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。     

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.     

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.     

紫杉醇生物合成相关酶类的研究进展

紫杉醇是红豆杉属植物次生代谢产物之一,是近20年来抗癌药物研究领域的重要发现。弄清楚紫杉醇合成途径和相关酶的反应可以从根本上大大提高紫杉醇的产量。综述近几年来紫杉醇生物合成途径中相关酶的研究工作,包括已经得到相应cDNA克隆的紫杉二烯合成酶,细胞色素P450氧化酶和3个紫杉烷的乙酰转移酶,由于GGPP是紫杉醇合成的必需前体,HMGR和GGPP合成酶的相关情况也有简述。     

胸径、构件和季节对南方红豆杉中紫杉醇和10-DAB含量的影响

系统采集了12个不同胸径的南方红豆杉当年生针叶和树皮样品,用超高效液相色谱(UPLC)测定其紫杉醇(taxol)和10-去乙酰基巴卡亭Ⅲ(10-deacetylbaccatin Ⅲ,10-DAB)含量,结果显示针叶的两种被测试物质含量的平均值分别为0.0127mg·g-1和0.0805mg·g-1,树皮的这两种物质含量平均值分别为0.1164mg·g-1和0.4842mg·g-1。当年生针叶、树皮中紫杉醇含量与胸径相关性不显著。对植株不同构件(枝条、针叶、树皮和根)中紫杉醇和10-DAB含量的测定表明,根中紫杉醇和10-DAB含量最高,当年生针叶紫杉醇含量最低。天然和人工栽培南方红豆杉当年生针叶中紫杉醇和10-DAB含量呈明显的季节变化,各月份间含量差异显著,人工栽培和天然植株针叶在生长速度明显减缓的10月份,紫杉醇和10-DAB含量均出现一个明显的峰值。     

Enhancement of Taxol production and excretion in Taxus chinensis cell culture by fungal elicitation and medium renewal

An endophytic fungus, Aspergillus niger, isolated from the inner bark of a Taxus chinensis tree, was used as an elicitor to stimulate the Taxol (paclitaxel) production in a Taxus chinensis cell suspension culture. Different elicitor doses and elicitation times were tested in a batch culture; and the highest volumetric Taxol yield was achieved when 40 mg of the fungal elicitor (carbohydrate equivalent) l(-1) was added to the culture during the late exponential-growth phase. The elicitation resulted in a more than two-fold increase in the Taxol yield and about a six-fold increase in total secretion. The Taxol yield was further improved substantially by applying medium renewal and re-elicitation to the culture. In particular, with repeated medium renewal (in a way similar to medium perfusion) and a second elicitation of the culture, the volumetric Taxol yield was increased to 67.1+/-7.5 mg l(-1), which was about seven times the amount obtained in the non-elicited batch culture. The Taxol productivity of the perfusion-like culture with repeated fungal elicitation was 1.5 mg l(-1) day(-1), which was about 40% higher than that of the elicitor-treated batch culture and three times the productivity of the non-elicited batch culture.     

从中国红豆杉细胞培养物中分离鉴定紫杉醇

中国红豆杉(Taxus chinensis(Pilger．)Rehd)细胞在培养过程中合成了紫杉醇,我们利用固液分离、大孔吸附树脂吸附富集、有机溶剂萃取、硅胶柱层析、低压硅胶柱层析、重结晶等方法,从中国红豆杉的细胞培养物中分离纯化了紫杉醇,用多种核磁共振波谱方法(^1H NMR,^13C NMR,DEPT,^1H-^1H-COSY,NOESY,HMQC,HMBC)结合质谱(FABMS),红外光谱、紫外光谱等方法鉴定了它的化学结构,证明与源于天然红豆杉植物材料提取的紫杉醇为同一物质。     

An endophytic taxol-producing fungus from Taxus x media, Aspergillus candidus MD3

An endophytic taxol-producing fungus (strain MD3) isolated from the inner bark of Taxus x media was identified as Aspergillus candidus according to its morphological characteristics, physiological and biochemical characteristics, and 18S rRNA gene sequence analysis. Taxol produced by A. candidus MD3 was shown to be identical to authentic taxol analyzed by UV, HPLC, MS and nuclear magnetic resonance spectroscopy. The gene encoding the 10-deacetylbaccatin III-10- O -acetyl transferase, which catalyzes formation of the last diterpene intermediate in the taxol biosynthetic pathway, has been cloned from A. candidus MD3 for the first time and possesses high homology to the same gene found in Taxus spp.     

Kinetics of taxol production, growth, and nutrient uptake in cell suspensions of Taxus cuspidata

Cell culture of Taxus cuspidata may represent an alternative to extraction of bark as a source of taxol and related taxanes. Cell suspensions of a cell line of T. cuspidata were grown for 44 days in shake flasks containing B5C2 medium. Throughout the growth cycle, fresh and dry weight accumulation, taxol yield on a dry weight basis, taxol accumulation in the medium, pH and pigmentation variation in the medium, as well as the uptake of sucrose, glucose, fructose, nitrate, and inorganic phosphate from the culture medium were examined. The results showed that the growth was relatively slow (doubling times of 17 and 20 days for fresh and dry weight, respectively), and taxol accumulation in the cells was non-growth related (higher in the stationary phase) and at relatively low levels (up to 4 mug/g of the extracted dry weight). Taxol concentration in the medium had two peaks: one during the early (0.4mug/mL) and another during the late (0.1-mug/mL) parts of the growth cycle. On a volumetric basis, the average total amount of taxol produced during the stationary phase (day 38) was 0.15 mug/mL, of which approximately 66% was in the medium and 34% was in the cells. Total carbohydrate uptake was closely associated with the increase in dry biomass. Sucrose was apparently extracellularly hydrolyzed after the first 6 days of culture; glucose was used before fructose. Nitrate was assimilated throughout the growth cycle, but phosphate was absorbed within the first week of culture. The pH variation showed an initial drop followed by a trend toward alkalinization for most of the growth period. Dark pigmentation in the medium increased progressively, particularly during the stationary phase. (c) 1994 John Wiley & Sons, Inc.     

基于定量PCR技术探讨紫杉醇生物合成的限速步骤

次生代谢产物牛物合成受到发育和诱导的调控,本实验研究了组织分化和诱导处理对紫杉醇生物合成的影响,并采用定量PCR技术分析了紫杉醇生物合成不同阶段关键酶基因的动态表达特征。结果表明。紫杉醇主要分布在中国红豆杉（Taxus chinensis）树皮和根皮组织中,针叶内含量很少,催化紫杉醇功能官能团连接的关键酶摹因也主要定位在树皮和根皮组织巾;茉莉酸甲酯（MJ）和真菌诱导子F5分别提高了中国红豆杉悬浮培养细胞HG-1紫杉醇得率8倍和10倍,同时有效诱导紫杉醇生物合成基因的表达。发现催化紫杉醇侧链连接的基因与紫杉醇生物合成早正相关。结果表明。紫杉醇生物合成的限速步骤是催化功能官能团连接的步骤。     

紫杉醇免疫检测方法的研究进展

紫杉醇是一种有效的抗肿瘤药物,广泛应用于治疗卵巢癌、乳腺癌和肺癌等癌症。紫杉醇在紫杉树皮中的含量极低（仅为0．01％）,而且紫杉醇是一种对蛋白质有着高亲和力的小分子,在体液中约有98％的分子与蛋白质结合,因此需要一种高灵敏度、高通量的检测方法对紫杉醇进行鉴定。在分析紫杉醇检测方法的基础之上,综述了紫杉醇免疫学检测方法的研究进展,包括紫杉醇半抗原的分子修饰、蛋白偶联物的构建和鉴定以及免疫学检测方法在植物组织和病人血浆中紫杉醇定性和定量中的应用。     

磷甘霉素和洛伐它汀处理对中国红豆杉悬浮培养细胞生物合成紫杉醇的影响

采用非甲羟戊酸途径抑制剂磷甘霉素和甲羟戊酸途径抑制剂洛伐它汀对中国红豆杉悬浮细胞培养物进行处理.在添加和未添加茉莉酸甲酯诱导的情况下,前者使紫杉醇产量减少了2/5和1/5,后者使紫杉醇产量减少了1/6和1/10,表明两种途径对紫杉醇的生物合成都具有贡献,其中非甲羟戊酸途径贡献较大;通过定量PCR技术分别检测两条途径的关键酶5-磷酸脱氧木酮糖还原异构酶(DXR)和3-羟基-3-甲基戊二酰辅酶A还原酶(HMGR)mRNA水平的变化,发现两种抑制剂都能够激活hmgr和dxr的转录,表明两种代谢途径之间存在协同作用,共同为紫杉醇的生物合成提供前体.     

产紫杉醇真菌的研究概况与紫杉醇工业生产的一个新思路

分离自太平洋红豆杉的天然抗癌药物紫杉醇(Taxol)已在临床上广泛应用,市场需求日益增强,但工业产量受原料红豆杉树木短缺的制约,供需存在巨大差距。1993年,Stierle等分离到一株与太平洋红豆杉共生的真菌安德烈紫杉菌,证实产生紫杉醇,为利用真菌发酵生产紫杉醇带来可能。通过分析目前工业生产技术存在的问题,总结了产紫杉醇真菌研究的进展及其重要意义。认为利用真菌大规模发酵生产紫杉醇或其中间体,是摆脱制约的一条新思路,具有广阔的应用前景,并且可以带动其他产品的开发。     

溶氧水平对红豆杉细胞悬浮培养的影响研究

紫杉醇 (Ｔａｘｏｌ)是源自红豆杉提取物的一种高度衍生化的二萜类化合物 ,临床实验结果表明紫杉醇对于卵巢癌、乳腺癌、胃肠道癌等具有明显的抗肿瘤活性[1] ,因而受到世界各国的广泛关注 ,并已被美国食品与药品管理局 (ＦＤＡ)批准用于卵巢癌与乳腺癌的治疗[2 ] 。到目前为止紫杉醇仍然主要从树皮中提取 ,但由于红豆杉生长缓慢 ,天然资源非常有限 ,加快其替代来源的研究势在必行。利用植物细胞悬浮培养生产紫杉醇作为一种可行的选择 ,近年来取得了较大的进展[3 ,4 ] 。本文研究了摇瓶及 2 0 Ｌ反应器培养过程的溶氧水平对细胞生长及紫杉醇…     

Supercritical fluid extraction of taxol and baccatin III from needles of Taxus cuspidata

Summary Taxol and baccatin III were extracted from the ground needles of Taxus cuspidata using supercritical carbon dioxide mixed with 3 wt % ethanol as a cosolvent. The pressure and temperature ranges used to attain supercritical fluid condition are 100300 bar and 4070 °C, respectively. However, the amount of taxol and baccatin III in the extract obtained at 100 bar was not noticeable, while the major portion of extract was found to be the waxy compounds. The highest selectivity of taxol and baccatin III were about 0.094 and 0.158 wt %, respectively, at 40 °C and 300 bar. At the same pressure and temperature condition, taxol and baccatin III selectivities in the extract obtained from the ground seeds of Taxus cuspidata was about 0.198 and 0.157 wt %, respectively.     

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.     

The shape of things to come: structural and synthetic studies of taxol and related compounds

The history of the development of Taxol (paclitaxel) as an anticancer drug is reviewed, and some aspects of the phytochemistry of Taxus species and of the medicinal chemistry of taxol are discussed. The nature of the taxol-tubulin interaction is then described, with an emphasis on studies that led to the discovery and experimental proof of the T-taxol conformation as the tubulin-binding conformation of taxol. The implications of this conformation for future drug development are also briefly covered.     

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.     

聚乙二醇对东北红豆杉培养细胞的

在改良的B