【作者】 矫文策；

【导师】 赵心清；

【作者基本信息】 大连理工大学 ， 生物化工， 2013， 博士

【摘要】 海洋链霉菌是多种新颖天然产物的重要来源。对海洋链霉菌进行分离、鉴定及其活性次级代谢产物分离纯化、结构解析,有助于深入挖掘其独特的基因资源和生物合成潜能,为开发在医药和农业等领域迫切需要的药物和先导化合物奠定基础。微生物影像质谱是近几年来发展起来的研究微生物与环境、微生物细胞间次级代谢产物相互作用而产生的新技术,目前国内相关的研究还较少。本课题组在大连周边海域沉积物样品中分离了几百株海洋放线菌,但对其分类鉴定和活性化合物的研究还没有深入进行。本研究针对其中的两株放线菌S187和M10,分别从菌株的分类鉴定、活性次级代谢产物的分离纯化和结构解析、发酵条件的优化等几个方面进行了研究,并利用影像质谱和基因组挖掘对两株放线菌产生的活性次级代谢产物进行了分析预测,所取得的研究结果如下：1.多相分类法鉴定菌株S187通过形态特征、培养特征、细胞化学组分、16S rRNA序列分析及基因组DNA-DNA随机杂交对菌株S187进行分类鉴定。菌株S187与其近缘菌株Streptomyces flavofuscus NRRL B-8036T和S. albiaxialis DSM在培养特征、生理生化特征等均呈现明显差异。DNA-DNA杂交试验表明,菌株S187同S. flavofuscus和S. albiaxialis的相关性仅为31.4%和46.9%,因此,鉴定S187为链霉菌属的新种,命名为星海链霉菌(S. xinghaiensis sp.nov.)。星海链霉菌对大肠杆菌、金黄色葡萄球菌、枯草芽孢杆菌和铜绿假单胞菌等细菌测试菌具有良好的拮抗活性,具有潜在的开发前景。2.星海链霉菌活性次级代谢产物研究对星海链霉菌在三种不同固体培养基TSB、Al和Marine agar上的纯培养和琼脂块法抗菌活性测试影像质谱分析,结果表明,星海链霉菌的次级代谢物中没有有效的质谱信号与抑菌圈完全吻合。以金黄色葡萄球菌为指示菌,通过乙酸乙酯萃取、硅胶柱层析、C18快速分离柱和半制备HPLC从25L星海链霉菌发酵液中分离得到两个化合物xinghaiamine A和B。运用质谱、红外光谱、紫外光谱和核磁共振(1H-NMR,13C-NMR,1H-1H cosy, HSQC, HMBC, DEPT135)等光谱方法对两个化合物进行表征,结构解析确定xinghaiamine A为一种含亚砜官能团的生物碱,文献检索确定其为一种新结构化合物且其特征的亚砜结构系首次从微生物中分离得到。抗菌活性测试表明,xinghaiamine A和B具有广谱抗细菌活性,且对耐药金黄色葡萄球菌(MRSA)具有较强的抑制作用,但对真菌没有抑制作用。细胞毒活性表明,xinghaiamine A对四株人类肿瘤细胞均有一定的抑制作用,但xinghaiamine B对四株细胞株的其抑制作用较弱。3.星海链霉菌发酵生产xinghaiamine A条件优化单因素实验确定星海链霉菌最佳发酵条件为：起始pH7,接种量6%(v/v),温度28℃,装液量为160mL/500mL。最佳碳源、有机氮源和无机氮源分别为可溶性淀粉、黄豆粉和(NH4)2SO4。均匀设计法优化发酵培养基组成为(g/L)：可溶性淀粉22,黄豆粉50,(NH4)2SO42.2,CaCO35, NaCl2和K2HPO40.5.利用原位固相萃取优化xinghaiamine A产量,结果表明：在发酵接种后72h后加入3%(w/v)的树脂HP-20,抗生素产量从0.35mg/L提升到3.4mg/L,发酵周期从18d缩短为9d。细胞自毒性实验表明,xinghaiamine A对其生产菌株星海链霉菌具有强烈的抑制作用,最小抑菌浓度为8μg/mL,推测自毒性的减弱或消除是树脂吸附增加抗生素产量的原因。4.基因组挖掘、影像质谱和串联质谱分析菌株M10活性次级代谢产物放线菌M10经过形态观察和16S rRNA序列分析初步鉴定为摩洛哥链霉菌(S.marokkonensis)的相似菌株。对M10进行全基因组测序,生物信息学分析表明基因组中含有20个与次级代谢产物相关的生物合成基因簇,包括3个PKS、4个NRPS、4个杂合PKS-NRPS、2个lantibiotic、4个萜类和3个siderophore基因簇。其中一个PKS基因簇(pks1)与杀念珠菌素生物合成基因簇具有一定的相似性,通过RNA反转录验证了该基因簇中的部分基因的转录。对链霉菌M10在三种不同固体培养基TSB、Al和Marine琼脂上的纯培养,琼脂块法抗菌活性测试和与测试菌共培养的影像质谱分析,结果表明：m/z1152和1168两个质谱信号在TSB和A1上纯培养、琼脂块法抗菌活性测试和与番茄叶霉菌共培养上均可检测到,推断其为M10产生的抗真菌化合物。对M10在A1培养基上粗提物进行串联质谱多肽分析,结果表明在900-1000Da范围内存在两个多肽类化合物家族MF1和MF2。5.链霉菌M10抗真菌物质分离纯化收集M10在A1固体培养基上的培养物,先后经过乙酸乙酯和丁醇萃取,得到乙酸乙酯和丁醇粗提物。对丁醇粗提物分别经过Sephadex LH-20层析柱,C18快速分离柱和半制备HPLC,以白色念珠菌为指示菌,以多烯大环内酯类物质为假定目标分离物,最终得到三个化合物9-03、9-04和9-05。紫外吸收表明,三个化合物具有典型的多烯大环内酯类化合物特征吸收：核磁共振光谱分析表明,化合物9-04具有特征的共轭双键和内酯结构。因此推断链霉菌M10能够产生多烯大环内酯类抗生素。同时,使用MALDI-TOF在M10乙酸乙酯粗提物流经Sephadex LH-20的组分中检测到了之前发现了两个多肽化合物家族MF1和MF2,表明菌株M10在A1琼脂培养基上能够产生多肽类化合物。更多还原

【Abstract】 Marine streptomyces are rich source of novel natural products. Isolation and characterization of marine streptomyces, purification and structure elucidation of bioactive secondary metabolites not only benefit the exploration of the unique genetic resources and the potential of biosynthesis of the new species, but also provide novel bioactive compounds for drug discovery. Imaging mass spectrometery (IMS) has been used as a new technology for microbial natural product using microbial interactions. However, related studies using marine actinomyces have been poorly reported. In our previous studies, marine-derived actinomycetes were isolated from various areas in Dalian, but studies on species characterization and their bioactive secondary metabolites were still limited. In this study, two marine-derived actinomycetes S187and M10were investigated on species characterization, purification and structure elucidation of bioactive compounds, and optimization of fermentation. Meanwhile, bioactive secondary metabolites from these two strains were explored. The results listed below were obtained.1. Identification of strain S187by polyphasic taxonomyStrain S187was investigated by polyphasic taxonomy including morphological characteristics, cultural characteristics, cell chemical composition,16S rRNA sequence analysis and genomic DNA-DNA hybridization. Compared with its phylogenetically closed strain S. flavofuscus NRRL B-8036T and S. albiaxialis DSM41799T, S187differed in the characteristics of phenotype, chemotype and genotype, and it was proposed that strain S187represented a novel species of the genus Streptomyces, for which the name S. xinghaiensis sp. nov. is proposed and the type strain is S187.2. Bioactive secondary metabolites from S. xinghaiensisIMS was performed using S. xinghaiensis growing on TSB agar, A1agar and Marine agar media, and the results indicated that no MS signals was in accordance with the inhibition zone. Xinghaiamine A and B were purified from25L S. xinghaiensis fermentation broth by ethyl acetate extraction, silica gel column, flash C18column and semi-preparative HPLC using S. aureus as an indicator. The sturcture elucidation was performed on the basis of ESI-MS, IR, UV and NMR spectrum (’H-NMR,13C-NMR,’H-’H COSY, HSQC, HMBC and DEPT135). Xinghaiamine A was identified as a novel alkaloid with sulfoxide functional group which has never been reported in microorganism. Xinghaiamine A and B exhibited excellent broad-spectrum antibacterial activities, especially to the MRSA isolates, but no inhibition to C. albicans was observed. Xinghaiamine A also showed cytotoxicity to the tested human tumor cell lines.3. Fermentation optimization of xinghaiamine A production from S. xinghaiensisThe optimum fermentation condition was determined to be initial pH7, inoculation rate6%(v/v), temperature28℃with the medium volume of160mL in500mL flask. The optimum organic carbon source, organic and inorganic nitrogen source were starch, soybean powder and (NH4)2SO4. An addition of3%(w/v) adsorbent resin HP-20at72h post-inoculation significantly improved xinghaiamine A production from0.35to3.4mg/L and shortened fermentation time from18to9days. Self-cytotoxicity of xinghaiamine A was verified and MIC value to its producing strain was estimated to be8μ/mL.4. Genome mining, IMS and MS/MS analysis of M10secondary metabolitesStrain M10was demonstrated to be most closely related to S. marokkonensis by16S rRNA sequence analysis, and bioinformatics analysis identified20secondary metabolites associated gene clusters involved in the genome of M10, including three PKS, four NRPS, four hybrid PKS-NRPS, two lantibiotics, four terpenoid and three siderophores biosynthesis gene clusters. Among the gene clusters, pksl exhibited85%sequence similarity to the candicidin gene cluster and selected genes in pksl gene cluster were demonstrated in transcriptional level by RT-PCR experiment. IMS signals of m/z1152and1168produced by M10were found on both TSB, A1agar and were in accordance with the inhibition zone and time-dependent cocultures of M10and Fulva fulva, which demonstrated that they could be the antifungal compounds produced by M10. Analysis of M10crude extract on A1agar by MS/MS revealed two peptide families (MF1and MF2) with the molecular weight ranging from900to1000Da.5. Purification of antifungal compounds produced by M10A total of630A1agar plates of M10were collected and extracted successively with ethyl acetate and butanol. Three compounds named9-03,9-04and9-05with the typical UV adsorption of polyene molecules were purified from the butanol extract by employing Sephadex LH-20column, flash C18column and semi-preparative HPLC. The NMR data of9-04also exhibited several conjugated double bonds and one lactone bond, indicating that it belongs to the polyene macrolides. In addition, the peptide molecule familes MF1and MF2were detected in the Sephadex LH-20column fractions using MALDI-TOF, indicating that M10produce peptide antibiotics on A1agar medium.更多还原