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 product η-rhodomycinone of the endophyte Streptomyces sp. SZC001 in Sinomenium acutum

摘要:

【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9 109 166 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(half maximal 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。

Abstract:

[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 of 9 109 166 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 compound ε-rhodomycinone and compound 2 is a new compound η-rhodomycinoe, which is derived from α₂-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 1.55–4.59 μmol/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 enriches ε-rhodomycinone derivatives, furnishing a foundation for the subsequent exploitation of the endophytic resources of S. acutum.