南极放线菌药用资源的调查及次级代谢产物研究
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摘要
放线菌是一类最重要的药源微生物,探索放线菌新资源,研究其药用价值对于创新药物及其先导化合物的发现具有重要意义。南极是重要的微生物种源地,但对其中的放线菌资源尚未有系统深入的研究。本论文以南极环境放线菌这一药用资源为研究对象,通过分离南极放线菌资源,研究南极环境放线菌资源的多样性、新颖性;采用生物活性筛选和化学筛选相结合的筛选模式筛选其代谢产物的抗肿瘤活性;通过放线菌次级代谢产物生物合成酶的基因筛选,考察其次级代谢产物的合成能力;选择2株放线菌分离其代谢产物,研究其代谢产物的新颖性、多样性。
从18个南极土壤样品中分离得到了105株放线菌,采用BOX-PCR法对分离的105株放线菌进行了聚类分析及排重,获得67株不同种属的南极放线菌。通过BOX-PCR的聚类分析,发现23株菌的BOX-PCR聚类分析相似度较低。对这23株放线菌的16S rRNA基因序列进行了扩增和blast比对,比对结果显示这些菌株分属于Streptomyces,Arthrobacter,Nocardiopsis,Saccharopolyspora等属的不同种,显示了南极放线菌物种丰富多样;比对结果还表明,有5株南极放线菌与已知标准菌株的16S rRNA同源性低于98%。通过构建系统进化树分析,这5株放线菌可能为放线菌的新种。
采用海虾生物致死法和P388细胞为模型的筛选模型,以细胞周期抑制、细胞凋亡诱导以及坏死性细胞毒为活性指标,结合化学筛选,获得具有活性且次级代谢产物丰富的活性菌株12株。
对南极放线菌次级代谢产物合成中的聚酮合成酶(PKSⅠ)、非核糖体肽合成酶(NRPS)和卤化酶(helogenase)基因进行了基因筛选,获得了具有产肽类化合物能力的菌株46株、产聚酮类化合物能力的菌株39株和产含卤素成分化合物能力的菌株11株。
采用正相和反相硅胶柱色谱、Sephadex LH-20凝胶柱色谱、半制备反相高压液相色谱等分离方法,从Sreptomyces sp.ZS22-3中分离得到6个化合物(1-6),从Sreptomyces sp.GW25-5中分离得到11个化合物(7– 17)。
采用现代波谱技术(UV、IR、NMR、MS),并结合其理化性质,阐明了17个化合物的化学结构(化合物结构参见Fig. 1);其中新化合物3个,为放线菌素类化合物(3-5)。
研究结果表明,南极地区放线菌资源新颖丰富,是新化合物及活性先导结构的重要来源。通过研究,对南极放线菌药用资源及其应用价值有了深入的了解,为开拓药用微生物资源的新领域,同时也为极地微生物资源的合理有效利用提供必要的依据。
Actinomycetes are the most economically and biotechnologically valuable prokaryotes. They are responsible for the production of about half of the discovered bioactive secondary metabolites. Because of the excellent track record of actinomycetes in this regard, it is significant that new groups of actinomycetes from unexplored or underexploited habitats be pursued as sources of novel bioactive secondary metabolites. As an important microorganism resource, Antarctica actinomycetes,howerver, have not been researched systematically till now. In this paper, the diversity and novelty of Antarctic actinomycetes have been studied. Antitumor bioactivity was been screening combined with chemical screening. The NRPS, PKS and helogenase genes of actinomycetes have been screening by PCR. The study of secondary metabolites produced by two Antactic actinomycetes strains was carried out to investigate the potential anti-tumor compounds.
105 strains were isolated from 18 Antarctic soil samples. After dereplicated by BOX-PCR, there are 67 different strains remained. According to BOX-PCR genomic fingerprinting cluster analysis, 23 strains were selected to be amplified the 16S rDNA sequences. The sequences blast results showed that the 23 strains belong to family Streptomyces,Arthrobacter,Nocardiopsis and Saccharopolyspora respectively. There are 5 strains that the similarities between their 16S rDNA sequences and those of standard strains in the NCBI database are less than 98%. According to phylogenetic analysis, these 5 strains are likely to be novel actinomycetes.
By combinatory use of brine shrimp bioassay testing, flow cytometry bioassay against P388 cell line, and chemical analysis, 12 bioactive strains have been screened from 67 strains.
The modular Polyketide synthase (PKS), nonribosomal Peptide synthetase (NRPS) and helogenase have been found to be involved in natural Products synthesis in many microorganisms. In this study, the PKS gene, NRPS gene and helogenase gene have been screening. There are 46 strains containing NRPS gene, 39 strains containing PKS gene and 11 strains containing helogenase gene.
Monitoring by bioassay-guided isolation, the active fraction is chromato- graphed on silica gel, Sephadex LH-20 and prepared TLC, and preparative reversed phase HPLC, respectively, 17 compounds were isolated from the two active strains. Six compounds (1-6) were isolated from Sreptomyces sp. ZS22-3, eleven compounds (7-17) from Sreptomyces sp. GW25-5.
By means of modern spectral analysis (UV, IR, NMR, MS) and physico-chemical properties, the structures (see Fig.1) of seventeen pure compounds were respectively determined. Among them there are three new compounds, which are actinomycin-type compounds(3-5). In addition, the chemical structure types of compounds are mainly involved in cyclic dipeptides (11-17),nucleoside (9,10), Benzene derivates (6,8), etc.
The results revealed the novelty and diversity of Anarctic actinomycetes and their secondary metabolites. Antarctic actinomycetes are important resources for bioactive secondary metabolites and they have great value for further study and development.
从18个南极土壤样品中分离得到了105株放线菌,采用BOX-PCR法对分离的105株放线菌进行了聚类分析及排重,获得67株不同种属的南极放线菌。通过BOX-PCR的聚类分析,发现23株菌的BOX-PCR聚类分析相似度较低。对这23株放线菌的16S rRNA基因序列进行了扩增和blast比对,比对结果显示这些菌株分属于Streptomyces,Arthrobacter,Nocardiopsis,Saccharopolyspora等属的不同种,显示了南极放线菌物种丰富多样;比对结果还表明,有5株南极放线菌与已知标准菌株的16S rRNA同源性低于98%。通过构建系统进化树分析,这5株放线菌可能为放线菌的新种。
采用海虾生物致死法和P388细胞为模型的筛选模型,以细胞周期抑制、细胞凋亡诱导以及坏死性细胞毒为活性指标,结合化学筛选,获得具有活性且次级代谢产物丰富的活性菌株12株。
对南极放线菌次级代谢产物合成中的聚酮合成酶(PKSⅠ)、非核糖体肽合成酶(NRPS)和卤化酶(helogenase)基因进行了基因筛选,获得了具有产肽类化合物能力的菌株46株、产聚酮类化合物能力的菌株39株和产含卤素成分化合物能力的菌株11株。
采用正相和反相硅胶柱色谱、Sephadex LH-20凝胶柱色谱、半制备反相高压液相色谱等分离方法,从Sreptomyces sp.ZS22-3中分离得到6个化合物(1-6),从Sreptomyces sp.GW25-5中分离得到11个化合物(7– 17)。
采用现代波谱技术(UV、IR、NMR、MS),并结合其理化性质,阐明了17个化合物的化学结构(化合物结构参见Fig. 1);其中新化合物3个,为放线菌素类化合物(3-5)。
研究结果表明,南极地区放线菌资源新颖丰富,是新化合物及活性先导结构的重要来源。通过研究,对南极放线菌药用资源及其应用价值有了深入的了解,为开拓药用微生物资源的新领域,同时也为极地微生物资源的合理有效利用提供必要的依据。
Actinomycetes are the most economically and biotechnologically valuable prokaryotes. They are responsible for the production of about half of the discovered bioactive secondary metabolites. Because of the excellent track record of actinomycetes in this regard, it is significant that new groups of actinomycetes from unexplored or underexploited habitats be pursued as sources of novel bioactive secondary metabolites. As an important microorganism resource, Antarctica actinomycetes,howerver, have not been researched systematically till now. In this paper, the diversity and novelty of Antarctic actinomycetes have been studied. Antitumor bioactivity was been screening combined with chemical screening. The NRPS, PKS and helogenase genes of actinomycetes have been screening by PCR. The study of secondary metabolites produced by two Antactic actinomycetes strains was carried out to investigate the potential anti-tumor compounds.
105 strains were isolated from 18 Antarctic soil samples. After dereplicated by BOX-PCR, there are 67 different strains remained. According to BOX-PCR genomic fingerprinting cluster analysis, 23 strains were selected to be amplified the 16S rDNA sequences. The sequences blast results showed that the 23 strains belong to family Streptomyces,Arthrobacter,Nocardiopsis and Saccharopolyspora respectively. There are 5 strains that the similarities between their 16S rDNA sequences and those of standard strains in the NCBI database are less than 98%. According to phylogenetic analysis, these 5 strains are likely to be novel actinomycetes.
By combinatory use of brine shrimp bioassay testing, flow cytometry bioassay against P388 cell line, and chemical analysis, 12 bioactive strains have been screened from 67 strains.
The modular Polyketide synthase (PKS), nonribosomal Peptide synthetase (NRPS) and helogenase have been found to be involved in natural Products synthesis in many microorganisms. In this study, the PKS gene, NRPS gene and helogenase gene have been screening. There are 46 strains containing NRPS gene, 39 strains containing PKS gene and 11 strains containing helogenase gene.
Monitoring by bioassay-guided isolation, the active fraction is chromato- graphed on silica gel, Sephadex LH-20 and prepared TLC, and preparative reversed phase HPLC, respectively, 17 compounds were isolated from the two active strains. Six compounds (1-6) were isolated from Sreptomyces sp. ZS22-3, eleven compounds (7-17) from Sreptomyces sp. GW25-5.
By means of modern spectral analysis (UV, IR, NMR, MS) and physico-chemical properties, the structures (see Fig.1) of seventeen pure compounds were respectively determined. Among them there are three new compounds, which are actinomycin-type compounds(3-5). In addition, the chemical structure types of compounds are mainly involved in cyclic dipeptides (11-17),nucleoside (9,10), Benzene derivates (6,8), etc.
The results revealed the novelty and diversity of Anarctic actinomycetes and their secondary metabolites. Antarctic actinomycetes are important resources for bioactive secondary metabolites and they have great value for further study and development.
引文
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