石磺来源海洋链霉菌Streptomyces ardesiacus SCSIO LO23中germicidin类化合物的分离鉴定及其生物合成分析

Isolation of germicidins and analysis of their biosynthetic pathways in Streptomyces ardesiacus SCSIO LO23, a marine-derived actinomycete from Onchidium sp.

摘要:

海洋共附生放线菌具有生产结构特殊和活性显著化合物的潜力。【目的】以海洋软体动物石磺Onchidium sp.来源共附生海洋链霉菌Streptomyces ardesiacus SCSIO LO23为研究对象，分离并鉴定其次级代谢产物结构，分析化合物的生物合成基因簇及其生物合成途径。【方法】采用多种培养基对该菌株进行发酵优化并放大发酵，利用多种柱色谱方法分离得到germicidin类化合物，并利用波谱学手段和X-Ray单晶衍射技术完成化合物的结构鉴定；利用Illumina HiSeq对目标菌株进行全基因组测序，结合antiSMASH和BLAST在线分析软件实现菌株中germicidin类化合物生物合成基因功能注释和生物合成途径分析，并通过异源表达进一步确定其生物合成基因。【结果】

从AM3培养基发酵产物中分离鉴定了6个吡喃酮类化合物germicidin A (1)、germicidin B (2)、germicidin D (3)、germicidin H (4)、isogermicidin A (5)和isogermicidin B (6)，其中化合物2和6对斑马鱼神经行为有显著兴奋作用。通过对Illumina HiSeq测序结果生物信息学分析，结合异源表达，验证了化合物生物合成的相关基因，并结合文献对化合物1–6的生物合成途径进行了推导。【结论】本研究阐明了菌株S.ardesiacus SCSIO LO23来源germicidin类化合物的结构、生物活性和其生物合成基因，夯实了该类化合物的研究基础。

关键词:germicidin;生物合成;Ⅲ型聚酮合酶;Streptomyces ardesiacus;海洋共附生放线菌

Abstract:

Marine epiphytic actinomycetes are potential sources of natural bioactive products with unique structures. [Objective] To isolate and identify the secondary metabolites of Streptomyces ardesiacus SCSIO LO23 which was derived from marine Onchidium sp. (Mollusca), and analyze their biosynthetic gene clusters and biosynthetic pathways. [Methods] A variety of media were used to optimize the fermentation of the strain. Further large-scale fermentation and extraction were performed to yield the germicidins. The structures of the germicidins were characterized by spectroscopy and single-crystal X-Ray diffraction. The whole genome of the strain was sequenced by Illumina HiSeq. antiSMASH and BLAST were combined for gene function annotation and biosynthetic pathway analysis of germicidins in the strain. The biosynthetic gene was further verified by heterologous expression. [Results] Six germicidins were isolated and identified from the fermentation products of AM3 medium, which were germicidin A (1), germicidin B (2), germicidin D (3), germicidin H (4), isogermicidin A (5), and isogermicidin B (6). Compounds 2 and 6 showed excitatory effect on zebrafish neurobehavior at the concentration of 30 μmol/L. The biosynthetic gene was verified by bioinformatic analysis and heterologous expression, and the biosynthetic pathways of compounds 1-6 were deduced. [Conclusion] The structures, bioactivity, and the biosynthetic genes of germicidins from S.ardesiacus SCSIO LO23 are clarified, which lays a solid foundation for the study of this type of compounds.

Key words:germicidin;biosynthesis;type Ⅲ polyketide synthase;Streptomyces ardesiacus;marine epiphytic actinomycetes