中国科学院海洋研究所机构知识库

海洋来源的丝状真菌（尤其是曲霉属和青霉属真菌）所产生的各种骨架类型的天然产物往往具有良好的生物活性。海藻及红树林来源真菌是海洋来源真菌的重要组成部分，也是天然产物研究的重要资源宝库。但是，随着研究的深入和已报道天然产物数量的增多，已知化合物的重复发现变得越来越频繁，新化合物的发现则变得越来越困难。鉴于许多微生物次级代谢产物的合成基因簇在常规培养条件下多是沉默的，近年来多种研究策略像共培养、表观遗传修饰和基因组发掘等被广泛应用于激活这些沉默代谢途径，也因此而发现、报道了若干新化合物。

本论文以海藻和红树林来源的三株曲霉属真菌（阿拉巴马曲霉Aspergillus alabamensis EN-547、聚多曲霉A. sydowii EN-534和构巢曲霉A. nidulans MA-143）和两株青霉属真菌（梅花状青霉Penicillium herquei MA-370和桔青霉P. citrinum EN-535）为研究对象，采用改变培养基类型（MA-370和EN-547）、共培养（EN-534 & EN-535）和添加诱导分子（MA-143）等策略来激活真菌的沉默代谢途径，从中共分离鉴定不同结构类型的化合物109个，其中新化合物29个（含1个新骨架化合物），新天然产物2个，结构类型涵盖了吲哚二酮吗啉类、喹啉酮类、喹诺唑啉酮类、桔青霉素类、蒽醌类、氧杂蒽酮类、α-吡喃酮类、甾体类、甘油酯类等多种骨架。对获得的所有新化合物和部分已知化合物进行了抗细菌、抗农业病害真菌和神经氨酸酶抑制活性的评价。

从梅花状青霉MA-370的发酵粗提物中分离鉴定了39个不同结构类型的化合物，其中新化合物22个，化合物HI 1为一个新骨架类型的非那烯衍生物，另外还有两个新的α-吡喃酮类化合物（HI 8和HI 9）被分离鉴定。首次对分离得到的非那烯类化合物进行了手性HPLC分析工作并对其异构化的机理进行了分析和讨论，完成了部分对映异构化合物的手性拆分工作并通过ECD计算确定了其绝对构型。从阿拉巴马曲霉EN-547的发酵粗提物中分离鉴定化合物21个，包括2个新的吲哚二酮吗啉类衍生物（AL 1和AL 2）、1个新的多羟基麦角甾酮类衍生物（AL 3）和1个新的生物碱类天然产物（AL 4）。对聚多曲霉EN-534和桔青霉EN-535进行了共培养研究，从其发酵粗提物中分离鉴定化合物33个，包括2个新的桔青霉素类衍生物（SC 1和SC 2）。从构巢曲霉MA-143的发酵粗提物中分离鉴定化合物16个，包括1个新的蒽醌类衍生物（ND 1）、1个新的氧杂蒽酮类衍生物（ND 2）和1个新的生物碱类天然产物（ND 3）。

抗菌活性测试结果表明，化合物HI 2对受试细菌中的藤黄微球菌Micrococcus luteus，铜绿假单胞菌Pseudomonas aeruginosa，溶藻弧菌Vibrio alginolyticus和副溶血性弧菌V. parahemolyticus有较好的抑制活性，部分活性与阳性对照氯霉素相当；化合物HI 3和HI 4对受试农业病害真菌中的小麦纹枯病菌Ceratobasidium cornigerum，苹果炭疽病菌 Colletotrichum gloeosporioides和苹果腐烂病菌Valsa mali表现出一定的抑制活性，最小抑菌浓度（MIC值）变化范围为16-64 μg/mL。化合物AL 1和AL 2对大肠埃希氏杆菌Escherichia coli，藤黄微球菌M. luteus，小麦纹枯病菌C. cornigerum，苹果炭疽病菌C. gloeosporioides，小麦赤霉病菌Fusahum graminearum，番茄枯萎病菌F. oxysporum和苹果腐烂病菌V. mali均表现出一定的抑制活性，MIC值变化范围为16-64 μg/mL；化合物AL 1和AL 3对溶藻弧菌V. alginolyticus表现出一定的抑制活性，MIC值均为64 μg/mL；化合物AL 2和AL 3对铜绿假单胞菌P. aeruginosa 表现出一定的抑制活性，MIC值变化范围为32-64 μg/mL。化合物SC 1和 SC 2对鲇鱼爱德华氏菌Edwardsiella ictaluri，铜绿假单胞菌P. aeruginosa，溶藻弧菌V. alginolyticus，小麦赤霉病菌F. graminearum，番茄枯萎病菌F. oxysporum和苹果腐烂病菌V. mali均表现出一定的抑制活性，MIC值变化范围为16-64 μg/mL。化合物ND 1和 ND 4对大肠埃希氏杆菌E. coli，鲇鱼爱德华氏菌E. ictaluri，藤黄微球菌M. luteus，溶藻弧菌V. alginolyticus和副溶血性弧菌V. parahemolyticus均表现出一定的抑制活性，MIC值变化范围为1-32 μg/mL，部分抑菌效果与阳性对照药物氯霉素相当；化合物ND 2对小麦赤霉病菌F. graminearum，番茄枯萎病菌F. oxysporum和苹果腐烂病菌V. mali均表现出一定的抑制活性，MIC值变化范围为8-64 μg/mL。神经氨酸酶抑制活性测试结果表明，化合物SC 1-SC 5对神经氨酸酶均具有一定的抑制作用，以化合物SC 3的抑制活性最好，IC₅₀值为12.9 nM。

本论文的研究内容不仅证实了改变培养基类型、添加诱导分子和共培养等策略在微生物天然产物研究工作中的有效性，也丰富了海洋来源天然产物的化合物类型，为海洋药物的研发提供了物质基础和数据支撑。

Marine derived filamentous fungi (especially genera Aspergillus and Penicillium) are luxuriant sources of natural products with diverse scaffolds and bioactivities. Among them, fungi associated with marine algae and mangrove are a promising source for secondary metabolites research. However, the discovery of new compounds is getting more difficult, while the re-isolating of known compounds is becoming more frequently. On the other hand, many microbial biosynthetic genes related to secondary metabolites are silent or in lower transcription under a standard/common laboratory fermentation conditions. Recently, multiple approaches, such as co-cultivation, epigenetic manipulation, and genome mining, have been developed to activate its cryptic metabolic pathways and a number of new compounds were therefore identified.

During our ongoing study of bioactive secondary metabolites from marine algae and mangrove derived filamentous fungi, P. herquei MA-370, A. alabamensis EN-547, A. sydowii EN-534, P. citrinum EN-535 and A. nidulans MA-143 were selected as our target fungal strains, and we carried out a series of strategies such as modified fermentation medium (MA-370 and EN-547), cocultivation (EN-534 and EN-535) and revulsant addition (MA-143) to activate their cryptic biosynthetic pathways. As a result, a total number of 109 metabolites with diverse scaffolds were isolated and identified from the extracts of the above fungal strains’ fermentation, and 29 of them (including a new skeleton phenalenone derivative) were new compounds. All of the new compounds and some of the known were evaluated for their bioactivities and some of them showed potent value for drug development.

Thirty-nine secondary metabolites were isolated from P. herquei MA-370, seven of them were racemic phenalenones (HI 1-HI 7 and HI 10-HI 11, including 18 new stereostructures); HI 1 was a new skeleton compound, and HI 8 and HI 9 were two new α-pyrone analogues. The racemic phenalenones were analysized and some of them were successfully separated by chiral HPLC columns for the first time, and we have discussed the probable mechanism for their racemation. The absolute configurations of some of the racemic phenalenones were determined by ECD calculation. Twenty-one secondary metabolites were isolated from A. alabamensis EN-547. Among them, AL 1 and AL 2 are rare diketomorpholine derivatives whereas AL 3 is a highly conjugated ergostane-type steroid. Thirty-three secondary metabolites, including a new citrinin dimer (SC 1) and a new citrinin monomer (SC 2), were isolated from the coculture of A. sydowii EN-534 and P. citrinum EN-535. Sixteen compounds, including a new anthraquinone derivative (ND 1), a new xanthone analog (ND 2), and a new amino acid derivative (ND 3), were isolated from culture broth and mycelia extracts of A. nidulans MA-143 under 0.1 % ethanol stress.

All of the identified new compounds were tested for their antimicrobial activities against two human pathogens, five aquatic bacteria, and nine agricultural fungal pathogens. HI 2 showed potent inhibition against M. luteu, P. aeruginosa, V. alginolyticus, and V. parahemolyticus, which were comparable to the positive control chloramphenicol. HI 3 and HI 4 showed some inhibition against C. cornigerum, C. gloeosporioides, and V. mali, with MICs ranging from 16 to 64 μg/mL. AL 1 and AL 2 showed some inhibition against C. cornigerum, C. gloeosporioides, E. coli, F. graminearum, F. oxysporum, M. luteus, and V. mali with MICs ranging from 16 to 64 μg/mL. SC 1 and SC 2 showed some inhibition against E. ictaluri, F. graminearum, F. oxysporum, P. aeruginosa, V. alginolyticus and V. mali, with MICs ranging from 16 to 64 μg/mL. ND 1 and ND 4 showed some inhibition against E. coli, E. ictaluri, M. luteus, V. alginolyticus, and V. parahemolyticus, with MICs ranging from 1 to 32 μg/mL. ND 2 showed some inhibition against F. graminearum, F. oxysporum, and V. mali, with MICs ranging from 8 to 64 μg/mL. SC 1-SC 5 were further assayed for anti-influenza neuraminidase (NA) (homologous protein of H₅N₁) activity in vitro. SC 3 showed the best inhibition activity with IC₅₀ 12.9 nM.

In conclusion, our study provided positive evidence of strategies such as coculture and medium modification may activate the cryptic biosynthetic pathways of marine fungi, and broadened the structures of marine natural products.