产紫杉醇内生真菌Z58的分离和鉴定

本研究从曼地亚红豆杉（Taxus x media）树皮内表皮分离得到一株产紫杉醇的内生真菌Z58,通过高效液相色谱法、质谱法和核磁共振波谱法对其紫杉醇提取物进行了分析. 结果表明,内生真菌Z58的紫杉醇提取物具有和紫杉醇标准品相近的色谱特征峰,其保留时间为10.2 min;也与紫杉醇标准品具有相同的质谱特征峰（（M+Na）+=876）和1H-NMR谱带.并通过形态学特征分析和18S rDNA序列分析,将内生真菌Z58初步鉴定为肉座菌属（Hypocrea sp.）真菌.肉座菌Z58的紫杉醇产量约为2.5～3.0 μg/g（紫杉醇/菌丝干重）,是一株具有潜在应用价值的产紫杉醇内生真菌.     

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.     

一株产紫杉醇内生真菌YN6的分离及鉴定

从云南红豆杉（Taxus yunnanensis）树皮内表皮中分离得到75株内生真菌,采用基于紫杉醇合成关键酶10-去乙酰巴卡亭Ⅲ-10-O-乙酰基转移酶（10-deacetylbaccatin Ⅲ-10-O-acetyl transferase,DBAT）和C-13苯丙氨基侧链CoA乙酰基转移酶（C-13-phenylpropanoid side chain-CoA acyltransferase,BAPT）基因为标志分子的快速筛选方法获得一株可产紫杉醇的内生真菌YN6,并通过高效液相色谱法和质谱法对其紫杉醇进行分析.同时,通过对内生真菌YN6的形态特征分析以及18S rDNA序列分析将其初步鉴定为拟盘多毛孢属（Pestalotiopsis sp.）真菌. 拟盘多毛孢YN6的紫杉醇产量约为120~140 μg/L,是目前已报道的紫杉醇产量较高的野生菌株之一. 拟盘多毛孢YN6的发现为微生物发酵生产紫杉醇提供了潜在的优良种质资源.     

从南方红豆杉中筛选和鉴定25株产紫杉烷的内生真菌

为从南方红豆杉(Taxus chinensis var.mairei)中分离产紫杉烷的内生真菌,从其幼茎、树皮和叶片中分离纯化了491株内生真菌,经筛选获得25株内生真菌具有产紫杉烷的能力,其中,4株可产紫杉醇、巴卡亭Ⅲ和10-去乙酰巴卡亭Ⅲ,8株能产紫杉醇和巴卡亭Ⅲ,1株能产紫杉醇和10-去乙酰巴卡亭Ⅲ,1株能产巴卡亭Ⅲ和10-去乙酰巴卡亭Ⅲ,6株仅产紫杉醇,5株仅产巴卡亭Ⅲ。根据内生真菌的来源,幼茎中有11株产紫杉烷的内生真菌,叶片中有9株,而树皮中仅有5株。这些菌株的紫杉醇、巴卡亭和10-去乙酰巴卡亭Ⅲ产量分别为0.64~9.87、0.48~3.42和0.20~1.00μg L~(–1)。因此,南方红豆杉中具有紫杉烷类代谢途径的内生真菌来源广,数量多,是研究真菌中紫杉烷类化合物代谢途径的良好材料,也为紫杉烷类抗癌药生产提供了潜在的真菌种源。     

内生真菌培养液对东北红豆杉细胞生长及紫杉醇合成的影响

产紫杉醇的内生真菌（Fusarium mairei）先培养在B5液体培养基中,然后制备成内生真菌培养液。在东北红豆杉（Taxus cuspidata）细胞悬浮培养的不同阶段（5、10和15天）,用不同剂量的内生真菌培养液（2、4和6mL）分别进行处理。结果表明,在用4mL内生真菌培养液处理的植物细胞中可获得最高的紫杉醇产量（5.88mg·L^-1）与释放率（67%）,分别是对照的1.9倍与5.6倍。添加时间方面,在植物细胞培养周期的第5天添加4mL内生真菌培养液,可获得最佳效果,紫杉醇产量与释放率分别为6.1mg·L^-1与75%,分别是对照的2倍与6.8倍。与其它诱导子相比,4mL内生真菌培养液不仅可提高紫杉醇的释放率,而且不会引起东北红豆杉细胞膜的明显伤害,说明内生真菌发酵液激活了紫杉醇主动运输过程中的相关酶类。     

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.     

一株产紫杉醇真菌的分离

从四川省阿坝地区生长的红豆杉树中,分离到一株内生丝状真菌.在液体培养基中, 该真菌于25℃经过3周发酵,其发酵液经HPLC和MALDI-TOF分析,结果表明,发酵液中含有紫杉醇.因此,这是一株产紫杉醇真菌, 暂定名为Taxomyces sp.     

1株产紫杉醇内生真菌LNUF014的鉴定及产物检测

从红豆杉的韧皮部组织中分离得到的360株内生真菌中,通过对发酵粗提的检测,共筛选到11株产紫杉醇的真菌。其中1株内生真菌LNUF014的发酵液采用有机试剂抽提紫杉醇,经薄层层析和高效液相色谱分析,初步表明该菌株的紫杉醇类物质含量为53.68μg/L。根据形态学研究和真核生物18S rDNA基因序列分析,将其鉴定为镰刀菌属(Fusarium)。产紫杉醇内生真菌的研究将对紫杉醇类抗肿瘤药物的研制具有重要意义。     

南方红豆杉产紫杉醇内生真菌的分离

对从广东乳源县的南方红豆杉(Taxus chinensis var. mairei)内生真菌中分离和筛选产紫杉醇的内生真菌进行了研究。从南方红豆杉的树皮、茎部、叶片及叶片研磨物中分离纯化了145株内生真菌,对其中的53株内生真菌采用摇瓶发酵培养的方法筛选产紫杉醇的内生真菌。发酵物和菌丝体经研磨、离心、乙酸乙酯萃取和浓缩,经硅胶薄层层析(TLC),高效液相色谱(HPLC)以及液相色谱-质谱(HPLC-MS)分析和检测,结果表明,从茎部分离的1株内生真菌能够产紫杉醇或其异构体, 产量达180 μg L^-1。通过对产紫杉醇内生真菌进行诱变、筛选以及优化培养条件等措施,大规模培养生产紫杉醇是具有可行性的。     

红豆杉中产紫杉醇内生真菌分离部位的比较研究

目的 探讨红豆杉不同部位在内生真菌分离效率以及产紫杉醇菌株筛选率方面的规律性,为红豆杉产紫杉醇内生菌菌株的分离与筛选提供一定的理论依据.方法 从根、茎、叶3个器官取大小和表面积相同的太行山野生红豆杉(Taxous chinensis)材料,用组织块法分离红豆杉内生真菌,计算各部位内生真菌的分离效率;用高效液相法时分离到的内生真菌发酵液提取物进行紫杉醇含量分析,计算各部位产紫杉醇内生真菌的筛选率.结果 共分离到109株红豆杉内生真菌,根部、茎部和叶部分离效率指数分别为0.90、0.63和0.28;其中有28株产紫杉醇,紫杉醇菌株筛选率分别为31.48%、21.05%和17.65%.结论 在内生真菌的分离效率及其产紫杉醇内生真菌的筛选率上,均为根部>茎部>叶部,即根部在内生真菌分离效率和筛选产紫杉醇内生真菌效率上均具有明显的优势.     

Will fungi be the new source of the blockbuster drug taxol?

Taxol (paclitaxel) is widely used for the treatment of various kinds of cancers. Originally, the major source of taxol was bark of the Pacific yew tree (Taxus brevifolia). However, this proved devastating to natural populations of the trees. To protect the Pacific yew, alternatives to the use of trees are sought. One solution is the use of taxol or its precursors derived from fungi. A large number of endophytic fungi that reside within healthy plants have been reported to be taxol producers. However, fungal epiphytes, pathogens and saprophytes have also been found to produce taxol. Several strains of fungi belonging to species Metarhizium anisopliae and Cladosporium cladosporioides MD2 are very promising, producing taxol at levels up to 800 μg/L. This review examines the potential for production of taxol from fungi. The biology of taxol synthesis in fungi and measures which may improve taxol yield are also discussed.     

河南太行红豆杉产紫杉醇内生真菌的筛选

目的：筛选出高产紫杉醇的内生菌株,为发酵生产紫杉醇提供菌种。方法：从不同来源的红豆杉根、茎、叶中分别分离内生真菌,并对其进行发酵,用HPLC法对菌丝体和发酵液中的紫杉醇含量进行检测,获得高产菌株。结果：获得54株产紫杉醇的内生真菌,其中根、茎、叶分别为29株、16株、9株。根、茎、叶三部位产紫杉醇菌株的平均产量分别为248.57μg/L、149.09μg/L、104.94μg/L;其中一株产量高达622.75μg/L。结论：从野生红豆杉的根部分离内生真菌效果较好,并获得了一株高产菌株。     

内生真菌产紫杉醇研究的回顾与展望

紫杉醇是一种高效、低毒、广谱的天然抗癌药物,是人类未来20年间最有效的抗癌药物之一。近年来的研究发现,从植物内生真菌中发酵生产紫杉醇被证明是解决药源问题的有效途径。本文概述了微生物发酵生产紫杉醇的研究进展,包括产紫杉醇内生真菌的分离、多样性和真菌产紫杉醇的优势,紫杉醇的提取和检测技术,同时对如何提高内生真菌紫杉醇产量进行了比较全面的综述。随着现代分子生物学技术和微生物发酵工程的发展,工业上大规模发酵生产紫杉醇将有望实现。     

内生真菌紫杉醇生物合成的研究现状与展望

紫杉醇是重要的抗癌药物之一,已经证明其对多种癌症具有显著疗效。目前,人们主要是从红豆杉的树皮中提取、分离和纯化紫杉醇,但由于红豆杉为生长缓慢、散生、濒危的珍稀植物,且随着紫杉醇临床用途的不断拓宽,市场需求的稳定增长,单纯依靠从红豆杉树皮中提取紫杉醇已经无法满足日益增长的市场需求。为了解决紫杉醇的药源不足,科学家已把目光从红豆杉树分离提取紫杉醇转向了其他替代方法,如化学全合成、半合成、组织培养与细胞培养、微生物发酵法生产紫杉醇等。因此,了解内生真菌紫杉醇生物合成的分子基础和遗传调控机制,对解析内生真菌紫杉醇生物合成机制、构建高产紫杉醇基因工程菌株和早日实现内生真菌紫杉醇工业化生产具有重要的科学意义和现实意义。结合本课题组多年来的科研工作,概述了红豆杉细胞紫杉醇生物合成途径、内生真菌发酵生产紫杉醇的优势、产紫杉醇内生菌的分离研究现状和生物多样性及紫杉醇生物合成相关基因的研究现状。内生真菌生物发酵合成紫杉醇是可以无限生产、大量获取紫杉醇、解决紫杉醇药源短缺问题的很有前景的方法之一。     

In vitro cell cultures obtained from different explants of Corylus avellana produce Taxol and taxanes

Background  

Taxol is an effective antineoplastic agent, originally extracted from the bark of Taxus brevifolia with a low yield. Many attempts have been made to produce Taxol by chemical synthesis, semi-synthesis and plant tissue cultures. However, to date, the availability of this compound is not sufficient to satisfy the commercial requirements. The aim of the present work was to produce suspension cell cultures from plants not belonging to Taxus genus and to verify whether they produced Taxol and taxanes. For this purpose different explants of hazel (Corylus avellana species) were used to optimize the protocol for inducing in vitro callus, an undifferentiated tissue from which suspension cell cultures were established.     

Taxol: biosynthesis, molecular genetics, and biotechnological applications

Over the past decade, Taxol and its closely related structural analogue Taxotere have emerged as very important antitumor agents. Their widespread use in the treatment of a variety of cancer types, their likely approval for the treatment of additional forms of cancer, and their use at earlier stages of intervention will lead to increased demand for these drugs in the future. Because of yield considerations, Taxol and Taxotere are currently derived via semisynthesis from the advanced taxoid 10-deacetylbaccatin III, which must be isolated from yew (Taxus) trees. Thus, efforts are underway to produce Taxol (and other advanced taxoids for use in semisynthesis) by alternate, biotechnological means. This article provides a current overview of research on taxoid biosynthesis and an assessment of bioengineering applications for taxoid production in yew cell culture.     

Downstream reactions and engineering in the microbially reconstituted pathway for Taxol

Taxol (a trademarked product of Bristol-Myers Squibb) is a complex isoprenoid natural product which has displayed potent anticancer activity. Originally isolated from the Pacific yew tree (Taxus brevifolia), Taxol has been mass-produced through processes reliant on plant-derived biosynthesis. Recently, there have been alternative efforts to reconstitute the biosynthetic process through technically convenient microbial hosts, which offer unmatched growth kinetics and engineering potential. Such an approach is made challenging by the need to successfully introduce the significantly foreign enzymatic steps responsible for eventual biosynthesis. Doing so, however, offers the potential to engineer more efficient and economical production processes and the opportunity to design and produce tailored analog compounds with enhanced properties. This mini review will specifically focus on heterologous biosynthesis as it applies to Taxol with an emphasis on the challenges associated with introducing and reconstituting the downstream reaction steps needed for final bioactivity.     

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.     

Abundant expression of the microtubule-associated protein, ensconsin (E-MAP-115), alters the cellular response to Taxol.

Correlation between expression level of a microtubule-associated protein called ensconsin (E-MAP-115) and degree of Taxol sensitivity in several cultured cell lines prompted us to investigate potential cause-and-effect relationships between ensconsin level and Taxol action. We used human MCF-7 or HeLa cells, which are sensitive to low Taxol concentrations (LD(50) of 30-35 and 3.5 nM, respectively) to prepare stably transfected populations of cells expressing heterogeneous levels of ensconsin chimeras, either green fluorescent protein (GFP) conjugated to full-length ensconsin (GFP-Ensc) or to ensconsin's microtubule-binding domain (GFP-EMTB). Both a subjective microscopic assay, i.e., scoring fluorescence of GFP-ensconsin chimeras following Taxol treatment, and a quantitative immunobiochemical assay, i.e., measuring level of GFP-ensconsin chimera in cells surviving treatment with Taxol, showed that cells expressing higher levels of GFP-ensconsin chimera were killed more readily by Taxol concentrations approaching the LD(50). In contrast, in TC-7 cells, which are relatively insensitive to Taxol (LD(50) > 600 nM), high-level expression of GFP-EMTB conferred no significant susceptibility to killing by Taxol. However, heightening the Taxol sensitivity of GFP-EMTB-TC-7 cells by pre-incubating cells with the p-glycoprotein inhibitor, verapamil, did result in selective killing of cells highly expressing GFP-EMTB. Taken together, results obtained in MCF-7, HeLa, and TC-7 cells suggest that elevated ensconsin level bestowed a selective disadvantage upon Taxol-sensitive cells. To probe potential mechanisms by which ensconsin could alter the Taxol response, we isolated microtubules from HeLa cells that were or were not pretreated with Taxol. In vivo Taxol treatment significantly tightened microtubule-binding of ensconsin, suggesting that Taxol alters ensconsin's microtubule-binding properties and may, in turn, alter the Taxol response of the microtubules. Our data support the hypothesis that Taxol works synergistically or in concert with microtubule-binding proteins in bringing about deleterious effects on the microtubule cytoskeleton.