多级联合筛选及表观遗传修饰策略下海洋真菌新颖抗青枯菌产物研究

Marine fungi have been a hotspot for study of new antibacterial biopesticides, as they can produce secondary metabolites with novel structures and potential bioactivities endowed by the unique marine environment. Bacterial wilt is an important soil-borne disease on plants caused by Ralstonia solanacearum, and it is desiderate to develop new type of antibacterial pesticides toward bacterial wilt, as there are no very effective agents to control the disease. In our previous work, a marine-derived fungal strain, Fusarium equiseti D39, attracted our attention because the extract of the fungal culture showed obvious antibacterial activity against R. solanacearum, and the structures of the secondary metabolites were novel. In this research proposal, based on the exiting work, bioassay-LCMS-NMR multistage-combinatorial screening and epigenetic modification will be applied to improve the discovery probability and efficiency of the antibacterial new compounds. Epigenetic modification and fermentation optimization should be performed on the target strain, and multistage-combinatorial screening should be used to choose the optimal fermentation conditions with the selected modification agent. The secondary metabolites will be isolated and identified from the fermentation broth, and be evaluated for their antibacterial activity. The bioactive compounds will be modified by chemical methods in order to discuss their structure-activity relationships. The antibacterial mechanisms of bioactive compounds will be preliminary investigated at last. The above research will provide basic material for the discovery of new antibacterial pesticides toward bacterial wilt with high efficiency and low toxicity.

海洋独特的水体环境赋予了栖息其中的海洋真菌能够产生结构新颖、活性多样的次级代谢产物，已成为抗细菌生物农药研发的新热点。青枯病是由青枯雷尔氏菌引起的一类十分严重的细菌性植物土传病害，目前尚无有效的防治药剂，亟需研发新型抗青枯病农药。我们在前期工作中筛选获得了一株海洋真菌Fusarium equiseti D39，其发酵粗提物具有显著的抗青枯菌活性，且次级代谢产物结构类型新颖。本项目拟在已有的工作基础上，采用bioassay-LCMS-NMR多级联合筛选及表观遗传修饰策略，提高活性新化合物的发现概率和效率。对目标菌株进行表观遗传修饰和发酵优化，运用多级联合筛选策略确定最优发酵条件；进而开展新颖抗菌活性化合物的分离鉴定和活性测定；对活性化合物进行结构修饰并系统分析构效关系；最后，初步探讨活性化合物的抗菌作用机制。本项工作可为新颖高效低毒抗青枯病农药的发现提供基础资料。

海洋独特的水体环境赋予了栖息其中的海洋真菌能够产生结构新颖、活性多样的次级代谢产物，已成为生物农药研发的新热点。本项目采用Bioassay-LCMS-NMR的多级联合手段，筛选出3株抗菌活性显著的海洋真菌Fusarium equiseti D39，Aspergillus sp. D40和Aspergillus versicolor D5，对其次级代谢产物进行研究，并评价其抗青枯菌等植物病原细菌、真菌及除草等农用生物活性。同时，对F. equiseti D39进行了表观遗传修饰，在添加最优试剂丁酸钠的条件下进行了规模化发酵。从3株海洋真菌及其表观遗传修饰发酵产物中共分离鉴定化合物60个，其中新化合物14个，结构类型包括聚酮、生物碱、甾体、脂肪酸和核苷等。首次获得了含有三环[7.2.1.02,7]十二烷骨架和6/6/5环体系的3-decalinoyltetramic acid（3DTA）类化合物decalintetracids A和B，并通过HPLC-ECD、ECD计算、NMR计算的方法确定了化合物的绝对构型。同时，推测了该类化合物的生物合成途径和互变机理。针对结构新颖的fusarisetins类化合物，采用VCD计算和X-Ray单晶衍射的方法确定了其极具挑战性的绝对构型。在生物活性测试中，14个化合物表现出了不同程度的抗菌和除草活性。其中，新骨架化合物decalintetracids A和B对绿苋和反枝苋的种子生长具有明显的抑制作用。Equisetin类化合物不仅具有显著的除草活性，同时能够破坏细菌细胞的超微结构，发挥抗菌活性。针对强活性equisetin，开展了OSMAC发酵优化，使其产量达到59.85 mg/L。呋喃酮类化合物penicillic acid具有显著的抗植物病原细菌活性，对烟草青枯病菌等6种细菌均有不同程度的抑制作用，IC50为11.6–58.2 μg/mL。喹啉生物碱3,6-O-dimethylviridicatin具有显著的抗植物病原细菌活性，对青枯雷尔氏菌和野油菜黄单胞菌的MIC分别为50和100 µg•mL-1。Xanthone类化合物versicone L对灰葡萄孢的最小抑菌浓度达到152 µM；Sterigmatocystin对灰葡萄孢的最小抑菌浓度达到38.3 µM，活性是多菌灵的30倍。本研究为新型生物农药的开发提供了化合物基础。

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