基于基因组挖掘的农抗120活性成分制霉菌素和丰加霉素的发现和鉴定

Genome mining guided discovery and identification of nystatin and toyocamycin in Streptomyces hygrospinosus var. beijingensis

摘要:

[目的] 分析刺孢吸水链霉菌北京变种（农抗120产生菌）基因组和次级代谢产物组分，研究并鉴定农抗120产生菌中未被发现的活性组分。[方法] 利用antiSMASH在线分析农抗120产生菌Streptomyces hygrospinosus var.beijingensis基因组信息，锁定可能的制霉菌素和丰加霉素生物合成基因簇。利用HPLC和LC-MS等分析方法对农抗120产生菌发酵产物进行分析，同时利用制霉菌素和丰加霉素标准品作为对照，以鉴定该菌株代谢组分中的次级代谢产物。此外，通过构建目标基因簇大片段缺失突变株，并对所得突变株发酵产物进行检测，以确定生物合成基因簇与目的代谢产物的对应关系。[结果] 本研究综合利用基因组序列分析、基因缺失突变株构建以及代谢产物检测方法，鉴定了农抗120产生菌中制霉菌素和丰加霉素两种活性成分，并确定了负责这些化合物合成的基因簇。[结论] 本研究所构建的多重基因簇失活突变株为挖掘刺孢吸水链霉菌北京变种更多的天然次级代谢产物奠定了基础。

Abstract:

[Objective] We aimed to discover and identify the uncharacterized active components of Agricultural Antibiotic 120 by comprehensive analysis of genome sequence and secondary metabolites of Streptomyces hygroscopicus var. beijingensis (Bacteria, which produced Agricultural Antibiotic 120).[Methods] The genome sequence of wild-type strain was analyzed by antiSMASH for the sake of identifying possible biosynthetic gene clusters of nystatin and toyocamycin. HPLC, LC-MS and Q-TOF were used to analyze the secondary metabolites of wild-type strains, using chemical standards as control. Biosynthetic gene cluster (BGC) deletion mutants were constructed, and the fermentation products were analyzed.[Results] Biosynthetic BGCs of nystatin and toyocamycin were identified, and these two compounds were proved to be another two bioactive ingredients of Agricultural Antibiotic 120, besides the known anisomycin and tetramycin.[Conclusion]] The multiple BGC deletion mutant deducted the effect of compounds with high abundance and could serve as a good material to mine more natural compounds originally produced by WT strain. Moreover, correspondence analysis of genome sequence and metabolite profile was an effective method to identify compounds encoded by the silent biosynthetic gene clusters hidden in the genome.