Streptomyces diastatochromogenes丰加霉素合成途径与调控机制解析

Toyocamycin (TM) is a member of the nucleoside antibiotic family. Because of similarity to the structure of nucleoside, TM exhibits various biological activities. Recently the TM was found to be highly efficient against a broad range of plant pathogenic fungi, including Rhizoctonia solani Kühn, Fusarium oxysporum f. sp. cucumerinum, F.oxysporum f. sp niveum and Colletotrichum lindemuthianum. Therefore, TM has been recognized as an ideal fungicide that can be utilized to control the occurrence of plant diseases in the agriculture field. Streptomyces diastatochromogenes 1628, which was originally isolated from soil sample, is a producer of TM. So far, the TM production of S. diastatochromogenes 1628 is only report of TM biosynthesis. As a result, S. diastatochromogenes 1628 has the important research value, however, the TM production is limited in this strain, it can’t meet the requirement of industrial production and limited its application. Therefore, analysis of biosynthetic pathway and regulatory mechanism of TM in S. diastatochromogenes 1628 is of theoretical and practical significance to further construct the higher-producer of TM by using metabolic engineering. In this research, the biological functions of the cloned TM biosynthesis gene cluster were investigated by PCR-targeting and detection of intermediates in order to elucidate the biosynthetic pathway of TM at the molecular level in S. diastatochromogenes 1628. Furthermore, on this basis, the effects of central pleiotropic transcriptional activator adpAsd on the transcriptional level of TM biosynthesis gene cluster and mode of its action were determined to reveal the regulatory mechanism involved in TM biosynthesis of S. diastatochromogenes 1628.

核苷类抗生素丰加霉素(Toyocamycin)可有效防治多种植物真菌病害，已成为农业植物病害防治领域中备受关注的新型抗生素品种。因此，作为目前唯一生物合成丰加霉素的菌株S. diastatochromogenes 1628具有重要的研究价值，但其产量较低，难以满足工业化生产需要，限制了其广泛应用。因此，从分子水平上阐明丰加霉素合成途径及其调控机制具有重要的理论和现实意义。本研究以S. diastatochromogenes 1628为研究对象，克隆生物合成丰加霉素基因簇，利用PCR-targeting技术和中间代谢产物检测等技术方法研究丰加霉素基因簇的生物学功能，阐明其生物合成丰加霉素的分子机制；进一步研究S. diastatochromogenes 1628中全局性调控因子adpAsd对丰加霉素合成基因簇的转录水平影响和作用方式，解析其对生物合成丰加霉素的调控机制。

核苷类抗生素丰加霉素(Toyocamycin,TM)可有效防治多种植物真菌病害，已成为农业植物病害防治领域中备受关注的新型抗生素品种。因此，作为目前唯一生物合成丰加霉素的菌株Streptomyces diastatochromogenes 1628具有重要的研究价值，但其产量较低，难以满足工业化生产需要，限制了其广泛应用。因此，从分子水平上阐明丰加霉素合成途径及其调控机制具有重要的理论和现实意义。.首先，以S. diastatochromogenes 1628 DNA为模板，设计特异性引物，利用PCR，Gibsen Assembly的方法克隆了大小为10.4 kb的丰加霉素基因簇toy cluster（GenBank：KY022432.1）。其与龟裂链霉菌（Streptomyces rimosus）和不吸水链霉菌（Streptomyces ahygroscopicus）中丰加霉素基因簇的核苷酸序列相似度分别为75%，99%。经生物信息学分析，基因簇内的toyA基因编码的TOYA蛋白属于LAL家族，该家族是链霉菌内的一类途径特异性调节因子。以S. diastatochromogenes 1628 DNA为模板，设计特异性引物，克隆toyA基因（GenBank：KT291433.1），大小为2877 bp。将其构建到穿梭质粒pIB139上，获得重组质粒pIB139-toyA，再利用接合转移的方式转入菌株1628获得重组菌株1628-TOYA。发酵后其丰加霉素的产量相对于原始菌株提高了80.6%；经荧光定量PCR检测，重组菌中toyB，toyM，toyG及toyF基因的转录水平均显著高于原始菌株。将1628菌株中的多效性调节基因adpAsd在原核宿主大肠杆菌E. coli Rosseta中表达，获得可溶性的ADPAsd蛋白。经过EMSA分析，ADPAsd能够直接结合toyA基因的启动子序列。.为大幅度提高丰加霉素的产量，利用核糖体工程技术筛选抗性突变株S. diastatochromogenes 1628。利福平抗性（Rifr）突变株1628-T15的TM产量最高达689.3 mg/L，为原始菌株产量的4.5倍；而Rifr突变株1628-T62的TM产量明显降低，只有原始菌株产量的20%。研究结果表明，adpAsd涉及S. diastatochromogenes 1628