实验海洋生物学重点实验室知识库

天然产物是药物先导化合物的重要来源，许多天然产物被研发、批准成为药物。据1981-2019年的统计数据表明约有65%的FDA批准上市的药物与天然产物相关。然而在经历了天然产物发现的黄金时期后，新天然产物的发现几率开始下降，重复发现率升高。为应对这些问题，本论文通过从深海极端环境中获取微生物资源，结合基因组及转录组等组学分析，深入挖掘菌株次级代谢潜力，获得了结构新颖且活性显著的天然产物。

本论文从南海冷泉及麦哲伦海山采集到的动物样品中分离得到内生真菌，并对其通过不同培养基进行筛选，采用高效液相色谱（HPLC）和薄层色谱（TLC）为检测、评估手段，最终选定菌株Curvularia verruculosa CS-129及Penicillium steckii AS-324为目标菌株，对其进行规模发酵。采用大米固体培养基静置发酵，后经乙酸乙酯处理得到粗提物，并对其粗提物进行分离纯化最终得到单体化合物。

从南海冷泉动物来源内生真菌C. verruculosa CS-129中共分离鉴定了20个单体化合物，其中8个为新化合物（CV1-CV8），2个为新天然产物（CV9-CV10），出新率约为50%，通过核磁共振、高分辨质谱、NOESY谱图、DP4+分析、ECD计算以及X射线单晶衍射等实验确定了新化合物的绝对构型。所分离获得的20个单体化合物中19个为细胞松弛素类化合物，1个为弯孢菌素类化合物。对所得化合物进行了抗肿瘤活性、血管紧张素转换酶抑制活性以及抗菌活性测试，发现化合物CV1具有较强的血管紧张素转化酶抑制活性，化合物CV6、CV13和CV16具有显著的细胞毒活性，大多数细胞松弛素类化合物对人类及水产病害细菌表现出较强的抑制活性。

从麦哲伦海山动物来源内生真菌P. steckii AS-324中分离得到34个单体化合物，均为聚酮类化合物，其中24个为新化合物（PS1-PS23和PS33），通过核磁共振、高分辨质谱、NOESY谱图、ECD计算以及X射线单晶衍射等实验确定了新化合物的结构。主要结构类型为tanzawaic acid类化合物及其衍生物，另外包括一个新的和一个已知的烯内酰胺类化合物。对所有化合物进行了抗肿瘤活性、神经氨酸酶抑制活性、抗菌活性及抗氧化活性测试，结果表明化合物PS1、PS9、PS18、PS26、PS29对水产病害细菌具有较强的抑制活性。

利用生物信息学的手段，结合基因组及转录组分析，对海藻来源真菌鲜红青霉P. chermesinum EN-480次级代谢产物进行了深入挖掘。实验室前期研究发现该菌株使用大米固体培养基进行培养时可产生一系列结构新颖的混源萜类化合物，且具有较强的抗菌活性。通过对菌株基因组测序及数据库比对发现，其具有合成萜类、聚酮类、混源萜类、多肽类等多种次级代谢产物的潜力。为了进一步激发其代谢潜力，促进沉默基因的表达，采用一菌多产物（OSMAC）策略，通过对不同培养基及发酵方式的筛选，转录组分析，最终确定其规模发酵方式。对改进的大米培养基及MH2培养基的发酵产物进行选择性的分离，从中分离得到8个新化合物（PCR1-PCR5以及PCM1-PCM3），其中4个为结构新颖的混源萜类化合物（PCR1-PCR4），1个为骨架类型新颖的甾体类化合物（PCR5），3个为法尼基酯化成环形成的特殊结构类型的倍半萜类化合物（PCM1-PCM3）。对新化合物进行了抗菌活性测试，所有化合物均表现出较强的抗人类及水产病害细菌、抗植物病害真菌活性。

本论文通过获取深海极端环境中不同动物来源内生真菌资源，结合多种组学手段及生物信息学分析，对海洋真菌次级代谢产物进行深入挖掘，从南海冷泉潜铠虾来源内生真菌C. verruculosa CS-129、麦哲伦海山珊瑚来源内生真菌P. steckii AS-324和海藻来源内生真菌P. chermesinum EN-480中共分离鉴定62个次级代谢产物，其中40个新化合物，2个新天然产物，提高了菌株次级代谢产物的出新率以及新天然产物的发现效率，活性筛选表明若干结构新颖的化合物具有显著的生物活性，为海洋真菌次级代谢产物的研究及应用提供了科学依据。

Natural products are important sources for the discovery of new drugs. Many natural products have been approved as drugs. Statistics from 1981 to 2019 showed that about 65% of all FDA-approved drugs were natural products or natural product origin. However, after the golden age of natural product discovery, the discovery rate of new natural products began to decline, and the repeated discovery rate increased. In order to deal with these problems, in this paper, microbial resources were obtained from the extreme deep-sea environment, and the secondary metabolic potential of fungal strains was further explored by combining genomic and transcriptome analysis. New natural products with novel structures and significant activities were obtained.

In this paper, endophytic fungi were isolated from animal samples collected from cold spring in the South China Sea and Magellan Seamount and screened through different media. HPLC and TLC were used as evaluation methods. Finally, Curvularia verruculosa CS-129 and Penicillium steckii AS-324 were selected as the target strains and scale fermentation was carried out on them. Rice solid medium was used for static fermentation, and then the crude extract was treated with ethyl acetate. The crude extract was isolated and purified, and finally the monomer compounds were obtained.

In this study, 20 compounds were isolated and identified from strain CS-129 (C. verruculosa). Among them, eight compounds were new compounds (CV1-CV8) and two were new natural products (CV9-CV10). The proportion of new compounds was about 50%. The absolute configurations of the new compounds were determined by NMR, MS and NOESY spectra as well as DP4+ possibility analyses, ECD calculations and X-ray single crystal diffraction experiments. Among the 20 compounds, 19 were cytochalasins and one was curcumin. Cytotoxicity, angiotensin converting enzyme (ACE) inhibitory activity and antimicrobial activities of all compounds were tested. Compound CV1 showed strong ACE inhibitory activity. Compounds CV6, CV13 and CV16 exhibited significant cytotoxic activities. Most compounds have strong antibacterial activities.

34 compounds were isolated from AS-324 (P. steckii) and all of them were polyketones. Among them, 24 compounds (PS1-PS23 and PS33) were new compounds. The structures of the new compounds were determined by NMR, MS, NOESY spectra, ECD calculations and X-ray single crystal diffraction experiments. The main structural types are tanzawaic acid compounds except one new and one known enolactam compounds. The antitumor, neuraminidase inhibitory, antimicrobial and antioxidant activities of all compounds were tested. The results showed that compounds PS1, PS9, PS18, PS26 and PS29 exhibited potent antibacterial activities against aquatic pathogenic bacteria.

By combining genome and transcriptome information, we explored the secondary metabolites of the algal-derived fungus P. Chermesinum EN-480 using bioinformatics methods. It was found that the strain could produc meroterpenoids with new structures and strong antibacterial activities. By sequencing the whole genome of the strain and comparing it with database, we found that it has the potential to synthesize terpenoids, polyketones, meroterpenoids, polypeptides and so on. In order to further stimulate its secondary metabolic potential and promote the expression of silenced genes, the strategy of OSMAC was used to determine the fermentation method through the screening of crude extracts from different media and transcriptome analysis. Finally, eight new compounds (PCR1-PCR5 and PCM1-PCM3) were obtained. Four of them were new meroterpenoids (PCR1-PCR4). One was a novel sterol (PCR5) and three of them were sesquiterpenoids (PCM1-PCM3) formed by esterification and cyclization of pentanolactone moiety. The antimicrobial activities of new compounds were tested and all of them showed strong antibacterial activities against human and aquatic pathogenic bacteria or plant pathogenic fungi.

This study obtained endophytic fungi from animals living in extreme environment of deep-sea, and combined genome and transcriptome analysis and bioinformatics methods to dig marine fungi secondary metabolites. A total of 62 natural products were obtained from C. verruculosa CS-129, P. steckii AS-324 and P. chermesinum EN-480, including 40 new compounds and two new natural products. The rate of new compounds discovery was improved efficiently. Besides, many new compounds with novel structures and significant activities were isolated, which provided basis for the study and application of secondary metabolites of marine fungi.

第1章  绪论... 1

1.1  引言... 1

1.2  深海来源真菌次级代谢产物研究进展... 2

1.2.1  深海来源真菌萜类次级代谢产物... 2

1.2.2  深海来源真菌聚酮类次级代谢产物... 6

1.2.3  深海来源真菌生物碱类次级代谢产物... 10

1.3  基因组及转录组学在海洋天然产物研究中的应用... 14

1.3.1  基因组学在海洋天然产物研究中的应用... 15

1.3.2  转录组学在海洋天然产物研究中的应用... 17

1.4  天然产物研究发展趋势... 18

第2章  冷泉动物来源真菌Curvularia verruculosa CS-129次级代谢产物及其生物活性研究... 19

2.1  实验部分... 20

2.1.1  菌株CS-129的分离纯化与鉴定... 20

2.1.2  菌株CS-129的筛选与发酵... 20

2.1.3  菌株CS-129发酵产物的分离纯化... 21

2.2  菌株CS-129化合物的结构解析... 22

2.2.1  菌株CS-129新化合物结构鉴定... 22

2.2.2  菌株CS-129新化合物物理性质与波谱数据... 49

2.2.3  菌株CS-129已知化合物结构鉴定... 51

2.3  菌株CS-129化合物的生物活性测试... 59

2.3.1  菌株CS-129化合物抗肿瘤活性测试... 59

2.3.2  菌株CS-129化合物血管紧张素转换酶抑制活性测试... 59

2.3.3  菌株CS-129化合物抗菌活性测试... 61

2.4  菌株CS-129原位培养转录组分析... 63

第3章  海山动物来源真菌Penicillium steckii AS-324次级代谢产物及其生物活性研究... 67

3.1  实验部分... 68

3.1.1  菌株AS-324的分离纯化与鉴定... 68

3.1.2  菌株AS-324的筛选与发酵... 68

3.1.3  菌株AS-324发酵产物的分离纯化... 68

3.2  菌株AS-324化合物的结构解析... 69

3.2.1  菌株AS-324新化合物结构鉴定... 69

3.2.2  菌株AS-324新化合物物理性质与波谱数据... 102

3.2.3  菌株AS-324已知化合物结构鉴定... 106

3.3  菌株AS-324化合物的生物活性测试... 113

3.3.1  菌株AS-324化合物抗肿瘤活性测试... 113

3.3.2  菌株AS-324化合物神经氨酸酶抑制活性测试... 113

3.3.3  菌株AS-324化合物抗菌活性测试... 114

第4章  基于基因组及转录组分析对海藻来源真菌Penicillium chermesinum EN-480次级代谢产物进行深入挖掘... 117

4.1  EN-480基因组分析... 117

4.2  EN-480转录组分析... 120

4.3  实验部分... 123

4.3.1  菌株EN-480的分离纯化与鉴定... 123

4.3.2  菌株EN-480的发酵... 124

4.3.3  菌株EN-480发酵产物的分离纯化... 124

4.4  EN-480改良大米培养基规模发酵产物结构解析... 125

4.4.1  新化合物PCR1结构鉴定... 125

4.4.2  新化合物PCR2结构鉴定... 127

4.4.3  新化合物PCR3结构鉴定... 128

4.4.4  新化合物PCR4结构鉴定... 132

4.4.5  新化合物PCR5结构鉴定... 133

4.4.6  新化合物物理性质与波谱数据... 136

4.5  EN-480 MH2培养基规模发酵产物结构解析... 138

4.5.1  新化合物PCM1结构鉴定... 138

4.5.2  新化合物PCM2结构鉴定... 140

4.5.3  新化合物PCM3结构鉴定... 141

4.5.4  新化合物物理性质与波谱数据... 144

4.6  菌株EN-480新化合物生物合成途径推测... 145

4.7  菌株EN-480化合物生物活性测试... 146

第5章  结论与展望... 149

参考文献... 151

附录  部分化合物NMR谱图... 159

致 谢... 165

作者简历及攻读学位期间发表的学术论文与研究成果... 167