Screening of Taxol-producing fungi based on PCR amplification from Taxus

Genes coding for 10-deacetylbaccatin III-10-O-acetyl transferase and C-13 phenylpropanoid side chain-CoA acyltransferase were used as molecular markers for screening of Taxol-producing endophytic fungi. Using PCR, three out of 90 endophytic fungi, isolated from Taxus x media and Taxus yunnanensis, gave positive results. These 3 strains, when grown in 300 ml potato/dextrose liquid medium at 25°C for 10 days, contained 100–160 μg Taxol/g dry wt of mycelium. Peng Zhang and Peng-peng Zhou contributed equally to this work.     

Paclitaxel production using co-culture of <Emphasis Type="Italic">Taxus</Emphasis> suspension cells and paclitaxel-producing endophytic fungi in a co-bioreactor

The co-culture of the suspension cells of Taxus chinensis var. mairei and its endophytic fungi, Fusarium mairei, in a 20-L co-bioreactor was successfully established for paclitaxel production. The co-bioreactor consists of two-unit tanks (10 L each) with a repairable separate membrane in the center, culturing Taxus suspension cells in one tank and growing fungi in another. By optimizing the co-culture conditions, there was a desirable yield of paclitaxel in Taxus cell cultures. The Taxus cell cultures by co-culture produced 25.63 mg/L of paclitaxel within 15 days; it was equivalent to a productivity of 1.71 mg/L per day and 38-fold higher than that by uncoupled culture (0.68 mg/L within 15 days). The optimum conditions for co-culture in the co-bioreactor were: B5 medium, inoculating fungi when Taxus cells had grown for 5 days in the co-bioreactor, hydrophilic separate membrane in the center of the co-bioreactor, and air flow rate of 1:0.85 v/v/m in fungus cultures.     

Cadinane sesquiterpenoids of Phomopsis cassiae, an endophytic fungus associated with Cassia spectabilis (Leguminosae)

Five cadinane sesquiterpenes derivatives were isolated by bioassay-guided fractionation from Phomopis cassiae, an endophytic fungus isolated from Cassia spectabilis. The structures of the two diastereoisomeric 3,9,12-trihydroxycalamenenes (1, 2); 3,12-dihydroxycalamenene (3); 3,12-dihydroxycadalene (4) and 3,11,12-trihydroxycadalene (5) were established on the basis of analyses of 1D and 2D NMR and HRTOFMS experiments. Antifungal activity of the isolates was evaluated against Cladosporium sphaerospermum and Cladosporium cladosporioides, revealing 5 as the most active compound.     

Benzopyrans from Curvularia sp., an endophytic fungus associated with Ocotea corymbosa (Lauraceae)

An isolate of Curvularia sp. was obtained from the leaves of Ocotea corymbosa, a native plant of the Brazilian Cerrado. The ethyl acetate extract from culture of this fungus afforded two benzopyran derivatives: (2'S)-2-(propan-2'-ol)-5-hydroxy-benzopyran-4-one (2) and 2,3-dihydro-2-methyl-benzopyran-4,5-diol (4); and two known benzopyrans: 2-methyl-5-methoxy-benzopyran-4-one (1) and (2R)-2,3-dihydro-2-methyl-5-methoxy-benzopyran-4-one (3). The structures of 2 and 4 were established on the basis of comprehensive spectroscopic analysis, mainly using 1D and 2D NMR experiments. The benzopyrans 1 and 2 showed weak in vitro antifungal activity against Cladosporium sphaerospermum and C. cladosporioides. Analyses of the biological activities were also carried out on HeLa (human cervix tumor) and CHO (Chinese hamster ovary) cells, aiming to evaluate their potential effects on mammalian cell line proliferation. Results from both cell lines indicated that compound 2 was able to induce cell proliferation: 70% on HeLa cells and 25% on CHO cells.     

First fungal community analyses of endophytic ascomycetes associated with Viscum album ssp. austriacum and its host Pinus sylvestris

The endophytic fungal communities in the hemi-parasitic epiphyte Viscum album and in its phorophyte Pinus sylvestris were compared to reveal the fungal distribution patterns in their hosts. The ITS nrDNA of 208 multiple-isolated fungal strains was sequenced and a newly designed process was applied for assigning taxon names to the obtained sequences. Furthermore, the isolates were grouped as clusters, by subjecting a sequence similarity matrix to various cluster analyses, the results of which were compared and verified by data from phylogenetic reconstructions. In contrast to a previously reported dominance of Leotiomycetes among Pinus inhabiting fungi, the endophytic communities of the two host plant species studied here were dominated by Xylariaceae (Sordariomycetes). This is in accordance with the finding that host selectivity was only a minor factor in explaining the distribution patterns of the endophytic fungi in Viscum and Pinus. Organ and, probably, tissue selectivity had a more pronounced effect. The composition and condition of the woods in the surrounding, however, are concluded to be the major determinants, due to the following circumstantial evidence: The highest similarities in fungal community compositions were found for the leaves of the two host plant species, especially when considering only the older leaves. The finding that the inhabitants of matured or senescent organs are less host-selective is in accordance with decreasing defence capabilities of ageing host plant tissue and an increased nutrient supply for saprobic taxa. Therefore, the composition of the fungal communities in ageing leaves seems to be predominantly ascribed to contagious spread and to depend on the spectrum of nearby sporulating fungal taxa. We suggest that because a broad range of suitable substrates for Xylariaceae was present in immediate vicinity of the study sites, these fungi also dominated among the recorded endophytic taxa.     

植物细胞工程技术生产紫杉醇研究进展

紫杉醇是一种二萜类生物碱,主要从红豆杉树皮中分离提取。目前采用植物细胞工程技术是提高紫杉醇产率、保护稀缺资源红豆杉、解决紫杉醇药源紧缺的一种最有效方法。该文对近十年来国内外有关采用植物细胞工程技术生产紫杉醇的研究进展,包括红豆杉细胞系的建立、悬浮培养条件的研究、生物反应器培养以及紫杉醇合成代谢调控方面的最新研究进展进行综述。并重点论述了前体、诱导子和代谢支路抑制剂对红豆杉细胞悬浮培养生产紫杉醇的影响,为植物细胞工程技术生产紫杉醇提供借鉴和参考。     

大戟内生菌的抑菌活性及其与宿主相关性研究

为了开发利用药用植物的内生菌资源,以大戟和其内生真菌作为材料,以一株乌桕来源的内生菌作为对照,研究自身具有抑菌作用的药用植物的内生菌在抑菌作用上和宿主的相关性.结果表明,大戟内生菌镰刀菌E5有抑制宿主受试病原菌的作用,并在活植物体内表现出对受试病原菌的拮抗作用;其抑菌能力受宿主成分刺激而加强,而宿主成分对受试病原菌无抑制作用,乌桕来源的内生菌链隔孢S12无抑制受试病原菌的作用.内生菌E5需要在氧气充足条件下培养才能产生抑菌作用,抑菌活性最大产生时间和生物量最大值不同时出现.内生菌E5的抑菌能力受到pH、温度、紫外线影响.大戟内生菌E以上特点,体现了其与宿主之间的互惠共生关系.     

Aromatic compounds produced by Periconia atropurpurea, an endophytic fungus associated with Xylopia aromatica

6,8-Dimethoxy-3-(2'-oxo-propyl)-coumarin (1) and 2,4-dihydroxy-6-[(1'E,3'E)-penta-1',3'-dienyl]-benzaldehyde (2), in addition to the known compound periconicin B (3), were isolated from the ethyl acetate extract of Periconia atropurpurea, an endophytic fungus obtained from the leaves of Xylopia aromatica, a native plant of the Brazilian Cerrado. Their chemical structures were assigned based on analyses of MS, 1D and 2D-NMR spectroscopic experiments. Biological analyses were performed using two mammalian cell lines, human cervix carcinoma (HeLa) and Chinese hamster ovary (CHO). The results showed that compound 1 had no effect when compared to the control group, which was treated with the vehicle (DMSO). Compound 2 was able to induce a slight increase in cell proliferation of HeLa (37% of increase) and CHO (38% of increase) cell lines. Analysis of compound 3 showed that it has potent cytotoxic activity against both cell lines, with an IC50 of 8.0 microM. Biological analyses using the phytopathogenic fungi Cladosporium sphaerospermum and C. cladosporioides revealed that also 2 showed potent antifungal activity compared to nystatin.     

Polyketides from Penicillium sp. JP-1, an endophytic fungus associated with the mangrove plant Aegiceras corniculatum

Four polyketides, leptosphaerone C (1), penicillenone (2), arugosin I (3) and 9-demethyl FR-901235 (4), as well as five known compounds, bacillosporin A (5), bacillosporin C (6), sequoiamonascin D (7), sequoiatone A (8), and sequoiatone B (9) were isolated from the Penicillium sp. JP-1, an endophytic fungus isolated from Aegiceras corniculatum. Their structures were determined by spectroscopic methods, mainly by 2D NMR spectroscopic analyses. Compound 1 showed cytotoxicity against A-549 cells with an IC50 value of 1.45 microM, while compound 2 showed cytotoxicity against P388 cells with an IC50 value of 1.38 microM.     

Interactions between host plants,endophytic fungi,and a phytophagous insect in an oak (Quercus grisea x Q. gambelii) hybrid zone

We examined interactions between host plants, endophytic fungi, and leaf-mining moths (Phyllonorycter sp.) in an oak (Quercus grisea x Q. gambelii) hybrid zone. The community of endophytic fungi and two common endophyte species examined responded to host plant hybridization. Total fungal frequency (TFF) and frequency of Gnomonia cerastis were lowest on hosts resembling Q. grisea, and increased linearly towards those resembling Q. gambelii. In contrast, Coccochorella quercicola was most frequently isolated from Q. grisea-like hosts and decreased in frequency across hybrids towards Q. gambelii. Frequency of G. cerastis and TFF covaried with Phyllonorycter density across the hybrid zone, but direct effects of endophytes on Phyllonorycter density were not detected. Associations between endophytes and unexplained mortality of Phyllonorycter varied according to endophyte species and state of Phyllonorycter development. In the sap-feeding stage, unexplained mortality was negatively associated with TFF and frequencies of Hormonema sp. and Preussia funiculata; whereas, in the tissue-feeding stage, unexplained Phyllonorycter mortality was positively associated with G. cerastis frequency. Three-way interactions between plant hybridization, endophytic fungi, and the insect herbivore were not significant.     

Production of taxoids with biological activity by plants and callus culture from selected Taxus genotypes

Twenty seven different yew trees belonging to various genotypes and hybrids have been screened for their capacity to produce significant amounts of taxoids provided with biological activity in the tubulin test. From the three best genotypes selected, Taxus x media "Sargentii" proved to be able to produce viable calluses from excised roots placed in vitro. Taxoid composition at various times of the in vitro culture was determined and the carcinostatic efficiency of the extracts was established using the KB cell cytotoxicity test. In leaves and calluses, respectively, 0.069 and 0.032% paclitaxel (taxol) contents were found. These contents were significantly higher than those previously reported for other genotypes.     

Naphthoquinone spiroketal with allelochemical activity from the newly discovered endophytic fungus Edenia gomezpompae

Chemical investigation of the mycelium of Edenia gomezpompae, a newly discovered endophytic fungus isolated from the leaves of Callicarpa acuminata (Verbenaceae) collected from the ecological reserve El Eden, Quintana Roo, Mexico, resulted in the isolation of four naphthoquinone spiroketals, including three new compounds and palmarumycin CP2 (4). We elucidated the structures of the metabolites by extensive NMR spectroscopy studies, including DEPT, COSY, NOESY, HSQC, HMBC, and chiroptical methods. The trivial names proposed for these compounds are preussomerin EG1 (1), preussomerin EG2 (2) and preussomerin EG3 (3). In addition, the X-ray data for 4 were obtained. The bioactivity of the mycelial organic extracts and the pure compounds was tested against three endophytic fungi (Colletotrichum sp., Phomopsis sp., and Guignardia manguifera) isolated from the same plant species (C. acuminata, Verbenaceae) and against four economically important phytopathogenic microorganisms (two fungoid oomycetes, Phythophtora capsici and Phythophtora parasitica, and the fungi Fusarium oxysporum and Alternaria solani). Spiroketals 1-3 displayed significant growth inhibition against all the phytopathogens. IC50 values for the four phytopathogens were from 20 to 170 microg/ml. Palmarumycin CP2 (4) was not bioactive against any of the fungi tested. Compound 1 showed the strongest bioactivity. The acetylated derivatives of preussomerin EG1 (1), 1a and 1b, were obtained and their biological activity was tested on endophytes and phytopathogens. Preussomerin EG1 1, 1a and 1b exhibited significant bioactivity against all microorganisms tested with the exception of Alternaria solani. This is the first report of allelochemicals with antifungal activity from the newly discovered endophytic fungus E. gomezpompae.     

红豆杉高产悬浮细胞系建立及其紫杉醇诱导的研究进展

紫杉醇是一种四环二萜酰胺类化合物,是从红豆杉科红豆杉属植物中提取分离出来的次生代谢物,是世界公认广谱、活性强的天然抗癌新药。但直接从植物中提取紫杉醇的传统生产方式,不仅产量低,且会对野生红豆杉资源造成严重破坏,同时紫杉醇的化学全合成也由于其结构复杂而不具备商业价值。与之相反,细胞培养技术具有受外界影响少、生产成本低、次生代谢产物多、细胞生长周期短的优势,是目前最具前景的紫杉醇生产方式。近年来随着科研水平的不断提升,紫杉醇无论在生理代谢调控、关键基因挖掘,还是新药物制剂与剂型及其类似物的开发和运用等方面,都取得了进展,但要建立紫杉醇商业化高产体系,还必须和前人的研究经验相结合。该文对红豆杉高产悬浮细胞系建立及其紫杉醇诱导的研究进展进行了综述,主要包括前人对红豆杉属植物组织与细胞培养相关的外植体、培养基、激素、培养条件、褐化等问题的研究,以及从代谢调节、培养方式、基因工程等多方面提高紫杉醇含量的最新进展,最后总结了当前研究的不足,并对今后通过多种组合方式来提高紫杉醇含量的生产途径进行了展望。以期促进红豆杉组织培养技术的进步,为药用资源保护和利用提供一定的理论基础与生产指导。     

NF kappa B inhibitors and antitrypanosomal metabolites from endophytic fungus Penicillium sp. isolated from Limonium tubiflorum

Chemical investigation of the endophytic fungus Penicillium sp. isolated from Limonium tubiflorum growing in Egypt afforded four new compounds of polyketide origin, including two macrolides, penilactone (1) and 10,11-epoxycurvularin (2), a dianthrone, neobulgarone G (7), and a sulfinylcoumarin, sulfimarin (14), along with twelve known metabolites (3-6, 8-13, 15 and 16). The structures of all compounds were assigned by comprehensive spectral analysis (1D and 2D NMR) and mass spectrometry. Compounds 3, 4, 13 and 16 showed pronounced antitrypanosomal activity with mean MIC values ranging from 4.96 to 9.75 ??M. Moreover, when tested against a panel of three human tumor cell lines compounds 3, 4, 6 and 12 showed selective growth inhibition against Jurkat and U937 cell lines with IC₅₀ values ranging from 1.8 to 13.3 ??M. The latter compounds also inhibited TNF??-induced NF-??B activity in K562 cells with IC₅₀ values ranging from 1.6 to 10.1 ??M, respectively.     

Flow cytometric analysis of protein content in Taxus protoplasts and single cells as compared to aggregated suspension cultures

Plant suspension cultures are highly aggregated, preventing the direct application of flow cytometry for the study of population dynamics. The utility of single cells to accurately represent aggregated suspension cultures was tested through the analysis of total protein content. Specifically, protein content of two Taxus cuspidata suspension culture lines was studied using the Bradford assay for aggregated suspension cultures, and flow cytometry with fluorescein isothiocyanate staining for protoplasts and single cells. Taxus protein levels were measured at 75–160 mg per gram dry weight via the Bradford assay. Aggregated suspension cultures, protoplasts, and single cells predicted the same trend of protein content over the culture period (21 days). Normalized protein content of isolated single cells was statistically equivalent to aggregated suspensions for both cell lines. However, normalized protein content of isolated protoplasts showed significant differences from aggregated suspensions for one of the two cell lines. Elicitation with methyl jasmonate (MJ) is commonly utilized to increase paclitaxel accumulation in suspension cultures, and therefore the effect of MJ elicitation on protein content in aggregated suspensions, isolated single cells and protoplasts was assessed. Aggregated suspension cultures, protoplasts, and single cells did not show any change in total protein content following elicitation with MJ at 200 M on day 7. This study illustrates the usefulness of flow cytometry for obtaining culture population information and the value of using intact single cells for the study of plant metabolism.     

Effects of paclitaxel-producing fungal endophytes on growth and paclitaxel formation of <Emphasis Type="Italic">Taxus cuspidata</Emphasis> cells

Effects of a fungal endophyte, Fusarium mairei, on growth and paclitaxel formation of Taxus cuspidata cells were investigated by adding fungal endophyte culture supernatant (FECS) to suspension cultures of T. cuspidata cells. The main effective chemical responsible for paclitaxel formation in FECS was an exopolysaccharide (EPS) of molecular weight ~2 kDa. FECS fractions except EPS stimulated growth of Taxus cells but had no effects on paclitaxel accumulation. Additionally, elicitation efficiency of FECS based on different culture conditions was studied. EPS content in FECS was related to FECS culture conditions. FECS with long cultivation and high-aeration cultivation contained higher EPS content and resulted in higher paclitaxel yield than that with short cultivation and low-aeration cultivation. The maximum yield of paclitaxel from Taxus cultures, elicited by FECS with 9-day cultivation, was 4.7-fold that of the control cultures.     

Reduced parasitism of a leaf-mining moth on trees with high infection frequencies of an endophytic fungus

We investigated relationships between endophytic fungi and a leaf-mining moth, Phyllonorycter sp., along an elevational gradient from 2255 to 2895 m. The fungi and moth larvae inhabit leaves of Quercus gambelii. Fungal frequencies and larval densities varied with elevation. However, larval densities were not associated with the frequencies of infection by endophytic fungi. Survival of larvae was positively associated with the most dominant fungus, Gnomonia cerastis, owing to reduced parasitism of moth larvae on trees with high frequencies of Gnomonia.     

Taxus metabolomics: methyl jasmonate preferentially induces production of taxoids oxygenated at C-13 in Taxus x media cell cultures

Cells from suspension cultures of Taxus cuspidata were extracted with pentane as a source of relatively non-polar taxoids. Of the 13 taxoids identified in this fraction, eight were oxygenated at C-14 and two had not been previously described. These taxoids, along with existing taxoid standards, were employed to profile the metabolites of Taxus x media cv. Hicksii cell suspension cultures induced with methyl jasmonate to produce paclitaxel (Taxol). The majority of the taxoid metabolites produced in these induced cultures were oxygenated at C-13, and not C-14.     

Chemistry and weak antimicrobial activities of phomopsins produced by mangrove endophytic fungus Phomopsis sp. ZSU-H76

Three metabolites named phomopsin A (1), B (2) and C (3), together with two known compounds cytosporone B (4) and C (5), were isolated from the mangrove endophytic fungus, Phomopsis sp. ZSU-H76 obtained from the South China Sea. Their structures were elucidated by spectroscopic methods, mainly by 1D and 2D NMR spectroscopic techniques. The medium-sized cyclic phenol ether based on 1 or 2 is rare in natural products. In bioassays, compounds 1, 2, and 3 had no significant antibiotic activities, but compounds 4 and 5 inhibited two fungi Candida albicans and Fusarium oxysporum with an MIC ranging from 32 to 64 microg/ml.     

Purification and characterization of a lectin from endophytic fungus Fusarium solani having complex sugar specificity

A lectin from the mycelial extract of an endophytic strain of Fusarium solani was purified. Its hemagglutinating activity was inhibited by glycoproteins possessing N-linked as well as O-linked glycans. The thermodynamics and kinetics of binding of glycans and glycoproteins to F. solani lectin was studied using surface plasmon resonance. The lectin showed high affinity for asialofetuin, asialomucin, asialofibrinogen, and thyroglobulin; and comparatively low affinity for mucin, fetuin, fibrinogen, and holotransferrin. Glycoproteins showed several fold higher affinity than their corresponding glycans with significant contribution from enthalpy and positive entropy, suggesting the involvement of non-polar protein-protein interaction. Moreover, the higher affinity of the glycoproteins was due to their faster association rates and low activation energy.