三株海洋放线菌聚酮类次级代谢产物的发现与过程优化
谢则平
学位类型博士
导师秦松
2012-05-16
学位授予单位中国科学院研究生院
学位授予地点北京
学位专业海洋化学
关键词海洋放线菌 聚酮类次级代谢产物 优化 分离纯化 结构鉴定
其他摘要     本研究从烟台附近潮间带的海泥样品中,分离到海洋放线菌348 株,并对其次级代谢产物进行了生物活性筛选,获得了它们对七株农业病原菌抗菌活性数据。发现具有选择性抗菌活性的比例约为34.8%。同时根据前人的实验数据,选取了本实验室前人分离的生物活性较好的30 株海洋放线菌进行了七株农业病原真菌的活性筛选。结果表明30 株放线菌的次级代谢产物均表现了选择性的抑菌活性,显示了海洋放线菌在筛选农用抗生素方面的巨大潜力。根据抗菌活性初筛结果,选取了活性较好的M268、M543、LM080、L242 及M086 五株放线菌,利用六种培养基,两种pH 和两种培养温度,共24 个培养条件对菌株进行了培养条件优化,通过生物活性筛选和化学筛选相结合,确定了优化范围内五株菌的最佳培养条件,分别为M543-ME-III、M268-FF-III、LM080-CL-III、L242-SM-IV 和M086-FF-II。与此同时,通过活性跟踪初步确定了优化条件下次级代谢产物的活性部位。
     通过对菌株优化条件下次级代谢产物的生物活性筛选及化学筛选结果,选取了M268、M543和LM080三株放线菌,分别按其最佳培养条件进行30 L或50 L发酵。综合利用正相、反相硅胶、Sephadex LH-20、PTLC、HPLC等各种色谱技术,共分离出33个化合物。通过综合波谱分析(1D、2D NMR和MS)、化学反应等方法鉴定了化合物的结构,包括3个新颖结构的聚酮化合物,Kiamycin(19),1,4-dimethoxy-3-(3-hydroxy-3-methyl-1-tetralone)-1(3H)-isobenzofuran A(20),1,4-dimethoxy-3-(3-hydroxy-3-methyl-1-tetralone)-1(3H)-isobenzofuran B(21);5个已知聚酮,Griseoviridin(1),Borrelidin(10),8-O-methyltetrangomycin(22),8-O-methylrabelomycin(23),X-14881 F(24);以及其他25个已知化合物如下:1-Acetyl-beta-carbolin(2),1-Methyluracil(3),
m-Hydroxybenzyl alcohol(4),Anthranilic acid(5),Vanillic acid(6),Poly-(hydroxybutyricacid)(7),(+)-Nonactic acid(8),(+)-Homononactic acid(9),1(10)E,5E-Germacradiene-3,11-diol(11),4S,7R-Germacra-1E,5E-dien-11-ol(12),3-Hydroxy-6-[(Z)-3'-hydroxy-2',4'-dimethyl-hept-4-enoylamino]-2,4-dimethyl-5-oxo-hex-anoic acid(13),p-Hydroxyphenylethyl alcohol(14),R-mevalonolactone(15),5'-Methylthioadenosine(16),(4S)-4,10,11-Trihydroxy-10-methyldodec-2-en-1,-olide(17),4,10-Dihydroxy-10-methyl-11-oxo-dodec-2-en-1,4-olide(18),E-1-(4'-hydroxyphenyl)-buten-3-one(25),Arbutin(26),Ferulic acid(27),3,4,5-Trihydroxybenzoicacid(28),4-methyl-2,6-dihydroxybenzaldehyde(29),2-Hydroxyphenyl-acetamide(30),thymine(31),β-carboline(32),1,2,3,4-tetrahydro-1-methyl-β-carboline-3-carboxylic acid(33)。
      对新结构化合物进行了生物活性筛选,其中化合物19 在100 μM 浓度时,对肿瘤细胞株HL-60、A549 和BEL-7402 的抑制率分别为68.2%,55.9%和31.7%;化合物20 在100 μM浓度时,对肿瘤细胞株HL-60、A549 和BEL-7402 的抑制率分别为4.4%,44.7%和30.7%,化合物20 无抑菌作用和抗乙酰胆碱脂酶活性。
      通过比较菌株M268 和W007 的16S rDNA 序列,发现来源于相同泥样的两株菌序列完全相同。因此基于W007 的全基因组序列,通过比较类似结构的生物合成基因的核苷酸序列,推测了来源于M268 的苯并蒽醌类聚酮化合物的生物合成途径。;       In this study, 348 isolateds had been isolated from coastal zone nearby Yantai, Shangdong Province. The bioactivities screening of the isolateds showed that the ratio of active strains
against 7 test agricultural pathogens applied in our experiments was around 34.8%. Meanwhile, another 30 marine actinomycetes saved in our lab were also selected to evaluate antifungal
activity against 7 agricultural pathogens, they all showed selective inhibitions. M268、M543、LM080、L242 and M086 were selected on the basis of the chemical screening and bio-screening for further optimization of fermentation research, and the optimum culture conditions were M268-FF-III、M543-ME-III、LM080-CL-III、L242-SM-IV and M086-FF-II,respectively.
      M268、M543 and LM080 were fermented for 30 L under optimum culture conditions according to optimization results, 33 compounds were obtained from the fermentation broth. And
the structures were established on the basis of extensive spectroscopic analyses including 1D and 2D NMR (1H-1H COSY, TOCSY, HSQC, HMBC, and NOESY) experiments, as well as
chemical degradations. The results were concluded as follows, three new polyketones:
Kiamycin(19), 1,4-dimethoxy-3-(3-hydroxy-3-methyl-1-tetralone)-1(3H)-isobenzofuran A(20), 1,4-dimethoxy-3-(3-hydroxy-3-methyl-1-tetralone)-1(3H)-isobenzofuran B(21); five known polyketones: Griseoviridin(1), Borrelidin(10), 8-O-methyltetrangomycin(22),
8-O-methylrabelomycin(23), X-14881F(24); and other known compounds: 1-Acetyl-beta-carbolin(2), 1-Methyluracil(3), m-Hydroxybenzyl alcohol(4), Anthranilic acid(5), Vanillic acid(6), Poly-(hydroxybutyric acid)(7), (+)-Nonactic acid(8), (+)-Homononactic acid(9), 1(10)E,5E-Germacradiene-3,11-diol(11), 4S,7R-Germacra-1E,5E-dien-11-ol(12), 3-Hydroxy-6-[(Z)-3'-hydroxy-2',4'-dimethyl-hept-4-enoylamino]-2,4-dimethyl-5-oxo-hex-anoic acid(13), p-Hydroxyphenylethyl alcohol(14), R-mevalonolactone(15),
5'-Methylthioadenosine(16), (4S)-4,10,11-Trihydroxy-10-methyldodec-2-en-1-olide(17), 4,10-Dihydroxy-10-methyl-11-oxo-dodec-2-en-1,4-olide(18), E-1-(4'-hydroxyphenyl)-buten-3-one(25), Arbutin(26), Ferulic acid(27), 3,4,5-Trihydroxybenzoicacid(28), 4-methyl-2,6-dihydroxybenzaldehyde (29),2-Hydroxyphenyl-acetamide(30), thymine(31), β-carboline(32), 1,2,3,4-tetrahydro-1-methyl-β-carboline-3-carboxylic acid(33).
      Compound 19 exhibited activity against human cell lines, namely HL-60 (leukemia), lung adenocarcinoma cells A549, and the hepatoma cell line BEL-7402with inhibition values of 68.2 %, 55.9 %, and 31.7 % at 100 μM; while compound 20 with inhibition values of 4.4%, 44.7 %, and 30.7 %; adriamycin as positive control showed inhibition rates of 70.0 %, 79.3 %, and 80.0 %, respectively, while compound 20 had no antimicrobial activities nor
acetylcholinesterase inhibition.
      Streptomyces sp. M268 was characterized according to the 16S rDNA gene sequence, and the gene sequence was identical to marine Streptomyces sp. W007 (Accession No. JN180126 in
GenBank. The biosynthetic pathway of angucyclines from M268 was speculated based on sequence comparison of amino acid sequences of angucycline synthetic gene.
文献类型学位论文
条目标识符http://ir.yic.ac.cn/handle/133337/5660
专题中国科学院烟台海岸带研究所知识产出_学位论文
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谢则平. 三株海洋放线菌聚酮类次级代谢产物的发现与过程优化[D]. 北京. 中国科学院研究生院,2012.
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