实验海洋生物学重点实验室知识库

  海洋拥有不同于陆地的特殊生境，是一个拥有巨大生物量的资源宝库。科学家已经从海洋中发现了大量结构新颖、活性良好的海洋天然产物。海洋真菌为了适应在特殊环境中生存而调整其代谢途径，从而产生独特的代谢产物，尤其是深海冷泉和红树林这两种特殊生境。

  本文使用不同培养基及不同的培养条件对采集到的冷泉及红树林样品在实验室条件下进行真菌的分离纯化，从中分离得到多种不同属的海洋真菌。采用化学筛选手段，依据培养基形态特征和化学多样性，结合分子生物学鉴定后的文献调研结果，对分离得到的真菌进行筛选，从中选定目标菌株CS-469和CS-289以及MA-325进行次级代谢产物研究。

  针对深海冷泉来源的沉积物样品中分离得到的篮状菌属真菌Talaromyces indigoticus CS-469，综合运用各种色谱学和波谱学手段从其发酵产物中分离鉴定42个化合物（28个为聚酮类化合物，9个为萜类化合物、5个为甾体类化合物），包括18个新的聚酮类化合物（TI4−TI13和TI17−TI24）、3个新的萜类化合物（TI1−TI3）及3个新的甾体类化合物（TI14−TI16）。其中化合物TI1是首次发现的具有二环[3.1.1]庚烷骨架的二萜类化合物，并推测了其可能的生物合成途径。化合物TI4−TI9是6个新的含有硫酯键的苯并呋喃类化合物。通过X射线单晶衍射法、ECD计算、NMR计算以及化学反应的方法，包括Mosher反应和糖的酸水解及衍生反应，确定了新化合物的绝对构型。对部分化合物进行了抗菌活性测试，其中化合物TI9和TI31对番茄枯萎病菌Fusarium oxysporum具有中等的抑制活性，化合物TI3和TI25对多株水产致病细菌表现出较强的广谱抑制活性，MIC值在4−16 µg/mL之间。此外，含硫酯的苯并呋喃类化合物TI9表现出了显著的抗PR8流感病毒活性。

  针对深海冷泉潜铠虾新鲜组织中分离得到的内生真菌Aspergillus jensenii CS-289，从其发酵产物中共分离鉴定了44个化合物（11个聚酮类化合物，4个混源萜类化合物，19个萜类化合物以及10个生物碱类化合物），其中6个为新化合物（AJ1−AJ6），包括2个新的混源萜类化合物（AJ1和AJ2），3个新的没药烷型倍半萜类化合物（AJ3−AJ5）以及1个新的生物碱类化合物（AJ6）。抗菌活性筛选结果显示，化合物AJ6a、AJ25−AJ27对水产致病菌哈氏弧菌V. harveyi表现出了中等的抑制活性，化合物AJ32和AJ33对植物病害菌穗状弯孢菌Curvularia spicifera表现出中等抑制活性。

  课题组前期从红树林植物木榄来源的青霉属真菌Penicillium sumatrense中分离得到一系列罕见的含硫弯孢霉菌素类化合物，抗肿瘤活性良好。为了进一步挖掘该类化合物的结构多样性，本论文选择不同红树林来源的同种真菌MA-325作为研究对象，以期从中分离得到结构新颖活性良好的弯孢霉菌素类化合物。针对从红树林来源的植物尖瓣海莲根际土壤样品中分离得到的青霉属真菌Penicillium sumatrense MA-325，从其发酵粗提物中分离鉴定得到14个化合物（13个弯孢霉菌素类化合物和1个生物碱类化合物），其中有4个新的弯孢霉菌素类化合物（PS1−PS4），通过X射线单晶衍射实验、NMR计算及ECD图谱比较的方法确定了其绝对构型。其中化合物PS1为首次报道的C-4位连接5-甲基-2-呋喃甲基基团的弯孢霉菌素类化合物，对其可能的生物合成途径进行了推测。抗菌活性和细胞毒活性测试结果显示，化合物PS1和PS6对水产致病菌溶藻弧菌V. alginolyticus和哈氏弧菌V. harveyi具有明显的抑制活性，MIC值在4−16 µg/mL之间。化合物PS6对多株肿瘤细胞株具有显著的细胞毒活性，且具有一定的选择性，其中对人膀胱癌细胞5637抑制活性的IC₅₀值达到3.5 μM，活性优于阳性对照顺铂（4.1 μM）。

  本论文以两株深海冷泉来源和一株海洋红树林来源真菌作为研究对象，对其次级代谢产物和生物活性进行了研究，共从中分离得到100个化合物，34个新化合物，含新聚酮化合物22个，丰富了海洋真菌来源聚酮类化合物的结构多样性。且部分化合物表现出了良好的生物活性，表明海洋真菌来源的天然产物具有研究价值和应用潜力，为海洋活性天然产物研究提供了一定的实验基础。

  The ocean has developed unique habitats different from land and is a treasure trove of resources with enormous biomass. A wide variety of natural products with intriguing structures and significant bioactivities have been discovered from the ocean. Marine-sourced fungi have adapted to evolve special metabolic pathways to survive in the extreme environments such as cold seep and mangroves.

  Marine fungi in multiple genera were isolated from the collected samples from cold seep and mangroves under laboratory conditions using different culture media and conditions. The marine-sourced fungi CS-469, CS-289, and MA-325 were screened for chemical investigations based on their morphological characteristics, chemical diversity, and related research results in literatures after molecular biology identification.

  Chemical investigation on cold seep sediment-derived Talaromyces indigoticus CS-469 resulted in isolation and identification of 42 compounds including 28 polyketides, 9 terpenoids, and 5 steroids using a combination of chromatographic and spectroscopic methods. Among these compounds, eighteen polyketides (TI4−TI13 and TI17−TI24), three terpenoids (TI1−TI3), and three steroids (TI14−TI16) are new compounds. Compound TI1 is the first diterpenoid with a bicyclo[3.1.1]heptane skeleton and its possible biosynthetic pathway was postulated. Compounds TI4−TI9 are new thioester-containing benzofuran derivatives. The absolute configurations of the new compounds were determined by X-ray single-crystal diffraction analysis, ECD calculations, NMR calculations, and chemical reaction methods, including the Mosher method, acid hydrolysis, and derivatization of sugars. Some of the compounds were assayed for antimicrobial activity, among which compounds TI9 and TI31 showed moderate inhibitory activity against Fusarium oxysporum, and compounds TI3 and TI25 showed strong broad-spectrum inhibitory activity against several strains of aquatic pathogenic bacteria with MIC values ranging from 4 to 16 µg/mL. Besides, thioester-containing benzoate derivative compound TI9 exhibited significant antiviral activity against PR8-infected cells.

  The fungus Aspergillus Jensenii CS-289 was isolated from fresh tissues of deep-sea cold seep squat lobster Shinkaia crosnieri. A total of 44 compounds were identified from its crude extract, including 11 polyketides, 4 meroterpenoids, 19 terpenoids, and 10 alkaloids. Six new compounds were discovered (AJ1−AJ6), including two new meroterpenoids (AJ1 and AJ2), three new bisabolane sesquiterpenes (AJ3−AJ5), and one new alkaloid (AJ6). The screening results of antimicrobial activity showed that compounds AJ6a and AJ25−AJ27 were effective against Vibrio harveyi and compounds AJ32 and AJ33 showed moderate inhibitory activities against Curvularia spicifera.

  In the previous study, a series of rare sulfur-containing curvularin derivatives with potent cytotoxic activity were isolated from Penicillium sumatrense, which was isolated from the marine mangrove plant Lumnitzera racemosa Willd. As a continuing research for structurally curvularin derivatives, this dissertation selected the same species of fungus MA-325 from different mangrove sources to discover novel and highly active curvularin derivatives. A total of 14 compounds, including 13 curvularin derivatives and one alkaloid, were isolated and identified from the crude extract of Penicillium sumatrense MA-325. The fungus Penicillium sumatrense MA-325 was isolated from the rhizosphere of the marine mangrove plant Bruguiera sexangular (Lour.) Poir. var. rhynchopetala Ko. The absolute configurations of four new curvularin derivatives (PS1−PS4) were determined by X-ray single crystal diffraction experiments, NMR calculations, and ECD spectra comparison. Among them, compound PS1 represents a unique example of curvularin derivative featuring a 5-methylfuran-2-yl-methyl group and its possible biosynthetic pathway has been speculated. Some of the isolated compounds were screened for their antibacterial and cytotoxic activity, compounds PS1 and PS6 showed inhibitory activity against aquatic pathogenic bacteria V. alginolyticus and V. harveyi with MIC values ranging from 4 to 16 µg/mL. Compound PS6 has significant cytotoxicity and selectivity to many tumor cell lines. Notely, PS6 displayed cytotoxic activity against human bladder cancer cell line 5637 with IC₅₀ value of 3.5 μM, which was superior to the positive control cisplatin (4.1 μM).

  This dissertation enriches the structural diversity of polyketide compounds derived from marine fungi by studying the secondary metabolites of the two deep-sea cold seep derived fungi and one mangrove-derived fungus. A total of 100 compounds were identified, including 34 new compounds, of which 22 were new polyketides. Some compounds showed explicit biological activity, indicating that natural products derived from marine fungi have research value and application potential, and providing a certain experimental basis for the study of marine active natural products.

目  录

第1章 深海来源真菌聚酮类化合物研究进展    1
1.1 引言    1
1.2 深海青霉属真菌来源聚酮类化合物研究进展    1
1.3 深海曲霉属真菌来源聚酮类化合物研究进展    10
1.4 深海篮状菌属真菌来源聚酮类化合物研究进展    12
1.5 深海其他属真菌来源聚酮类化合物研究进展    15
1.6 小结与讨论    20
第2章 冷泉沉积物来源真菌Talaromyces indigoticus CS-469次级代谢产物及其生物活性研究    23
2.1 前言    23
2.2 实验部分    23
2.2.1 实验仪器与材料    23
2.2.2 菌株CS-469的来源、分离纯化与鉴定    24
2.2.3 菌株CS-469的规模发酵    25
2.2.4 菌株CS-469发酵产物的提取与分离    25
2.2.5 改进的Mosher法    28
2.2.6 糖的酸水解反应及绝对构型的确定    28
2.2.7 菌株CS-469化合物生物活性测试    28
2.3 结果与讨论    30
2.3.1 菌株CS-469新化合物结构鉴定    30
2.3.2 菌株CS-469新化合物理化性质    66
2.3.3 菌株CS-469萜类新化合物生物合成途径推测    70
2.3.4 菌株CS-469已知化合物结构鉴定    72
2.3.5 菌株CS-469化合物的生物活性测试结果    84
第3章 冷泉潜铠虾来源真菌Aspergillus jensenii CS-289次级代谢产物及其生物活性研究    89
3.1 前言    89
3.2 实验部分    89
3.2.1 实验仪器与材料    89
3.2.2 菌株CS-289的来源、分离纯化与鉴定    89
3.2.3 菌株CS-289的规模发酵    90
3.2.4 菌株CS-289发酵产物的提取与分离    90
3.2.5 菌株CS-289化合物生物活性测试    92
3.3 结果与讨论    93
3.3.1 菌株CS-289新化合物结构鉴定    93
3.3.2 菌株CS-289新化合物理化性质    102
3.3.3 菌株CS-289混源萜类新化合物生物合成途径推测    103
3.3.4 菌株CS-289已知化合物结构鉴定    104
3.3.5 菌株CS-289化合物的生物活性测试    126
第4章 红树林来源真菌Penicillium sumatrense MA-325次级代谢产物及其生物活性研究    129
4.1 前言    129
4.2 实验部分    129
4.2.1 实验仪器与材料    129
4.2.2 菌株MA-325的来源、分离纯化与鉴定    129
4.2.3 菌株MA-325的规模发酵    130
4.2.4 菌株MA-325发酵产物的提取与分离    130
4.2.5 菌株MA-325化合物生物活性测试    132
4.3 结果与讨论    132
4.3.1 菌株MA-325新化合物结构鉴定    132
4.3.2 菌株MA-325新化合物理化性质    140
4.3.3 菌株MA-325中化合物PS1生物合成途径推测    141
4.3.4 菌株MA-325已知化合物结构鉴定    142
4.3.5 菌株MA-325化合物的生物活性测试    148
第5章 总结与展望    151
5.1  总结    151
5.2  创新点    152
5.3  展望    152
参考文献    155
附录  部分化合物核磁谱图    169
致  谢    177
作者简历及攻读学位期间发表的学术论文与研究成果    179