用链霉素抗性筛选技术拓展放线菌药用菌株资源的研究
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摘要
在微生物活性菌株筛选过程中,分离得到的绝大部分菌株往往因在所采用的活性指标下不显示任何生物活性而被闲置或销毁,造成前期投入的极大浪费和资源开发利用效率严重低下。如何二次开发这些菌株资源是具有实际意义的重要研究课题。微生物对抗生素的抗性突变不仅诱导了核糖体结构的改变,使突变株获得了对某些抗生素的抗性,同时,也改变或激活了其次级代谢产物的某些生物合成调控系统,使突变株生产某些代谢产物的能力大幅度提高或者使突变株代谢生产母株原不生产的代谢产物。鉴于此,本研究基于这些发现利用抗生素抗性筛选技术,开展了二次开发无活性放线菌菌株的相关方法学探索研究。
首先本文从渤海湾的海洋样品中分离得到127株海洋微生物,其中真菌80株,放线菌47株,并以人白血病K562细胞为受试细胞,采用MTT法结合显微镜下细胞形态学检测的方法,筛选了这些菌株发酵样品的抗肿瘤活性,经初筛和复筛,得到100μg/mL浓度下对K562细胞的抑制率大于40%的活性菌株8株(占菌株总数的6.3% ,其中放线菌4株,占放线菌数的8.5%,真菌4株,占真菌数的5.0%)。抑制率在20%~40%的放线菌6株(占放线菌数的12.7%)和大量无活性菌株,这些活性菌株为寻找抗肿瘤活性先导化合物的研究提供了活性菌株资源,同时得到的无活性菌株为核糖体工程拓展微生物药源菌株资源的研究提供了菌株来源。
继而,以2株无活性菌株HLF-39和HLF-43为出发菌,在分别测定链霉素MIC的基础上。利用在会大于MIC的不同浓度链霉素的固体平板培养基,进行了抗性筛选,结果得到了232株对不同浓度链霉素产生抗性的突变株。突变株的菌落形态、孢子颜色以及代谢产物与母体菌株有显著的差异。利用上述相同抗肿瘤活性模型对突变株进行了抗肿瘤活性筛选,获得100μg/mL样品浓度下对K562细胞的抑制率大于40%的活性突变株4株。
对其中一株具有显著抗肿瘤活性的突变株CHS-21101采用活性追踪的方法对其发酵产物的活性成分进行了初步分离,得到2个单体化合物。利用波谱学方法阐明了2个化合物的结构为环(4-羟基-脯氨酸-亮氨酸)和Phencomycin,其中化合物1在100μg/mL对K562细胞有明显的增殖抑制作用。
综上,本论文利用链霉素抗性筛选技术对2株没有抗肿瘤活性的海洋放线菌进行了抗性筛选,得到突变株232株,经对所得突变株发酵产物的活性筛选,得到具有显著抗肿瘤活性的突变株4株。并经对其中一株活性突变株的代谢产物进行初步研究,得到了2个单体化合物,分别鉴定为环(4-羟基-脯氨酸-亮氨酸)和Phencomycin。
这些研究结果表明链霉素抗性筛选技术用于开拓药源微生物活性菌株资源是基本可行的。该方法简单易行,利用该方法对无活性放线菌菌株进行二次开发,将会有效拓展药源微生物菌株来源。
When we screen the activity of microorganism, a majority of strains don’t show any activity under the available model. The improvement of the productivity of microbial strains is an important field in secondary metabolites from microorganisms. That how to make good use of the non-active microorganisms under the available model is also interesting. It was found that antibiotic productions could be activated dramatically by a certain ribosomal mutation and an idea occurred that bacterial gene expressions may be changed dramatically by modulating the ribosomal proteins or rRNA, which could eventually lead to the activation of inactive (silent) genes. One of the most conventional ways to modulating the ribosome is the introduction of mutations conferring resistance to antibiotics that attack the ribosome. The research focused on exploiting new microbial resources for medicinal uses based on ribosome-engineering.
Total 127 microbial strains, including 80 fungi and 47 actinomycetes, were isolated from soil samples collected at the tideland of Bohai Bay around Lüjühe in Tanggu district of Tianjin, China. The anti-tumor activity was assayed by MTT method using K562 cells together with morphological observation of K562 cells under inversed light microscope. Among them, samples from 4 actinomycetic and 4 fungal strains inhibited the proliferation of K562 cells with the inhibition rates over 40% at the 100μg/mL of concentration, while the samples from another 6 actinomycetic strains showed anti-tumor activity with inhibition rates ranging in 20% to 40% at the 100μg/mL concentration. The 80 fungal and 47 actinomycetic strains were isolated from the Bohai marine-environment samples, from which, the 4 fungal and 10 actinomycetic strains that produce metabolites with anti-tumor activity have been obtained. The frequency of obtaining the bioactive metabolite producing strains is obviously higher in actinomycetes than in fungi. These microbial strains including both the bioactive metabolite producing and non-producing strains have provided microbial strain resources for further studies on the bioactive metabolites and on the alteration of metabolic capacity of microbes to produce bioactive metabolites by ribosome-engineering, respectively.
A mutation CHS-21101 (streptomycin-resistant) showed stronger cytoclasis bioactivity on K562 cells It was selected to be fermented. The whole fermentation broth was separated by the means of modern chromatographic methods in a bioassay-guided separation manner. From the bioactive part, EtOH extract, tow compounds were isolated by silica gel, Sephadex LH-20 and reverse-phase high performance liquid chromatragraphy. The structures of one compounds were elucidated mainly by use of spectroscopic methods. Among them, two compounds showed the antitumor activities by the use of MTT and flow cytometry using mammalian cancer K562 cells.
In summary, Ribosome-engineering technology was stochastically carried out on 2 non-active strains by inducing the antibiotic resistance and 232 mutated strains were obtained. 4 mutated strains, which showed anti-tumor activity with inhibition rates ranging over 40% at the 100μg/mL concentration.
This research proves that it is feasible to exploit new microbial resources for medicinal usage by applying the ribosome-engineering technology. This method is economical and simple to be carried out. The silent genes of microorganisms can be activated and more and more metabolites with novel structures can be obtained by using this ribosome-engineering technology.
首先本文从渤海湾的海洋样品中分离得到127株海洋微生物,其中真菌80株,放线菌47株,并以人白血病K562细胞为受试细胞,采用MTT法结合显微镜下细胞形态学检测的方法,筛选了这些菌株发酵样品的抗肿瘤活性,经初筛和复筛,得到100μg/mL浓度下对K562细胞的抑制率大于40%的活性菌株8株(占菌株总数的6.3% ,其中放线菌4株,占放线菌数的8.5%,真菌4株,占真菌数的5.0%)。抑制率在20%~40%的放线菌6株(占放线菌数的12.7%)和大量无活性菌株,这些活性菌株为寻找抗肿瘤活性先导化合物的研究提供了活性菌株资源,同时得到的无活性菌株为核糖体工程拓展微生物药源菌株资源的研究提供了菌株来源。
继而,以2株无活性菌株HLF-39和HLF-43为出发菌,在分别测定链霉素MIC的基础上。利用在会大于MIC的不同浓度链霉素的固体平板培养基,进行了抗性筛选,结果得到了232株对不同浓度链霉素产生抗性的突变株。突变株的菌落形态、孢子颜色以及代谢产物与母体菌株有显著的差异。利用上述相同抗肿瘤活性模型对突变株进行了抗肿瘤活性筛选,获得100μg/mL样品浓度下对K562细胞的抑制率大于40%的活性突变株4株。
对其中一株具有显著抗肿瘤活性的突变株CHS-21101采用活性追踪的方法对其发酵产物的活性成分进行了初步分离,得到2个单体化合物。利用波谱学方法阐明了2个化合物的结构为环(4-羟基-脯氨酸-亮氨酸)和Phencomycin,其中化合物1在100μg/mL对K562细胞有明显的增殖抑制作用。
综上,本论文利用链霉素抗性筛选技术对2株没有抗肿瘤活性的海洋放线菌进行了抗性筛选,得到突变株232株,经对所得突变株发酵产物的活性筛选,得到具有显著抗肿瘤活性的突变株4株。并经对其中一株活性突变株的代谢产物进行初步研究,得到了2个单体化合物,分别鉴定为环(4-羟基-脯氨酸-亮氨酸)和Phencomycin。
这些研究结果表明链霉素抗性筛选技术用于开拓药源微生物活性菌株资源是基本可行的。该方法简单易行,利用该方法对无活性放线菌菌株进行二次开发,将会有效拓展药源微生物菌株来源。
When we screen the activity of microorganism, a majority of strains don’t show any activity under the available model. The improvement of the productivity of microbial strains is an important field in secondary metabolites from microorganisms. That how to make good use of the non-active microorganisms under the available model is also interesting. It was found that antibiotic productions could be activated dramatically by a certain ribosomal mutation and an idea occurred that bacterial gene expressions may be changed dramatically by modulating the ribosomal proteins or rRNA, which could eventually lead to the activation of inactive (silent) genes. One of the most conventional ways to modulating the ribosome is the introduction of mutations conferring resistance to antibiotics that attack the ribosome. The research focused on exploiting new microbial resources for medicinal uses based on ribosome-engineering.
Total 127 microbial strains, including 80 fungi and 47 actinomycetes, were isolated from soil samples collected at the tideland of Bohai Bay around Lüjühe in Tanggu district of Tianjin, China. The anti-tumor activity was assayed by MTT method using K562 cells together with morphological observation of K562 cells under inversed light microscope. Among them, samples from 4 actinomycetic and 4 fungal strains inhibited the proliferation of K562 cells with the inhibition rates over 40% at the 100μg/mL of concentration, while the samples from another 6 actinomycetic strains showed anti-tumor activity with inhibition rates ranging in 20% to 40% at the 100μg/mL concentration. The 80 fungal and 47 actinomycetic strains were isolated from the Bohai marine-environment samples, from which, the 4 fungal and 10 actinomycetic strains that produce metabolites with anti-tumor activity have been obtained. The frequency of obtaining the bioactive metabolite producing strains is obviously higher in actinomycetes than in fungi. These microbial strains including both the bioactive metabolite producing and non-producing strains have provided microbial strain resources for further studies on the bioactive metabolites and on the alteration of metabolic capacity of microbes to produce bioactive metabolites by ribosome-engineering, respectively.
A mutation CHS-21101 (streptomycin-resistant) showed stronger cytoclasis bioactivity on K562 cells It was selected to be fermented. The whole fermentation broth was separated by the means of modern chromatographic methods in a bioassay-guided separation manner. From the bioactive part, EtOH extract, tow compounds were isolated by silica gel, Sephadex LH-20 and reverse-phase high performance liquid chromatragraphy. The structures of one compounds were elucidated mainly by use of spectroscopic methods. Among them, two compounds showed the antitumor activities by the use of MTT and flow cytometry using mammalian cancer K562 cells.
In summary, Ribosome-engineering technology was stochastically carried out on 2 non-active strains by inducing the antibiotic resistance and 232 mutated strains were obtained. 4 mutated strains, which showed anti-tumor activity with inhibition rates ranging over 40% at the 100μg/mL concentration.
This research proves that it is feasible to exploit new microbial resources for medicinal usage by applying the ribosome-engineering technology. This method is economical and simple to be carried out. The silent genes of microorganisms can be activated and more and more metabolites with novel structures can be obtained by using this ribosome-engineering technology.
引文
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[1] 王长云,耿美玉,管华诗.海洋药物研究进展与发展趋势[J].中国新药杂志,2005,14(3):278.
[2] 邵顺波.近年来海洋药物化学成分及功效的研究概况[J].安徽医药,2005,9(11):861.
[3] Faulkner D.J..Marine Natural Products,Nat.Prod.Rep.,1998,15(2):113~189
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[5] Crews P.,Farias J.J.,Emrich R.et al..Milnamide A, anunusual cytotoxic tripeptide fromthe marine sponge Auletta of constricta,J.Org.Chem.,1994,59:2 932~2 934
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[1] 王长云,耿美玉,管华诗.海洋药物研究进展与发展趋势[J].中国新药杂志,2005,14(3):278.
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[3] Faulkner D.J..Marine Natural Products,Nat.Prod.Rep.,1998,15(2):113~189
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[5] Crews P.,Farias J.J.,Emrich R.et al..Milnamide A, anunusual cytotoxic tripeptide fromthe marine sponge Auletta of constricta,J.Org.Chem.,1994,59:2 932~2 934
[6]董志峰、李新萍等。基因工程与海洋药物研究,海洋科学,1998,1:16~20
[7] Che Ct..海洋产物作为抗病毒导向药物的来源,国外医学药学分册,1992,19(3):133~135
[8] Osawa R.etal..An investigationofqauatic bacteria capable of utilizing chitin as the sole source of nutrients, Lett.Appl.Microbiol.,1995, 21(5): 288~291.
[9] LzumidaH.etal..Anovel chitinase inhibitorforma marinebacterium, Pseudomonas sp., J .Antibiot.,1996,49(1): 76~80
[10] 刘志鸿、程力、牟海津。海洋微生物活性物质的研究概况,中国水产科学,1999,6(4):99~103
[11]许实波等。几种不饱和内酯二萜类海洋天然产物的抗肿瘤作用,中国海洋药物,1994,1(49):1~5
[12] 陈菊娣等。花刺参粘多糖的分离研究,中国海洋药物,1994,1(49):24~26
[13] 汤国枝、张鹤云.。一种具有刺激红系细胞集落生成的螺旋藻蛋白,南京大学学报,1994,30(2):337~380
[14] Orjala.J., Gerwick W.H..Barbamide,a chlorinatedmetabolite with molluscida activity from the Caribbean cyanbacterium Lyngbyamajuscula,J. Nat.Prod., 1996,59(4): 427~430
[15] 韦成礼、李玲、游丽云。国内外从海洋微生物开发新型药物的研究概况,生物技术通报杂志,1998,2:1~4
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