified from the transcriptome of Euphorbia fischeriana Steud, and the function of its product EfTPS12 was characterized by in vitro biochemical experiments and synthetic biology approaches. EfTPS12 catalyzed conversion of farnesyl diphosphate into three products, including cedrol (1) and eupho-acorenols A (2) and B (3) (two diastereoisomers of tricho-acorenol), thereby being named EfCAS herein. The structures of 2 and 3 were determined by spectroscopic methods and comparison of experimental and calculated electronic circular dichroism spectra. EfCAS is the first example of a plant-derived sesquiterpene synthase that is capable of synthesizing acorane-type alcohols. This study also documents that synthetic biology approaches enable large-scale preparation of volatile terpenes and thereby substantially facilitate characterization of corresponding terpene synthases and elucidation of the structures of their products.

Mining of the Catharanthus roseus Genome Leads to Identification of a Biosynthetic Gene Cluster for Fungicidal Sesquiterpenes.

Characterization of cryptic biosynthetic gene clusters (BGCs) from microbial genomes has been proven to be a powerful approach to the discovery of new natural products. However, such a genome mining approach to the discovery of bioactive plant metabolites has been muted. The plant BGCs characterized to date encode pathways for antibiotics important in plant defense against microbial pathogens, providing a means to discover such phytoalexins by mining plant genomes. Here is reported the discovery and characterization of a minimal BGC from the medicinal plant Catharanthus roseus, consisting of an adjacent pair of genes encoding a terpene synthase (CrTPS18) and cytochrome P450 (CYP71D349). These two enzymes act sequentially, with CrTPS18 acting as a sesquiterpene synthase, producing 5-epi-jinkoheremol (1), which CYP71D349 further hydroxylates to debneyol (2). Infection studies with maize revealed that 1 and 2 exhibit more potent fungicidal activity than validamycin. Accordingly, this study demonstrates that characterization of such cryptic plant BGCs is a promising strategy for the discovery of potential agrochemical leads. Moreover, despite the observed absence of 1 and 2 in C. roseus, the observed transcriptional regulation is consistent with their differential fungicidal activity, suggesting that such conditional coexpression may be sufficient to drive BGC assembly in plants.

Green production of silybin and isosilybin by merging metabolic engineering approaches and enzymatic catalysis.

Silymarin extracted from milk thistle seeds, is used for treating hepatic diseases. Silybin and isosilybin are its main components, and synthesized from coupling of taxifolin and coniferyl alcohol. Here, the biosynthetic pathways of taxifolin and coniferyl alcohol were reconstructed in Saccharomyces cerevisiae for the first time. To alleviate substantial burden caused by a great deal of genetic manipulation, expression of the enzymes (e.g. ZWF1, TYR1 and ARO8) playing multiple roles in the relevant biosynthetic pathways was selectively optimized. The strain YT1035 overexpressing seven heterologous enzymes and five native enzymes and the strain YC1053 overexpressing seven heterologous enzymes and four native enzymes, respectively produce 336.8 mg/L taxifolin and 201.1 mg/L coniferyl alcohol. Silybin and isosilybin are synthesized from taxifolin and coniferyl alcohol under catalysis of APX1t (the truncated milk thistle peroxidase), with a yield of 62.5%. This study demonstrates an approach for producing silybin and isosilybin from glucose for the first time.

Biotechnological production of betulinic acid and derivatives and their applications.

Betulinic acid (BA), a lupane-type triterpenoid, mainly distributes in birch plants. It has been reported that BA and its derivatives possess potent anticancer and anti-HIV activities. Commercial production of BA to date depends on phytochemical extraction and semi-synthesis from betulin (a biosynthetic precursor of BA). The biosynthetic pathway of BA has been completely revealed so far. The relevant enzymes involved in BA biosynthesis are summarized in this review. The studies on construction of biotechnological platforms for production of BA and other related triterpenoids are subsequently reviewed. The engineering strategies include overexpression of rate-limiting enzymes of triterpenoid biosynthesis, balancing flux between triterpenoid biosynthetic pathway and others, engineering endoplasmic reticulum, and improving cofactor availability. At the end, this review also attempted to provide future perspectives on potential strategies for further optimizing biosynthesis of BA and other triterpenoids in microbial hosts. KEY POINTS: • Summarizes the relevant enzymes involved in betulinic acid (BA) biosynthesis. • Highlights recent advances in biotechnological production of BA-related compounds. • Provides future perspectives on strategies for optimizing triterpenoid biosynthesis.

Control of Stereoselectivity in Diverse Hapalindole Metabolites is Mediated by Cofactor-Induced Combinatorial Pairing of Stig Cyclases.

Stereospecific polycyclic core formation of hapalindoles and fischerindoles is controlled by Stig cyclases through a three-step cascade involving Cope rearrangement, 6-exo-trig cyclization, and a final electrophilic aromatic substitution. Reported here is a comprehensive study of all currently annotated Stig cyclases, revealing that these proteins can assemble into heteromeric complexes, induced by Ca2+ , to cooperatively control the stereochemistry of hapalindole natural products.

Molecular Diversity of Terpene Synthases in the Liverwort Marchantia polymorpha.

Marchantia polymorpha is a basal terrestrial land plant, which like most liverworts accumulates structurally diverse terpenes believed to serve in deterring disease and herbivory. Previous studies have suggested that the mevalonate and methylerythritol phosphate pathways, present in evolutionarily diverged plants, are also operative in liverworts. However, the genes and enzymes responsible for the chemical diversity of terpenes have yet to be described. In this study, we resorted to a HMMER search tool to identify 17 putative terpene synthase genes from M. polymorpha transcriptomes. Functional characterization identified four diterpene synthase genes phylogenetically related to those found in diverged plants and nine rather unusual monoterpene and sesquiterpene synthase-like genes. The presence of separate monofunctional diterpene synthases for ent-copalyl diphosphate and ent-kaurene biosynthesis is similar to orthologs found in vascular plants, pushing the date of the underlying gene duplication and neofunctionalization of the ancestral diterpene synthase gene family to >400 million years ago. By contrast, the mono- and sesquiterpene synthases represent a distinct class of enzymes, not related to previously described plant terpene synthases and only distantly so to microbial-type terpene synthases. The absence of a Mg2+ binding, aspartate-rich, DDXXD motif places these enzymes in a noncanonical family of terpene synthases.

Identification of RoCYP01 (CYP716A155) enables construction of engineered yeast for high-yield production of betulinic acid.

Betulinic acid (BA) and its derivatives possess potent pharmacological activity against cancer and HIV. As with many phytochemicals, access to BA is limited by the requirement for laborious extraction from plant biomass where it is found in low amounts. This might be alleviated by metabolically engineering production of BA into an industrially relevant microbe such as Saccharomyces cerevisiae (yeast), which requires complete elucidation of the corresponding biosynthetic pathway. However, while cytochrome P450 enzymes (CYPs) that can oxidize lupeol into BA have been previously identified from the CYP716A subfamily, these generally do not seem to be specific to such biosynthesis and, in any case, have not been shown to enable high-yielding metabolic engineering. Here RoCYP01 (CYP716A155) was identified from the BA-producing plant Rosmarinus officinalis (rosemary) and demonstrated to effectively convert lupeol into BA, with strong correlation of its expression and BA accumulation. This was further utilized to construct a yeast strain that yields > 1 g/L of BA, providing a viable route for biotechnological production of this valuable triterpenoid.

Probing the promiscuity of ent-kaurene oxidases via combinatorial biosynthesis.

The substrate specificity of enzymes from natural products' metabolism is a topic of considerable interest, with potential biotechnological use implicit in the discovery of promiscuous enzymes. However, such studies are often limited by the availability of substrates and authentic standards for identification of the resulting products. Here, a modular metabolic engineering system is used in a combinatorial biosynthetic approach toward alleviating this restriction. In particular, for studies of the multiply reactive cytochrome P450, ent-kaurene oxidase (KO), which is involved in production of the diterpenoid plant hormone gibberellin. Many, but not all, plants make a variety of related diterpenes, whose structural similarity to ent-kaurene makes them potential substrates for KO. Use of combinatorial biosynthesis enabled analysis of more than 20 such potential substrates, as well as structural characterization of 12 resulting unknown products, providing some insight into the underlying structure-function relationships. These results highlight the utility of this approach for investigating the substrate specificity of enzymes from complex natural products' biosynthesis.

[Limonoids from seeds of Azadirachta indica and their antibacterial activity].

Fifteen limonoids were isolated from 95% ethanol extracts of the dry seeds of neem( Azadirachta indica) by various column chromatography techniques including silica gel,Pharmadex LH-20 gel and ODS resin. Based on spectroscopic analysis,their structures were determined as nimbocinol( 1),17ß-hydroxynimbocinol( 2),1α,3α,7α-triacetylvilasinin( 3),7α-benzoyltrichilinin( 4),1,3-diacetyl-7-tigloyl-12-hydroxyvilasinin( 5),3-deacetylsalannin( 6),1-O-acetyl-1-detigloylsalannin( 7),2'( R),3'-dihydrosalannin( 8),2'( S),3'-dihydrosalannin( 9),2,3-dihydronimbolide( 10),6-homodesacetylnimbin( 11),gedunin( 12),7-deacetyl-7-epi-dihydrogedunin( 13),7-deacetoxy-7α-hydroxygedunin( 14) and nimbinene( 15). Compound 7 is a new natural product. 4,8,9,13 and 14 are isolated from the genus Azadirachta for the first time. Compound 2 showed inhibitory activity against Escherichia coli and Staphylococcus epidermidis,with MIC values of 32 and 128 mg·L~(-1),respectively. Compound 10 showed moderate inhibitory activity against S. epidermidis with a MIC value of 64 mg·L~(-1). Compound 11 inhibited the growth of E. coli and Pseudomonas aeruginosa,both with MIC values of 128 mg·L~(-1). Compound 15 exhibited inhibitory activity against P. aeruginosa,with a MIC value of128 mg·L~(-1).

Investigation of terpene diversification across multiple sequenced plant genomes.

Plants produce an array of specialized metabolites, including chemicals that are important as medicines, flavors, fragrances, pigments and insecticides. The vast majority of this metabolic diversity is untapped. Here we take a systematic approach toward dissecting genetic components of plant specialized metabolism. Focusing on the terpenes, the largest class of plant natural products, we investigate the basis of terpene diversity through analysis of multiple sequenced plant genomes. The primary drivers of terpene diversification are terpenoid synthase (TS) "signature" enzymes (which generate scaffold diversity), and cytochromes P450 (CYPs), which modify and further diversify these scaffolds, so paving the way for further downstream modifications. Our systematic search of sequenced plant genomes for all TS and CYP genes reveals that distinct TS/CYP gene pairs are found together far more commonly than would be expected by chance, and that certain TS/CYP pairings predominate, providing signals for key events that are likely to have shaped terpene diversity. We recover TS/CYP gene pairs for previously characterized terpene metabolic gene clusters and demonstrate new functional pairing of TSs and CYPs within previously uncharacterized clusters. Unexpectedly, we find evidence for different mechanisms of pathway assembly in eudicots and monocots; in the former, microsyntenic blocks of TS/CYP gene pairs duplicate and provide templates for the evolution of new pathways, whereas in the latter, new pathways arise by mixing and matching of individual TS and CYP genes through dynamic genome rearrangements. This is, to our knowledge, the first documented observation of the unique pattern of TS and CYP assembly in eudicots and monocots.

Effect of Aspergillus flavus Fungal Elicitor on the Production of Terpenoid Indole Alkaloids in Catharanthus roseus Cambial Meristematic Cells.

This study reported the inducing effect of Aspergillus flavus fungal elicitor on biosynthesis of terpenoid indole alkaloids (TIAs) in Catharanthus roseus cambial meristematic cells (CMCs) and its inducing mechanism. According to the results determined by HPLC and HPLC-MS/MS, the optimal condition of the A. flavus elicitor was as follows: after suspension culture of C. roseus CMCs for 6 day, 25 mg/L A. flavus mycelium elicitor were added, and the CMC suspensions were further cultured for another 48 h. In this condition, the contents of vindoline, catharanthine, and ajmaline were 1.45-, 3.29-, and 2.14-times as high as those of the control group, respectively. Transcriptome analysis showed that D4H, G10H, GES, IRS, LAMT, SGD, STR, TDC, and ORCA3 were involved in the regulation of this induction process. The results of qRT-PCR indicated that the increasing accumulations of vindoline, catharanthine, and ajmaline in C. roseus CMCs were correlated with the increasing expression of the above genes. Therefore, A. flavus fungal elicitor could enhance the TIA production of C. roseus CMCs, which might be used as an alternative biotechnological resource for obtaining bioactive alkaloids.

[Limonoids from seeds of Azadirachta indica and their cytotoxic activity].

Eight limonoids were isolated from 95% ethanol extracts of neem(Azadirachta indica) seeds by various chromatographic methods. By comparison of their spectroscopic data with those reported in the literatures, these limonoids were determined as salannin(1), 1-detigloyl-1-isobutylsalannin(2), salannol-3-acetate(3), salannol(4), spirosendan(5), 1-detigloyloxy-3-deacetylsalannin-1-en-3-one(6), nimbin(7) and 6-deacetylnimbin(8). Compounds 2 and 5 were firstly isolated from this genus and 5 represented the only example of its type. And 6 is a new natural product. 6 showed inhibitory activity against HeLa and HL-60 cells, with IC50 of(21.61±4.37) and(27.33±5.74) µmol·L⁻¹, respectively. Both 7 and 8 mildly inhibited the growth of HeLa cells, with IC50 of (33.15±5.24) and (38.56±6.41) µmol·L⁻¹, respectively.

Investigating inducible short-chain alcohol dehydrogenases/reductases clarifies rice oryzalexin biosynthesis.

Rice (Oryza sativa) produces a variety of labdane-related diterpenoids as phytoalexins and allelochemicals. The production of these important natural products has been partially elucidated. However, the oxidases responsible for production of the keto groups found in many of these diterpenoids have largely remained unknown. Only one short-chain alcohol dehydrogenase/reductases (SDRs), which has been proposed to catalyze the last step in such a pathway, has been characterized to date. While rice contains >220 SDRs, only the transcription of five has been shown to be induced by the fungal cell wall elicitor chitin. This includes the momilactone A synthase (OsMAS/SDR110C-MS1), with the other four all falling in the same SDR110C family, further suggesting roles in diterpenoid biosynthesis. Here, biochemical characterization with simplified substrate analogs was first used to indicate potential functions, which were then supported by further analyses with key biosynthetic intermediates. Kinetic studies were then employed to further clarify these roles. Surprisingly, OsSDR110C-MS2 more efficiently catalyzes the final oxidation to produce momilactone A that was previously assigned to OsMAS/SDR110C-MS1, and we speculate that this latter SDR may have an alternative function instead. Conversely, two of these SDRs clearly appear to act in oryzalexin biosynthesis, with OsSDR110C-MI3 readily oxidizing the 3α-hydroxyl of oryzalexin D, while OsSDR110C-MS3 can also oxidize the accompanying 7ß-hydroxyl. Together, these SDRs then serve to produce oryzalexins A-C from oryzalexin D, essentially completing elucidation of the biosynthesis of this family of rice phytoalexins.

Four New Compounds Obtained from Cultured Cells of Artemisia annua.

Four new compounds obtained from cultured cells of Artemisia annua were reported. Products were detected by HPLC-ELSD/GC-MS and isolated by chromatographic methods. The structures of four new compounds, namely 6-hydroxy arteannuin I (1), 1-hydroxy arteannuin I (2), 2-hydroxy arteannuin J (3), and 14-hydroxy arteannuin J (4), were elucidated using their physico-chemical properties by NMR and MS data analyses. The results from the spontaneous oxidative experiment indicated that the biosynthesis of the new compounds was enzyme-catalyzed. Interestingly, the enzymes in the cultured cells of A. annua showed the abilities of substrate-selective and region-selective hydroxylation of the sesquiterpene lactone. Furthermore, the artemisinin contents were increased by 50% and 80% compared to the control group after the addition of arteannuin I/J to the suspension-cultured cells of A. annua under light and dark culture conditions, respectively.

Blocking Deprotonation with Retention of Aromaticity in a Plant ent-Copalyl Diphosphate Synthase Leads to Product Rearrangement.

Substitution of a histidine, comprising part of the catalytic base group in the ent-copalyl diphosphate synthases found in all seed plants for gibberellin phytohormone metabolism, by a larger aromatic residue leads to rearrangements. Through a series of 1,2-hydride and methyl shifts of the initially formed bicycle predominant formation of (-)-kolavenyl diphosphate is observed. Further mutational analysis and quantum chemical calculations provide mechanistic insight into the basis for this profound effect on product outcome.

Investigation of the Chemical Interface in the Soybean-Aphid and Rice-Bacteria Interactions Using MALDI-Mass Spectrometry Imaging.

Mass spectrometry imaging (MSI) is an emerging technology for high-resolution plant biology. It has been utilized to study plant-pest interactions, but limited to the surface interfaces. Here we expand the technology to explore the chemical interactions occurring inside the plant tissues. Two sample preparation methods, imprinting and fracturing, were developed and applied, for the first time, to visualize internal metabolites of leaves in matrix-assisted laser desorption ionization (MALDI)-MSI. This is also the first time nanoparticle-based ionization was implemented to ionize diterpenoid phytochemicals that were difficult to analyze with traditional organic matrices. The interactions between rice-bacterium and soybean-aphid were investigated as two model systems to demonstrate the capability of high-resolution MSI based on MALDI. Localized molecular information on various plant- or pest-derived chemicals provided valuable insight for the molecular processes occurring during the plant-pest interactions. Specifically, salicylic acid and isoflavone based resistance was visualized in the soybean-aphid system and antibiotic diterpenoids in rice-bacterium interactions.

Effects of ß-cyclodextrin and methyl jasmonate on the production of vindoline, catharanthine, and ajmalicine in Catharanthus roseus cambial meristematic cell cultures.

Long-term stable cell growth and production of vindoline, catharanthine, and ajmalicine of cambial meristematic cells (CMCs) from Catharanthus roseus were observed after 2 years of culture. C. roseus CMCs were treated with ß-cyclodextrin (ß-CD) and methyl jasmonate (MeJA) individually or in combination and were cultured both in conventional Erlenmeyer flasks (100, 250, and 500 mL) and in a 5-L stirred hybrid airlift bioreactor. CMCs of C. roseus cultured in the bioreactor showed higher yields of vindoline, catharanthine, and ajmalicine than those cultured in flasks. CMCs of C. roseus cultured in the bioreactor and treated with 10 mM ß-CD and 150 µM MeJA gave the highest yields of vindoline (7.45 mg/L), catharanthine (1.76 mg/L), and ajmalicine (58.98 mg/L), concentrations that were 799, 654, and 426 % higher, respectively, than yields of CMCs cultured in 100-mL flasks without elicitors. Quantitative reverse transcription (RT)-PCR showed that ß-CD and MeJA upregulated transcription levels of genes related to the biosynthesis of terpenoid indole alkaloids (TIAs). This is the first study to report that ß-CD induced the generation of NO, which plays an important role in mediating the production of TIAs in C. roseus CMCs. These results suggest that ß-CD and MeJA can enhance the production of TIAs in CMCs of C. roseus, and thus, CMCs of C. roseus have significant potential to be an industrial platform for production of bioactive alkaloids.

A novel terpenoid indole alkaloid derived from catharanthine via biotransformation by suspension-cultured cells of Catharanthus roseus.

OBJECTIVE: Although catharanthine (1) is well known as a biosynthetic precursor of the anticancer alkaloid, vinblastine, its alternative metabolic pathways are unclear. RESULTS: Biotransformation of 1 by suspension-cultured cells of Catharanthus roseus gave a new oxidative-cleavage product (2). The structure of 2 was determined as 3-hydroxy-4-imino-catharanthine by spectroscopic methods. Maximum conversion (9.75 %) of 2 was observed after 120 h adding 6 mg of 1/100 ml to 12-day-old suspension-cultured cells of C. roseus. Furthermore, qRT-PCR experiment was performed to reveal the effect of 1 on the expression of the genes in the biosynthetic pathway of TIA 1 up-regulated the transcript level of D4H whilst down-regulating the transcript levels of G10H, LAMT, GES, and IRS. CONCLUSION: A new metabolite of catharanthine, 3-hydroxy-4-imino-catharanthine, is reported.

Chemical constituents from branch of Fraxinus sieboldiana.

Using a combination of various chromatographic techniques including column chromatography over silica gel, Sephadex LH-20, macroporous adsorbent resin, and reversed-phase HPLC, 115 compounds including diterpenes, sesquiterpenes, treterpenes, coumarins, lignans, fatty acid derivatives, and simple aromatic derivatives were isolated from an ethanol extract of branch of Fraxinus sieboldiana (Oleaceaue), and their structures of the compounds were elucidated by spectroscopic methods including 1 D, 2D NMR and MS techniques. Among them, 41 compounds were new. In previous reports, we have been described the isolation, structure elucidation, and bioactivities of the 41 new compounds and 22 known orii including 8 coumarins, 4 phenolic and 12 phenylethanoidal glycosides. As a consequence, we herein reported the isolation and structure elucidation of the remaining 50 known compounds including 8- hydroxy-12-oxoabieta-9(11),13-dien-20-oic 8, 20-lactone(1), 6beta-hydroxyfcrruginol(2),(+)-pisiferic acid(3), (+)-pisiferal(4),(+)-7-dehydroabiet6none(5), 1-oxomiltirone(6), subdigitatone(7), linarionoside B(8), (9S)-linarionoside B(9), (3R,9R)-3-hydroxy-7,8-dihydro-beta-ionol 9-O-beta-D-apiofuranosyl-(1-->6)-beta-D-glucopyranoside(10), ursolic acid(11), betulinic acid(12), euscaphic acid(13), (+)-syringaresinol(14), (+)-fraxiresinol(15), (+)-1-hydroxysyringaresinol(16), pinoresinol(17), medioresinol(18), 8-acetoxypinoresinol(19), epipinoresinol(20), (-)-olivil(21), (+)-cyclo-olivil(22), 3,3'-dimethoxy-4,4',9-trihydroxy-7,9'-epoxylignan-7'-one(23),(+)-1-hydroxypinoresinol 4'-O-beta-D-glucopyranoside (24), (+)-1-hydroxypinoresinol 4"-O-beta-D-glucopyranoside(25),(+)-syringaresinol O-beta-D-glucopyranoside (26), liriodendrin (27), ehletianol D(28), icariside E5(29) (-)-(7R, 8R)-threo-1-C-syringylglycerol(30),(-)-(7R, 8S)-erythro-guaiacylglycerol (31),(-)-(7R, 8R)-threo-guaiacylglycerol(32), 3-(4-beta-D-glucopyranosyloxy-3-methoxy)-phenyl-2E-propenol(33),2,3-dihydroxy-l-(4-hydroxy-3,5-dimethoxyphenyl)-1-propanone(34), 2,3-dihydroxy-1-(4-hydroxy-3-methoxyphenyl)-1-propanone (35), 3-hydroxy-l-(4-hydroxy-3,5-dimethoxyphenyl)-1-propanone(36), omega-hydroxypropioguaiacone(37), sinapyladehyde(38), trans-p-hydroxycinnamaldehyde(39), syringic acid(40), vanilic acid(41), vanillin(42), 4-hydroxy-benzaldehyde (43), (24R)-24-ethyl-5alpha-cholestane-3beta,5,6beta-triol(44), beta-sitosterol(45), daucosterol(46), 2,6-dimethoxy-I,4-benzoquinone(47), 2,6-dimethoxy-pyran-4-one(48), 1-(beta-D-ribofuranosyl)uracil(49), and mannitol(50). Compouds 1-7,12,18,28-37,44 and 48 were obtained from the genus Fraxinus for the first time.