产生埃博霉素的纤维堆囊菌的分子筛选

为寻找潜在的产生埃博霉素(epothilone)的纤维堆囊菌(Sorangium cellulosum),以埃博霉素生物合成基因簇中的epoA,epoC和epoK基因为探针,分别对60株不同土壤样品来源的纤维堆囊菌的DNA样品进行PCR筛选.筛选结果得到了7株阳性菌株,进一步对其进行发酵和粗提物的HPLC-MS分析,检测结果显示3株菌XMU-Nc-03,XMU-So-64和XMU-So-112能产生埃博霉素B,其中XMU-So-112的产量最高.本实验在筛选产埃博霉素活性菌株的同时,还建立了一个快速可靠的发现埃博霉素产生菌的方法.

Screening of Epothilones Producing Strains Sorangium cellulosum

Epothilones are a new class of anticancer compounds produced by the Gram-negative myxobacterium Sorangium cellulosum.It shows high cytotoxicity for animal cells and mimic biological effect of taxol.In order to obtain epothilone producing strains,three pairs of primers were used to amplify fragments of epoA,epoC and epoK which encoding loading module,polyketide synthases(PKS) and P450 enzyme respectively in epothilone biosynthesis cluster from sixty S.cellulosum strains,and the PCR products were sequenced to avoid the false-positive strains.Seven isolates XMU-So-58,XMU-So-64,XMU-So-70,XMU-So-72,XMU-So-112,XMU-So-264 and XMU-Nc-03 were found to have at least one fragment of epoA,epoC and epoK.Then they were successfully purified and their secondary metabolites were analyzed using epothilone B as positive control by HPLC-MS.The result showed that XMU-So-58,XMU-So-70,XMU-So-72 and XMU-So-264 didn′t produce epothilone.While epothilone B were detected in the fermentation products of three strains,XMU-Nc-03,XMU-So-64 and XMU-So-112 with intensity of 2.7×105,6.2×105 and 7.0×106 respectively,at the retention time of 9.8-9.9 min.Thus,XMU-So-112 was proved to be the best producer.In summary,the best producer of epothilone B not only was obtained from 60 Sorangium strains,but also it was established that an efficient method for screening antibiotic producers based on PCR screening coupled with HPLC-MS analysis.