实验海洋生物学重点实验室知识库

      深海冷泉是典型的化能合成生态系统，具有高浓度的甲烷有机质流体、独特的硫氧化还原过程以及特殊的元素组成等环境特征，孕育了丰富而且独特的生物多样性以及生物资源。冷泉真菌在适应深海冷泉极端环境的过程中，进化出特殊的生理结构和代谢机制，能够直接或间接利用环境中独特的元素成分，从而产生和积累许多有趣的天然产物分子。但自1983年深海冷泉被发现以来，深海冷泉真菌天然产物相关研究报道较少，目前尚处于研究起步阶段。南海因其独特的地理位置以及海底地貌，成为我国“深海冷泉天然研究实验室”，受到越来越多的关注。

       本论文聚焦于南海冷泉真菌资源，结合HPLC和TLC分析等化学筛选结果，综合菌株发酵产量、次级代谢产物丰富度、化合物类型多样性以及文献调研情况等，选取了两株分别来源于南海冷泉贻贝和沉积物的真菌Talaromyces sp. CS-258 和Periconia byssoides CS-862，开展次级代谢产物的系统研究。利用多种色谱分离手段以及各种现代波谱学和量子化学计算技术，从两株真菌中共分离和鉴定了113个化合物，包括95个聚酮、8个生物碱、6个萜类以及4个脂肪族化合物。其中发现新化合物46个，新天然产物1个，培养化合物单晶9个。

      具体地，从我国南海台西南冷泉动物贻贝内生真菌Talaromyces sp. CS-258的发酵物中获得了70个单体化合物，包括 21个新化合物和1个新天然产物。部分化合物具有新颖的化学结构。其中，化合物TS1是首次分离得到具有7/6独特成环方式的倍半萜；化合物TS3是首次通过X射线单晶衍射方法确定的苯酰氨酸-鸟氨酸二肽；化合物TS7和TS10为鲜有报道的含碘原子聚酮；30个altenusin衍生物TS13–TS19，TS23–TS39，TS48–TS49以及TS51–TS54推测是由同一个聚酮合成中间体衍生而来。抗氧化活性测试（DPPH法、ABTS法和FRAP法）结果表明，5个酚类化合物TS4，TS48–TS50以及TS53表现出良好的抗氧化活性，其中TS4、TS48和TS49对4种食用油的氧化酸败过程具有明显的抗氧化保护活性，热力学参数计算结果表明它们很大程度上通过HAT机制来清除自由基。抗菌活性实验结果表明11个化合物TS24–TS26，TS30–TS34，TS37以及TS42–TS43对11种致病细菌表现出不同程度的抑制活性。

        从我国南海海马冷泉沉积物真菌Periconia byssoides CS-862的发酵粗提物中发现了43个单体化合物，包括新化合物25个。其中，4个差向异构体PB1–PB4为首次报道的具有14个碳原子骨架的新型koninginin类聚酮化合物；化合物PB8–PB11是芳香聚酮precochlioquinol D （PB34）碘原子或氯原子取代的衍生物；化合物PB14–PB18均属于三环苯酚类化合物（分子骨架中B环表现出丰富的结构多样性，能形成五元烯酮环以及高度饱和的多羟基环己烷）；化合物PB25是由4个聚酮前体分子经过羟醛缩合反应形成的聚酯类化合物；通过X射线单晶衍射手段确定了具有戊烷脂肪侧链结构的聚酮化合物PB1和PB5的绝对构型。在生物活性评价方面，4个聚酮化合物PB2、PB8、PB11和PB15在20 μM浓度下表现出对人胃癌细胞（MKN-45）较强的抗增殖活性。

       综上所述，本研究初步揭示了南海冷泉真菌次级代谢产物具有丰富的化学多样性和生物活性，为深海冷泉极端环境微生物资源的开发与利用提供了一定的物质基础和理论指导。

       Cold-seep in deep sea regions are the typical chemosynthetic-driven ecosystem with methane-rich fluid, unique sulfurate-redox process and special elemental composi-tions, which give birth to high abundance and biomass of specialized organisms. In the adaption to such extreme environment, cold-seep derived fungi have evolved special physiological structures and metabolic mechanisms, directly or indirectly utilizing unique elemental components from the environment to produce or accumulate many interesting natural molecules. However, since the discovery of cold-seep in 1983, there have been few research related to the natural product of cold-seep derived fungi, and which is currently in the initial stage. Due to unique geographical location and seafloor geomorphology, the South China Sea has become a natural research laboratory for cold-seep, receiving increasing attention.

     This research focuses on the fungal resources derived from cold-seep in the South China Sea. The chemical methods such as HPLC and TLC analysis were conducted and four following elements on strain production, abundance of secondary metabolites, diversity of chemical structures, and literature research results were taken into consideration for strains screening. As a result, two fungi of Talaromyces sp. CS-258 and Periconia byssoides CS-862, respectively isolated from cold-seep derived mussel and sediment samples collected in the South China Sea, were selected to carry out systematic chemical investigation on their secondary metabolites. By comprehensive utilization of various chromatographic separation methods as well as analysis of modern spectroscopic and quantum chemical calculation techniques, a total of 113 compounds were isolated and identified, consisting of 95 polyketides, 8 alkaloids, 6 terpenoids, and 4 aliphatic compounds. Among them, 46 new compounds together with one naturally occurred metabolite were discovered, and 9 single crystals of compounds were obtained.

      Specifically, 70 compounds, including 21 new molecules and a new natural product, were extracted and separated from the fermentation broth of the endophytic fungus Talaromyces sp. CS-258 sourced from a cold-seep mussel sample collecting in the northeast of the South China Sea. Some compounds have novel chemical structures. Compound TS1 represents the first isolated sesquiterpene with a unique 7/6 cyclization mechanism. Compound TS3 is the first phenylalanine ornithine dipeptide determined by X-ray single crystal diffraction. Compounds TS7 and TS10 are rarely reported iodine substituted polyketide compounds. Thirty altenusin derivatives of TS13–TS19, TS23–TS39, TS48–TS49 and TS51–TS54 are derived from the same polyketide synthesis intermediate. The antioxidant activity assays of DPPH, ABTS, and FRAP revealed that five phenolic compounds TS4, TS48–TS50 and TS53 exhibited relative antioxidant activity. Among them, TS4 and TS48–TS49 displayed antioxidant protective activities against the rancidity of four edible oils during the storage. The results of thermodynamics parameter calculation indicated that they could scavenge free radicals through the HAT mechanism. Eleven compounds of TS24–TS26, TS30–TS34, TS37 and TS42–TS43 showed significant antibacterial activity against 11 pathogenic bacteria.

      The number of 43 compounds were found in the crude extract of fungus Periconia byssoides CS-862 originating from the Haima cold-seep sediment sample and 25 new ones were identified. Four epimers PB1–PB4 represent the first example of koninginins with 14 carbon atoms. Compounds PB8–PB11 are identified as halogenated derivatives of aromatic polyketide precochlioquinol D (PB34) containing iodine and chlorine atoms. Compounds PB14–PB18 belong to tricyclic phenolic compounds and the B ring in their molecular skeleton possesses abundant structural diversity, which can form penta-ketene rings or highly saturated cyclohexane. Compound PB25 is designated as a polyester formed by the polymerization of four polyketide precursors. The absolute configurations of PB1 and PB5 with pentane fatty side chains were determined by X-ray single-crystal diffraction. In the bioassay, four polyketides of PB2, PB8, PB11 and PB15 exhibited definite antiproliferative activity against MKN-45 cell line (human gastric cancer cells) at 20 μM.

   In general, this paper preliminarily reveals the chemical and biological diversity of secondary metabolites obtained from cold-seep derived fungal resources in the South China Sea, providing a certain material basis and theoretical guidance for the develop-ment and utilization of microbial resources in extreme environments of cold-seeps in deep sea.

第1章 冷泉微生物天然产物化学研究进展... 1

1.1 前言... 1

1.1.1 冷泉环境特征... 2

1.1.2 冷泉微生物生态学意义... 3

1.2 冷泉微生物次级代谢产物研究... 4

1.2.1 冷泉微生物次级代谢产物化学多样性和生物活性研究... 4

1.2.2 冷泉微生物次级代谢产物研究现状... 22

1.3 冷泉微生物次级代谢产物研究方法... 28

1.3.1 OSMAC研究策略... 29

1.3.2 表观遗传修饰策略... 31

1.3.3 基因组挖掘与宏基因组学策略... 32

1.4 小结与讨论... 32

第2章 南海冷泉真菌分离与筛选... 35

2.1 前言... 35

2.2 实验与方法... 35

2.2.1 实验材料与仪器... 35

2.2.2 实验方法... 36

2.3 结果与讨论... 37

2.3.1 南海冷泉真菌分离... 37

2.3.2 南海冷泉真菌筛选... 38

2.4 小结... 44

第3章 南海冷泉贻贝内生真菌Talaromyces sp. CS-258次级代谢产物及其生物活性研究... 45

3.1 前言... 45

3.2 实验与方法... 45

3.2.1 实验仪器与试剂... 45

3.2.2 真菌CS-258来源与鉴定... 46

3.2.3 真菌CS-258发酵与分离... 47

3.2.4 实验方法... 48

3.3 结果与讨论... 54

3.3.1 真菌CS-258新化合物结构解析... 54

3.3.2 真菌CS-258新化合物理化性质... 82

3.3.3 真菌CS-258已知化合物结构鉴定... 87

3.3.4 真菌CS-258聚酮类化合物生源合成途径推测... 113

3.3.5 真菌CS-258化合物生物活性研究... 117

3.4 小结... 129

第4章 南海冷泉沉积物来源真菌Periconia byssoides CS-862次级代谢产物及其生物活性研究... 131

4.1 前言... 131

4.2 实验与方法... 131

4.2.1 实验仪器与试剂... 131

4.2.2 真菌CS-862来源与鉴定... 131

4.2.3 真菌CS-862发酵与分离... 132

4.2.4 实验方法... 133

4.3 结果与讨论... 134

4.3.1 真菌CS-862新化合物结构解析... 134

4.3.2 真菌CS-862新化合物理化性质... 165

4.3.3 真菌CS-862已知化合物结构解析... 168

4.3.4 真菌CS-862化合物生物活性研究... 178

4.4 小结... 181

第5章 总结与展望... 183

5.1 总结... 183

5.2 创新点... 184

5.3 展望... 184

参考文献... 187

附录 部分化合物NMR谱图... 205

致 谢... 223

作者简历及攻读学位期间发表的学术论文与其他相关学术成果... 225