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放线菌野生株代谢功能的核糖体工程改造与新产抗肿瘤活性产物研究
引用本文:韩晓,崔承彬,韩小贤,李长伟,杨明.放线菌野生株代谢功能的核糖体工程改造与新产抗肿瘤活性产物研究[J].国际药学研究杂志,2009,36(6):435-442,446.
作者姓名:韩晓  崔承彬  韩小贤  李长伟  杨明
作者单位:军事医学科学院毒物药物研究所,北京,100850
基金项目:国家自然科学基金,国家863计划,国家"重大新药创制"科技重大专项,中国大洋协会国际海底区域研究开发项目,军事医学科学院科研创新基金重大专项 
摘    要:目的 利用核糖体工程技术,改造放线菌次级代谢功能,筛选获得抗肿瘤活性突变株,并对突变株新产活性产物进行研究。方法 以海洋来源无活性放线菌野生株HLF-39和HLF-43为出发菌,通过单菌落挑选与平板划线培养,分离纯化链霉素抗性突变株,通过摇床液态发酵和发酵提取物乙酸乙酯萃取样品的抗肿瘤活性筛选,获得抗肿瘤活性突变株;采用活性跟踪与微量预试先导 放大实验制备组合的实验模式,与原始菌样品对照比较,在快速确定差异活性斑点基础上,组合各种色谱技术,分离纯化突变株新产抗肿瘤活性产物;根据理化性质和波谱数据鉴定化合物结构;采用MTT法测评抗肿瘤活性。结果 链霉素对HLF-39和HLF-43的最低抑菌浓度分别为3和10 mg·L-1。经抗性筛选,得到对链霉素产生抗性的HLF-39突变株28株、HLF-43突变株204株。在所得突变株中,3株HLF-39突变株和14株HLF-43突变株有抗肿瘤活性,100 mg·L-1样品对K562细胞的抑制率大于20%;其中,4株分别对11、100、200和300 mg·L-1链霉素产生抗性的HLF-43突变株CHS-21101、CHS-210010、CHS-220002和CHS-230001活性显著,100 mg·L-1样品对K562细胞的抑制率分别达59.5%、50.0%、44.1%和59.6%。从CHS-21101发酵物中分离得到2个该突变株新产抗肿瘤活性产物,并分别鉴定为环(4-羟脯-亮)二肽(1)和phencomycin(2)。化合物1和2对K562细胞呈抑制活性,100 mg·L-1抑制率分别为33.9%和21.4%。结论 利用核糖体工程抗性筛选技术,从2株无活性放线菌野生菌株成功筛选得到17株具有抗肿瘤活性的链霉素抗性突变株,从其中1株发酵物中分离鉴定了2个该突变株新产抗肿瘤活性产物。用核糖体工程技术改造无活性放线菌野生株的次级代谢功能,可筛选获得活性突变株并提供深入研究新产活性产物,从中筛选新药及其先导结构,从而拓展药源放线菌活性菌株新资源。

关 键 词:海洋微生物  放线菌  核糖体工程  链霉素抗性  抗肿瘤活性  环(4-羟脯-亮)二肽  phencomycin
收稿时间:2009-8-7

Alteration of metabolic function of wild-type actinomycete strain by ribosome engineering and the metabolites newly produced with antitumor activity
HAN Xiao,CUI Cheng-bin,HAN Xiao-xian,LI Chang-wei,YANG Ming.Alteration of metabolic function of wild-type actinomycete strain by ribosome engineering and the metabolites newly produced with antitumor activity[J].Foreign Medical Sciences(Section of Pharmarcy),2009,36(6):435-442,446.
Authors:HAN Xiao  CUI Cheng-bin  HAN Xiao-xian  LI Chang-wei  YANG Ming
Institution:(Institute of Pharmacology and Toxicology, Academy of Military Medical Sciences, Beijing 100850, China)
Abstract:Objective To alter the secondary metabolic function of wild-type actinomycete strains for investigating metabolites newly produced with antitumor activity by ribosome engineering. Methods Two marine-derived actinomycete strains HLF-39 and HLF-43 were used as initial strains, which do not produce metabolites showing antitumor activity under the given condition in present study. The streptomycin-resistant (str) mutants from HLF-39 and HLF-43 could be selected by single colony isolation on streptomycin-containing plates according to the method for obtaining drug-resistant mutants in ribosome engineering. The two initial stains and their str mutants were fermented in a liquid medium with the same composition without (for HLF-39 and HLF-43) or with (for str mutants) the relevant concentration of streptomycin and the samples for bioassay could be obtained by extraction of the whole fermentation broth with 66% aqueous acetone, followed by extraction of the aqueous acetone solution obtained, with the same volume of EtOAc, after removal of the acetone through evaporating under reduced pressure. Bioactive metabolites produced by a str mutant, CHS-21101 from HLF-43, were isolated by a bioassay-guided separation procedure with various chromatographic means, combined with direct comparison with the sample from initial strain HLF-43. Structures of the compounds obtained were identified by spectral analysis. The antitumor activity was assayed by MTT method using K562 cells for the crud EtOAc extracts and the compounds obtained. Results The minimal inhibitory concentration (MIC) of streptomycin for HLF-39 and HLF-43 was 3 and 10 mg·L-1 respectively. Total 28 and 204 str mutants were obtained from HLF-39 and HLF-43, respectively, which were resistant to different concentrations of streptomycin ranging 1 to 30 fold of MIC. In bioassay, samples from HLF-39 and HLF-43 showed almost no inhibitory effect on the proliferation of K562 cells even at the high concentration of 1000 mg·L-1 with the inhibition rates lower than 5.6% (HLF-39) and 3.9% (HLF-43) in repeated independent experiments. However, samples from 3 str mutants of HLF-39 and from 14 str mutants of HLF-43 inhibited the proliferation of K562 cells with the inhibition rates over 20% at 100 mg·L-1. Among them, the samples from four str mutants, CHS-21101, CHS-210010, CHS-220002 and CHS-230001, which all were obtained from HLF-43 and resistant to 11, 100, 200 and 300 mg·L-1 streptomycin respectively, showed significant inhibitory capacities with the inhibition rates 59.5%, 50.0%, 44.1% and 59.6% at 100 mg·L-1, respectively. From the fermentation broth of str mutant CHS-21101, two bioactive metabolites 1 and 2 being newly produced by the mutant were isolated and they were identified as cyclo (4-hydroxy-Pro-Leu) (1) and phencomycin (2). Compounds 1 and 2 inhibited the proliferation of K562 cells with the inhibition rates, 33.9% and 21.4% at 100 mg·L-1, respectively. Conclusion Present results instantiated a feasible direction for the development of applied ribosome engineering, that is, the metabolic function of wild-type actinomycetes that do not produce bioactive metabolites can be altered by ribosome engineering using convenient antibiotic-resistant mutation technique to obtain the bioactive metabolites (even those with novel structure) being newly produced. And thus, it is reasonable to consider that the ribosome engineering technique might be applicable to the exploitation of new microbial strain resource for drug screening from the actinomycete strains that do not produce bioactive metabolites.
Keywords:marine-derived microorganism  actinomycete  ribosome engineering  streptomycinresistance  antitumor activity  cyclo-(4-hydroxy-Pro-Leu)  phencomycin
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