激光诱变玫瑰孢链霉菌结合链霉素抗性筛选法选育达托霉素高产菌株

将经过20 mW激光辐照20 min的达托霉素(Daptomycin)生产菌株-玫瑰孢链霉菌(Streptomyces roseosporus)D-38的孢子悬液倾注在含有1．9λg／mL链霉素的高氏一号培养平板上。通过链霉素抗性法筛选获得了10％正变率的突变株,其中突变株LC-54摇瓶发酵单位为81．2 mg／L,比出发菌株提高了39％。     

达托霉素耐药分子机制研究进展

环脂肽抗生素达托霉素抗菌活性强,致病菌不容易产生耐药性,已成为治疗革兰氏阳性菌特别是耐药菌感染的一线药物。但由于广泛使用,仍然出现了达托霉素耐药菌。细胞膜磷脂代谢和细胞壁结构动态与致病菌达托霉素耐药密切相关。文中综述了达托霉素作用机制和耐药机制,以期对药物研发和临床用药有所裨益。     

达托霉素生物合成研究进展

达托霉素是由玫瑰孢链霉菌(Streptomyces roseosporus)生产的一种环脂肽类抗生素, 具有强大的抗革兰氏阳性致病细菌的作用, 是继“抗生素最后一道防线”万古霉素后的新型抗生素。本文主要对达托霉素的结构、作用机制、合成基因簇及合成机制等当前的研究成果进行综述, 并且总结了利用组合生物学对达托霉素进行结构改造的策略, 以此来研究结构与活性之间的关系, 并寻找更广谱高效的抗生素。最后, 本文总结了提高达托霉素产量的策略, 为工业上降低达托霉素生产成本提供理论参考。     

达托霉素作为新的胰蛋白酶激活剂的初探

首次发现达托霉素是一种新的胰蛋白酶激活剂,当胰蛋白酶与达托霉素的物质的量的比在反应时达到34.05时,达托霉素对胰蛋白酶比活力的激活率平均达到32.92%。通过计算机模拟技术对达托霉素与胰蛋白酶以及达托霉素对胰蛋白酶底物复合物分别进行分子对接,并利用等温滴定量热法(Isothermal titration calorimetry,ITC)验证模拟结果。研究发现达托霉素与胰蛋白酶的活性中心位置很靠近,达托霉素链状中的天冬氨酸的R基与酶活性中心的组氨酸-57发生了氢键相互作用,达托霉素使酶和底物复合物结构更加稳定,从而有利于催化作用。ITC结果表明胰蛋白酶上具有1个达托霉素结合位点,解离常数Kd为17.83μM,摩尔结合焓△H为237.9±28.17 cal/mol,摩尔结合熵△S为22.5 cal/mol·deg,这些结果从热力学角度支持了分子对接结果。研究发现达托霉素除具有抗革兰氏阳性耐药性细菌的功能外,还能促进胰蛋白酶比活这一新的功能,是一种新的胰蛋白酶激活剂。     

达托霉素生产工艺及其衍生物组合生物合成

作为首个进入临床应用的环脂肽类抗生素,达托霉素自2003年上市以来,适应症不断增加,市场前景良好。达托霉素的制备工艺与结构修饰,已经成为了近年抗感染用药研发的一个热点。追溯了达托霉素的发现历程,生产工艺沿革。重点介绍了达托霉素的发酵工艺,合成基因簇研究进展,以及组合生物技术在达托霉素结构修饰中的应用。     

应用响应面法优化发酵培养基提高达托霉素产量

[背景]达托霉素来自玫瑰孢链霉菌NRRL 11379的发酵产物,是重要的临床用抗生素.其原始产生菌发酵周期长,影响达托霉素的生产效率.本实验室前期在天蓝色链霉菌中重构了达托霉素的生物合成途径,有效地缩短了发酵周期,但重组菌株K10中达托霉素发酵产量很低,制约了后续的研究和开发.[目的]利用响应面法优化产达托霉素的重组菌...     

以达托霉素产生菌菌株改造为主线的微生物工程综合实验的探索和实践

为使本科学生更好地掌握微生物工程的基本实验操作技能和前沿研究技术,探索将教师的科研成果转化为教学实验,构建了以达托霉素产生菌菌株改造为主线的微生物工程综合实验。在教学模式、实验内容设计、教学安排和考核方式等方面进行实践,取得了较好的教学效果,培养了本科学生的科研技能、科研思维和研究素质。     

亚硝基胍诱变选育林肯霉素高产菌株

以林肯链霉菌９４７－８（Ｓｔｒｅｐｔｏｍｙｃｅｓｌｉｎｃｏｌｎｅｎｓｉｓ９４７－８）为出发菌株（产林肯霉素９４０γ／ｍｌ）。采用孢子热处理方法处理出发菌株孢子,得到变异株９４７－８ｓ,产林肯霉素１０８０γ／ｍｌ。对９４７－８ｓ菌株进行ＮＴＧ诱变处理,得变异株９４７－８ｘ,产林肯霉素为１２１８γ／ｍｌ,且生产能力稳定。     

链霉菌AC-57及其产生的阿克拉霉素A的研究

During the course of screening for new antitumor antibiotics, a new anthracycline antibiotic--aclacinomycin A was separated from the broth and mycelium of Streptomyces AC-57. The strain AC-57 was isolated from the soil collected in the Shanghai suburbs. According to its culture and physiological characteristics the producer was identified as Str. galilaeus AC-57. The broth and mycelium were extracted and treated with solvents as usual way. The aclacinomycin A was separated by silica-gel column chromatography eluted with chrolo-form-methanol. Aclacinomycin A, its aglycone and sugar components were identified by comparison of their physico-chemical and spectral data (MS, UV, IR, 1H-NMR, and 13C-NMR) with authentic compound, purified from the market sample.     

一株妥布拉霉素高产菌株特性的研究

用高温和NTG处理暗黑链霉菌410-II(Streptomyces tenebrarius410-II)的孢子,得到六株无色突变株。所产抗生素只有两个组分,经分析为氨甲酰妥布拉霉素(carb…yltobramycin)和阿普拉霉素(Apramycin),不再含有出发菌株产生的氨甲酰卡那霉素(Carbamoylkanamycin)。在适宜的发酵条件下,所含两个组分的比例约为I：1。突变株基本不产生可溶性紫色素,发酵液离心所得上清液虽加入Fe冲,也不变成紫色,有利于提取工艺。六株突变株中W1028一M,所产抗生紊总效价比原株410-Il高20％以上,发酵液中氨甲酰妥布拉霉素含量比4100l提高约45—50％。由f组分减少,提取分离效率提高,在实验室用10升发酵液进行提取实验,妥布拉霉素单组分的收率达到25％o突变株w1028一M5是一株产量增高、收率提高、适用于生产的菌株。     

Rapid detection of `rare' actinomycetes in environmental samples

Natural products continue to be a useful source of new leads for the pharmaceutical industry. Actinomycetes are prolific producers of natural products and one strategy to increase the possibility of discovering novel chemical entities is to screen actinomycetes considered 'rare' in the environment and previously under-represented in natural product screening collections. We describe a method using bacteriophage as a marker to detect these actinomycetes in environmental samples. This method allows samples to be pre-screened for the presence of target actinomycetes before lengthy isolation programmes are undertaken.     

Characterization and analysis of antibiotic activity of some aquatic actinomycetes

Nine different isolates of aquatic actinomycetes identified as Streptomyces spp. were studied for their morphological and cultural characteristics. One of these isolates (C4-S, Streptomyces violaceusniger) was extensively studied for its inhibitory effect against a wide range of Gram-positive, Gram-negative bacteria, Mycobacterium vaccae ATCC 29678, Candida albicans and several food associated filamentous fungi and yeasts. Most of these were characterized by flexous sporophore morphology and their inability to produce cultural pigments. Bioassay results indicated that S. violaceusniger of 10 days culture age was highly active against Gram-positive cocci and bacilli with an inhibition zone of 16-25 mm, and slightly active against M. vaccae ATCC 29678 with an inhibition zone of 5-10 mm. The inhibitory effect was slight against Escherichia coli, Aspergillus niger 1 and C. albicans with an inhibition zone of 8-10 mm for each of them. There was no inhibitory effect of S. violaceusniger against other Gram-negative bacteria, filamentous fungi and yeast. The nature of the active molecule produced by S. violaceusniger showed a maximum absorption in the UV region at 210-260 nm.     

抗逆放线菌的多样性、功能特性及其在环境修复中的应用

放线菌(actinomycetes)是一类可生存于各种极端环境的特殊菌群,具备较强的抗逆特性。其种类丰富,功能多样,适应性强,已被广泛用于抗生素开发、生物防治和环境修复等领域。放线菌可调节土壤微生物群落结构、介导营养元素转化和植物吸收、催化有机污染物降解及重金属的氧化还原过程,该特性赋予其在土壤改良、地力维持和污染物去除等方面广阔的环境应用前景。本文综述了放线菌的多样性、环境分布以及放线菌对环境改良和污染物去除的特性与机制,结合在环境修复领域的应用现状,总结了放线菌修复技术的优势及发展方向。     

Characterization of selected actinomycetes degrading polyaromatic hydrocarbons in liquid culture and spiked soil

Summary Five strains of the Rhodococcus and Gordonia genera were evaluated for their potential use in bioremediation of polycyclic aromatic hydrocarbons (PAH) with or without another substrate (co-substrate). Their ability to produce biosurfactants or to degrade phenanthrene when growing on glucose, hexadecane and rapeseed oil was tested in liquid medium at 30 °C. All strains showed biosurfactant activity. The highest reduction in surface tension was recorded in whole cultures of Rhodococcus sp. DSM 44126 (23.1%) and R. erythropolis DSM 1069 (21.1%) grown on hexadecane and Gordonia sp. APB (20.4%) and R. erythropolis TA57 (18.2%) grown on rapeseed oil. Cultures of Gordonia sp. APB and G. rubripertincta formed emulsions when grown on rapeseed oil. After 14 days of incubation, Rhodococcus sp. DSM 44126 degraded phenanthrene (initial concentration 100 μg ml⁻¹) as sole carbon source (79.4%) and in the presence of hexadecane (80.6%), rapeseed oil (96.8%) and glucose (below the limit of detection). The other strains degraded less than 20%, and then with a co-substrate only. Rhodococcus sp. DSM 44126 was selected and its performance evaluated in soil spiked with a mixture of PAH (200 mg kg⁻¹). The effect of the addition of 0, 0.1 and 1% rapeseed oil as co-substrate was also tested. Inoculation enhanced the degradation of phenanthrene (55.7% and 95.2% with 0.1% oil and without oil respectively) and of anthracene (29.2% with 0.1% oil). Approximately 96% of anthracene and 62% of benzo(a)pyrene disappeared from the soil (inoculated and control) after 14 days and anthraquinone was detected as a metabolite. Rhodococcus sp. DSM 44126 was identified as Rhodococcus wratislaviensis by 16S rRNA sequencing and was able to degrade anthracene as sole carbon source in liquid culture.     

Characterization in Micromonospora inyoensis of aminoglycoside acetyltransferase activity not previously encountered among actinomycetes

Extracts of Micromonospora inyoensis contain aminoglycoside acetyltransferase activity not previously encountered among actinomycetes. Neomycin and its derivatives (excluding butirosin) are good substrates as is the novel aminoglycoside apramycin, whereas the gentamicins and kanamycins are utilized poorly if at all. Sisomicin, which is produced by M. inyoensis, is not acetylated. When neomycin was modified by extracts of M. inyoensis the product, most probably 3-N-acetylneomycin, was inactive against cell-free protein synthesis.     

Mycothiol-dependent proteins in actinomycetes

The pseudodisaccharide mycothiol is present in millimolar levels as the dominant thiol in most species of Actinomycetales. The primary role of mycothiol is to maintain the intracellular redox homeostasis. As such, it acts as an electron acceptor/donor and serves as a cofactor in detoxification reactions for alkylating agents, free radicals and xenobiotics. In addition, like glutathione, mycothiol may be involved in catabolic processes with an essential role for growth on recalcitrant chemicals such as aromatic compounds. Following a little over a decade of research since the discovery of mycothiol in 1994, we summarize the current knowledge about the role of mycothiol as an enzyme cofactor and consider possible mycothiol-dependent enzymes.