基于活性和基因从海洋微生物中筛选烯二炔类抗生素

【目的】高质量的海洋微生物菌种库及其天然产物库是新药开发的重要来源。在本研究中我们通过筛选新的烯二炔类抗生素来对已经构建的海洋微生物天然产物库进行质量评价。【方法】首先我们根据烯二炔类抗生素能引起DNA断裂的活性构建了活性筛选模型,并对我们的天然产物库进行筛选;其次根据合成烯二炔核心结构的独特且保守的重复I型聚酮合成酶设计了引物,通过PCR扩增的方法对海洋微生物库进行序列筛选。【结果】通过活性筛选从我们的海洋微生物天然产物库中获得一个阳性的发酵产物。对该阳性菌(LS481)的系统发育学分析表明该菌属于能产生烯二炔类化合物—Dynemicin的Micromonospora chersina,对其发酵产物TLC分析证明该菌确实产生Dynemicin类化合物。通过基因筛选得到了2个具备合成烯二炔核心结构聚酮合成酶的菌株,16S rRNA基因分析显示其中一个很可能为灰色链霉菌(MS098),另外一株菌则同Streptomyces vinaceus NBRC 13425T和Streptomyces cirratus NRRLB-3250T最相近。【结论】我们的活性筛选模型能够有效获得烯二炔类物质,结合基因筛选能够进一步获得可能产生烯二炔物质的菌株。初筛结果也再一次验证了我们海洋微生物天然产物库的质量较好。

Exploiting bioactive Enediynes from marine microbe based on activity and gene screening

Abstract: Objective] A high quality library of marine microbes and their associated natural products is critical for successful drug discovery. In this research a method to assess the quality of our marine microbial natural product library through screening for novel enediyne-like compounds from this library was set up. Methods] A high throughput screening assay based on the unique DNA-damage activity of enediyne-like compounds has been constructed and our marine microbial natural product library was screened. Because the polyketide synthase responsible for the biosynthesis of enediyne core is a conserved iterative type I polyketide synthase, a sequence-based screening method was built to get the strains that harbored enediyne biosynthesis gene clusters. Results] Through activity-based screening, a positive hit from our marine natural product library was acquired. 16S rRNA analysis has revealed that this strain-LS481 was 99% similar to Micromonospora chersina DSM 44151T that could produce enediyne compound-Dynemicins. The crude extract of LS481 showed similar characteristics with Dynemicin A. In addition, two strains, MS098 and LS2004, were acquired through sequence-based screening. The 16S rRNA of MS098 was 100% the same as Streptomyces griseus NBRC 13350, and LS2004 was most close to Streptomyces vinaceus NBRC 13425T and Streptomyces cirratus NRRL B-3250T. Conclusion] We can successfully isolate enediyne compounds through the activity-based screening and assess the potential of producing enediyne compounds through sequence-based screening.