青风藤内生链霉菌SZC001生物合成潜力分析及抗肿瘤天然产物η-rhodomycinone的鉴定

Biosynthetic potential and anti-tumor natural productη-rhodomycinone of the endophyte Streptomyces sp.SZC001 in Sinomenium acutum

【目的】探究青风藤内生链霉菌SZC001的次级代谢产物生物合成潜力,挖掘未知的活性天然产物。【方法】表面消毒法分离SZC001并利用三代联合二代测序获得完整基因组,结合antiSMASH分析评估其天然产物合成潜力;使用4种培养基发酵初筛代谢谱,通过硅胶柱层析、高效液相制备、高分辨质谱以及核磁共振仪等对化合物进行分离鉴定;利用CCK-8法进行目标化合物的细胞毒性测定。【结果】鉴定研究菌株为链霉菌SZC001,基因组总长度9109166 bp,G+C含量71.08%;antiSMASH显示该菌株共含有31个潜在的生物合成基因簇,有17个基因簇与已知基因簇的相似度小于40%;在4种培养基中可产生多个蒽环类天然产物,经分离鉴定得到含量最高的2个化合物1和2。化合物1为已知化合物ε-rhodomycinone;化合物2为一个新化合物η-rhodomycinone,相对已知化合物α2-rhodomycinone其在C-10位羟基衍生化并且骨架6位羟基变为11位。化合物1与2对2种肿瘤细胞系均有较好的抑制活性,半抑制浓度(halfmaximal inhibitory concentration,IC50)为1.55-4.59μmol/L。【结论】SZC001是一株有挖掘活性天然产物潜力的菌株,从中获得的蒽醌类天然产物具有较好的抗肿瘤活性,后续值得进一步开发和利用。本研究增加了ε-rhodomycinone衍生物的种类,为青风藤内生菌资源的后续开发提供了基础。

[Objective]To investigate the biosynthetic potential of the endophyte Streptomyces sp.SZC001 in Sinomenium acutum and explore the unknown active natural products.[Methods]SZC001 was isolated by surface sterilizing method and its full-length genome sequence was obtained by third-generation and second-generation sequencing.Then,the biosynthetic potential of SZC001 was evaluated by antiSMASH analysis.Fermentation was carried out with four media,and the compounds were separated and identified by silica gel column chromatography,high-performance liquid chromatography,high-resolution mass spectrometry and nuclear magnetic resonance.The CCK-8 assay was employed to examine the cytotoxicity of the target compounds.[Results]The strain was identified as Streptomyces sp.SZC001,with a genome length of9109166 bp and the G+C content of 71.08%.The antiSMASH analysis showed that the genome of the strain contained a total of 31 biosynthetic gene clusters(BGCs),among which 17 BGCs had less than 40%similarity with the known BGCs.The strain produced several anthracyclines in four media,and the two most abundant compounds 1 and 2 were isolated and identified.Compound 1 is the known compoundε-rhodomycinone and compound 2 is a new compoundη-rhodomycinoe,which is derived fromα2-rhodomycinone by derivatization of the hydroxyl group at the C-10 position and transfer of the hydroxyl group at the 6-position to the 11-position of the backbone.Both compounds 1 and 2 showed relatively strong inhibitory effects on two tumor cell lines,with IC50 of 1.55–4.59μmol/L.[Conclusion]SZC001 is a strain that holds the potential for the exploration of active natural products.The anthracyclines produced by the strain exhibit anti-tumor activities and warrant further development and utilization.This study enrichesε-rhodomycinone derivatives,furnishing a foundation for the subsequent exploitation of the endophytic resources of S.acutum.