Separation of a tungus producing taxol

An endogenous filariform fungus has been separated from a treenamed Taxus growing in the Aba region, Sichuan, China. The fungus is fermented in fluid medium for 3 weeks at 25℃, then the HPLC and MALDI-TOF analysis of the zymotic fluid show that the zymotic fluid contains taxol. So it is a fungus which can produce taxol. It is named Taxomyces sp. temporarily.     

In vitro screening of taxol,an anticancer drug produced by the fungus,Colletotrichum capsici

The fungus Colletotrichum capsici was isolated from the diseased fruits of Chilli plant, Capsicum annuum. The isolated test fungus was identified by its morphological and molecular characteristic features. For the first time, the fungus was screened for the production of taxol on modified liquid medium. The presence of taxol was confirmed by the spectroscopic and chromatographic methods of analyses. The amount of taxol produced by this fungus was quantified by HPLC. The maximum amount of fungal taxol production was recorded as 687 μg/L. The production rate was 13 740‐fold higher than that, previously reported for the fungus Taxomyces andreanae. The extracted fungal taxol showed a strong cytotoxic activity in an in vitro culture of human cancer cells indicating that the increase in taxol concentration induces increased cell death. A PCR‐based screening for taxadiene synthase (ts), a unique gene in the formation of the taxane skeleton, confirmed the molecular blueprint for taxol biosynthesis. The results show that the fungus C. capsici is an excellent candidate for an alternate source of taxol supply and can serve as a potential species for genetic engineering to enhance the production of taxol to a higher level.     

Isolation and identification of an anticancer drug,taxol from <Emphasis Type="Italic">Phyllosticta tabernaemontanae</Emphasis>, a leaf spot fungus of an angiosperm, <Emphasis Type="Italic">Wrightia tinctoria</Emphasis>

Phyllosticta tabernaemontanae, a leaf spot fungus isolated from the diseased leaves of Wrightia tinctoria, showed the production of taxol, an anticancer drug, on modified liquid medium (MID) and potato dextrose broth (PDB) medium in culture for the first time. The presence of taxol was confirmed by spectroscopic and chromatographic methods of analysis. The amount of taxol produced by this fungus was quantified using high performance liquid chromatography (HPLC). The maximum amount of taxol production was recorded in the fungus grown on MID medium (461 μg/L) followed by PDB medium (150 μg/L). The production rate was increased to 9.2 × 10³ fold than that found in the culture broth of earlier reported fungus, Taxomyces andreanae. The results designate that P. tabernaemontanae is an excellent candidate for taxol production. The fungal taxol extracted also showed a strong cytotoxic activity in the in vitro culture of tested human cancer cells by apoptotic assay.     

Taxol,an anticancer drug produced by an endophytic fungus <Emphasis Type="Italic">Bartalinia robillardoides</Emphasis> Tassi,isolated from a medicinal plant, <Emphasis Type="Italic">Aegle marmelos</Emphasis> Correa ex Roxb.

Taxol is an important anticancer drug widely used in the clinic. An endophytic fungus Bartalinia robillardoides (strain AMB-9) was isolated from Aegle marmelos, a medicinal plant and screened for taxol production. The fungus was identified based on the morphology of the fungal culture and the characteristics of the spores. This fungus was grown in MID liquid medium and analyzed chromatographically and spectrometrically, for the presence of Taxol. The amount of taxol produced by this endophytic fungus was quantified by HPLC. It produced 187.6 μg/L of taxol which suggests that the fungus can serve as a potential material for genetic engineering to improve the production of Taxol. This fungal taxol isolated from the organic extract of this fungal culture, has strong cytotoxic activity towards BT 220, H116, Int 407, HL 251 and HLK 210 human cancer cells in vitro, tested by Apoptotic assay.     

Production of Taxol from Phyllosticta spinarum,an endophytic fungus of Cupressus sp.

Taxol production during the cultivation on a modified liquid and potato dextrose broth medium was indicated for the first time to occur in Phyllosticta spinarum, an endophytic fungus isolated from the needles of Cupressus sp. The presence of taxol in the fungal culture filtrate was confirmed by chromatographic and spectroscopic methods of analysis. The amount of taxol produced by this fungus was quantified by high performance liquid chromatography. The maximum amount of taxol production was obtained in this fungus when grown on M1D medium (235 μg/L) followed by PDB medium (125 μg/L). The results indicate that P. spinarum is an excellent candidate for taxol production . The production rate was 4.7 × 10³‐fold higher than that found in the culture broth of an earlier reported fungus, Taxomyces andreanae. The fungal taxol extracted also showed a strong cytotoxic activity in the in vitro culture of human cancer cells tested in an apoptotic assay.     

Production of taxol from Phyllosticta dioscoreae, a leaf spot fungus isolated from Hibiscus rosa-sinensis

Taxol is a highly functionalized anticancer drug widely used in hospitals and clinics. The leaf spot fungus, Phyllosticta dioscoreae was isolated from diseased leaves of Hibiscus rosa-sinensis and screened for extracellular production of taxol in M1D (Modified liquid medium) and PDB (Potato dextrose broth) medium for the first time. The fungus was identified by its morphological and conidial features in the culture growth. The presence of taxol in the fungal culture filtrate was confirmed by different spectroscopic and chromatographic analyses. The amount of taxol produced was quantified by HPLC. The maximum amount of taxol produced was found to be 298 μg/L in M1D medium. Production rate was 5.96 × 10³ times faster than that found in culture broth of earlier reported fungus, Taxomyces andreanae. The extracted fungal taxol also showed strong cytotoxic activity in vitro in the cultures of human cancer cells tested by apoptotic assay. The results indicate that P. dioscoreae is an excellent source of taxol production, which suggests that the fungus has potential to undergo genetic engineering in order to improve its production level.     

The induction of taxol production in the endophytic fungus—Periconia sp from Torreya grandifolia

A Periconia sp was isolated from Torreya grandifolia (a relative of yew that does not synthesize taxol) near Huangshan National Park in the People’s Republic of China. This fungus, not previously known as a tree endophyte, was isolated from the inner bark of a small lower limb. When freshly isolated from the tree and placed in a semi-synthetic medium, the fungus produced readily detectable quantities of the anticancer drug taxol. Other taxol-producing endophytes were also isolated from this source. The production of taxol by Periconia sp was demonstrated unequivocally via spectroscopic and immunological methods. However, successive transfers of the fungus in semi-synthetic medium resulted in gradual attenuation until low production occurred even though fungal growth was relatively unaffected. Several compounds, known previously as activators of microbial metabolism, including serinol, p-hydroxybenzoic acid, and a mixture of phenolic acids, were capable of fully or partially restoring taxol production to otherwise taxol-attenuated cultures. The compound with the most impressive ability to activate taxol production was benzoic acid at 0.01 mM. Benzoic acid was not a taxol precursor. Received 19 December 1997/ Accepted in revised form 19 February 1998     

A new endophytic taxane production fungus from <Emphasis Type="Italic">Taxus chinensis</Emphasis>

More than 50 kinds of endophytic fungi associated with Taxus chinensis were isolated and examined as a potential source of the imposing anticancer drug taxol. Of these, four isolates show ability to produce taxane when measured with the competitive inhibition enzyme immunoassay method. The most promising clone, DA10, identified as Mucor rouxianus sp., is the first rouxianus reported as taxol production fungus. The presence of taxol and its important precursors, such as 10-diacetyl baccatinIII (10-DAB) and baccatinIII, in theculture of this fungus was confirmed by reactivity with a taxane-specific monoclonal antibody, comparative chromatographic and mass spectrometric behavior, cytotoxity to liver carcinoma 7402, and molecular cloning of kernel fragment of taxadiene synthase gene. Published in Russian in Prikladnaya Biokhimiya i Mikrobiologiya, 2009, Vol. 45, No. 1, pp. 92–96. The text was submitted by the authors in English.     

Screening of taxol-producing endophytic fungi from <Emphasis Type="Italic">Taxus chinensis</Emphasis> var. <Emphasis Type="Italic">mairei</Emphasis>

A total of 38 endophytic fungus strains were isolated from Taxus chinensis var. mairei by the aseptic technique. Genomic DNA was extracted from isolated endophytic fungi and subjected to polymerase chain reaction (PCR) analysis for the presence of the Taxus taxadiene synthase (TS) gene, a rate-limiting enzyme gene in the taxol biosynthetic pathway. Twelve out of 38 isolated endophytic fungus strains showed PCR positive for the ts gene. Subsequently, taxol and its related compounds were extracted from culture filtrates and mycelia of the PCR positive strains, separated by column chromatography, and analyzed by High Performance Liquid Chromatography and Mass Spectrum. The analysis result showed that 3 strains could produce taxol and its related compounds at the detectible level. This study indicates that molecular detection of the ts gene is an efficient method for primary screening of taxol or its related compound-producing endophytic fungi, which can improve prominently screening efficiency. Published in Russian in Prikladnaya Biokhimiya i Mikrobiologiya, 2007, Vol. 43, No. 4, pp. 490–494. The text was submitted by the authors in English.     

Screening and breeding of high taxol producing fungi by genome shuffling

To apply the fundamental principles of genome shuffling in breeding of taxol-producing fungi, Nodulisporium sylviform was used as starting strain in this work. The procedures of protoplast fusion and genome shuffling were studied. Three hereditarily stable strains with high taxol production were obtained by four cycles of genome shuffling. The qualitative and quantitative analysis of taxol produced was confirmed using thin-layer chromatography (TLC), high performance liquid chromatography (HPLC) and LC-MS. A high taxol producing fungus, Nodulisporium sylviform F4-26, was obtained, which produced 516.37 μg/L taxol. This value is 64.41% higher than that of the starting strain NCEU-1 and 31.52%–44.72% higher than that of the parent strains.     

Identification of taxol biosynthesis stage-enriched transcripts in <Emphasis Type="Italic">Nodulisporium sylviforme</Emphasis>, using suppression subtractive hybridization

The endophytic fungus Nodulisporium sylviforme produces taxol, a diterpene alkaloid compound that has unique anti-neoplastic activity. In this study, suppression subtractive hybridization was performed to identify genes in N. sylviforme that were involved in the up-regulation of taxol production. A total of 91 clones represented the differentially expressed genes were selected based on reverse blotting results. These clones were then sequenced and analyzed using BLAST-X comparisons that revealed that about 37.8% of the ESTs were homologous to 17 different proteins with known function, 20.7% were homologous to nine hypothetical proteins and the remaining 41.5% had no identifiable homology; these may be novel genes. Two enzymatic genes involved in taxol production were found. The data presented in this study provide the first primary overview of a set of genes that are differentially expressed in N. sylviforme during taxol biosynthesis and form a solid foundation for the construction of high taxol-yielding genetically engineered strains.     

Taxol Determination from Pestalotiopsis pauciseta, a Fungal Endophyte of a Medicinal Plant

Taxol is the most effective antitumor agent developed in the past three decades. It has been used for effective treatment of a variety of cancers. A taxol-producing endophytic fungus Pestalotiopsis pauciseta (strain CHP-11) was isolated from the leaves of Cardiospermum helicacabum and screened for taxol production. The fungus was identified based on the morphology of the fungal culture and the characteristics of the spores and screened for taxol production. The amount of taxol produced by this endophytic fungus was quantified by HPLC and it produced 113.3 mg/L, thus the fungus can serve as a potential material for fungus engineering to improve taxol production. This fungal taxol also had strong anticancer activity against some cancer cells viz., BT 220, H116, Int 407, HL 251 and HLK 210 tested by Apoptotic assay and it is indicated that with the increase of taxol concentration from 0.005–0.05 mmol/L, taxol induced increased cell death through apoptosis.     

天然药物紫杉醇的研究与开发综述

本文对近年来紫杉醇(Taxol)的研究与开发进行了综述,其中包括:1.对紫杉醇研究的历史回顾;2.独特的药理作用机制;3.在植物界的分布;4.前药设计;5.半合成;6.全合成;7.组织培养;8.真菌发酵;9.构效关系等。     

内生真菌发酵生产紫杉醇的研究现状与展望

抗癌药物紫杉醇已在临床上广泛应用,但受原料红豆杉树木短缺的制约,存在巨大的供需差距,而内生真菌发酵生产紫杉醇是解决紫杉醇药源问题的很有前景的途径之一.结合课题组多年来开展的科学试验研究工作,概述了内生真菌发酵生产紫杉醇的优势、产紫杉醇内生真菌的分离研究现状和生物多样性及提高内生真菌生物合成紫杉醇量的途径.     

<Emphasis Type="Italic">Fusarium solani</Emphasis>, Tax-3, a new endophytic taxol-producing fungus from <Emphasis Type="Italic">Taxus chinensis</Emphasis>

An endophytic fungus, Tax-3, was isolated from barks of Taxus chinensis grown in the Qinba mountains, China. The strain was classified into Fusarium solani based on the morphological characteristics and the molecular phylogenetics inferred from the nuclear ribosomal DNA ITS sequences with the sequence similarity values of 100%. High performance liquid chromatography (HPLC) assay showed the F. solani, Tax-3, produced taxol with a higher yield of 163.35 μg/L in the reformative potato dextrose liquid medium (d), revealing its potential applications for taxol production. Bai Wan Deng and Kai Hui Liu contributed equally to this work.     

Screening and breeding of high taxol producing fungi by genome shuffling

To apply the fundamental principles of genome shuffling in breeding of taxol-producing fungi, Nodulisporium sylviform was used as starting strain in this work. The procedures of protoplast fusion and genome shuffling were studied. Three hereditarily stable strains with high taxol production were obtained by four cycles of genome shuffling. The qualitative and quantitative analysis of taxol produced was confirmed using thin-layer chromatography (TLC), high performance liquid chromatography (HPLC) and LC-MS. A high taxol producing fungus, Nodulisporium sylviform F4-26, was obtained, which produced 516.37 μg/L taxol. This value is 64.41% higher than that of the starting strain NCEU-1 and 31.52%―44.72% higher than that of the parent strains.     

一株产紫杉醇罗汉松内生真菌的分离和鉴定

[目的]紫杉醇是重要的抗癌药物,主要从罗汉松等植物中提取,为了保护罗汉松等种质资源,本文从罗汉松植株中分离产紫杉醇内生真菌,并对内生真菌所产紫杉醇的抗肿瘤活性进行了分析.[方法]采用组织块法自罗汉松的根、茎、叶等组织中分离内生真菌;通过四唑蓝(Methyl ThiazolylTetrazolium,MTT)比色法筛选有抗肿瘤活性的内生真菌菌株,通过薄层层析(Thin Layer Chro-matography,TLC)和高效液相色谱(High Performance Liquid Chromatography,HPLC)对内生真菌所产活性物质进行鉴定;采用抽提法抽提内生真菌所产紫杉醇,应用Vero细胞对抽提的紫杉醇的活性进行了分析.[结果]从罗汉松属(Podocrapus)植物中分离到155株内生真菌,其中28株内生真菌具有较高的抑癌活性.将其中一株菌株A2命名为EPTP-1,经形态学和分子分类学分析鉴定为烟曲霉(Aspergillus fumigatus).菌株EPTP-1中抽提的紫杉醇5.553μg/L～555.3 μg/L作用24h表现出明显的致细胞凋亡作用.菌株EPTP-1发酵5天时紫杉醇的产率为0.5578±0.0294 mg/L.[结论]从罗汉松中分离到了一株产紫杉醇内生真菌EPTP-1,可作为紫杉醇类药物工业化生产的候选菌株.     

产紫杉醇内生真菌MHZ-32的鉴定

从曼地亚红豆杉树皮内表皮分离获得一株内生真菌MHZ-32,通过高效液相色谱法检测发现,内生真菌MHZ-32的紫杉醇提取物中含有与紫杉醇标品 (15.02 min）、巴卡亭Ⅲ标品 (7.07 min)保留时间相近的色谱特征峰. 进一步通过质谱法检测发现,MHZ-32的紫杉醇提取物中具有与紫杉醇标品((M+Na)+=876)、巴卡亭Ⅲ标品((M+Na)+=609)相同的质谱特征峰,表明内生真菌MHZ-32可以产紫杉醇和巴卡亭Ⅲ. 其紫杉醇和巴卡亭III的产量分别约为0.6 μg/g和0.2 μg/g（紫杉醇或巴卡亭Ⅲ/菌丝干重）.并通过18S rRNA序列分析和形态学鉴定,将内生真菌MHZ-32初步鉴定为拟茎点霉属（Phomopsis sp.）真菌.     

产紫杉醇内生真菌TMS-26的分离和鉴定

为丰富产紫杉醇植物内生真菌资源库,从曼地亚红豆杉Taxus media茎中分离得到一株产紫杉醇的内生真菌TMS-26。通过对TMS-26的发酵提取物进行高效液相色谱分析,发现其具有与紫杉醇标准品(4.545 min)相近的色谱特征峰。进一步通过液质联用仪检测发现,内生真菌TMS-26的发酵提取物中具有与紫杉醇标准品((M+Na)+=876)相近的质谱特征峰,表明内生真菌TMS-26能够产生紫杉醇。同时通过传统形态学分类鉴定方法和18S r DNA序列分析、Internal-transcribed spacer(ITS)序列分析等现代分子生物学分类鉴定方法,最终将内生真菌TMS-26鉴定为曲霉属烟曲霉Aspergillus fumigatus,并命名为"烟曲霉TMS-26"。     

产紫杉醇(Taxol)内生真菌的生物多样性

主要阐述了产紫杉醇内生真菌的分离和生物多样性的研究状况 ,紫杉醇产生菌的生物多样性意义及微生物发酵法生产紫杉醇的研究进展 ,并结合内生真菌的特点 ,阐述了紫杉醇产生菌的宿主、生境和生态以及物种的生物多样性