壮观链霉菌CPCC 200148产生的新次级代谢产物系统挖掘

Streptomyces spectabilis CPCC 200148 is a known producer of spectinomycin and streptovaricin, and used to be an industrial strain for spectinomycin production. Our recent LC-MS analysis revealed the presence of prodigiosine, spectinabilin, SNF4435 and an un-identified new compound in the secondary metabolites of the strain. Bioinformatics analysis (antiSMASH)of Streptomyces spectabilis CPCC 200148 draft genome sequence revealed an enormous number of gene clusters for secondary metabolite biosynthesis. Streptomyces spectabilis CPCC 200148 must be a talented organism for secondary metabolite production, and new compounds remain to be identified from its secondary metabolites.. The present proposal aims first to maximize secondary metabolite diversity by activating silent gene clusters through random mutation, fermentation medium screening, optimal culture parameter searching and solid/liquid state fermentation, and then identify new compound(s) appeared in Streptomyces spectabilis CPCC 200148. The present proposal aims second to obtain the whole genome sequence of Streptomyces spectabilis CPCC 200148 for antiSMASH analysis, and discover new compound(s) of PKS or NRPS type by genome mining silent gene cluster(s) of interest. Three to five new compounds should be discovered from Streptomyces spectabilis CPCC 200148, some with the potential as lead compound(s) for drug development. A close and systematic relationship shall be established between all the secondary metabolites identified from Streptomyces spectabilis CPCC 200148 and the gene clusters responsible for their biosynthesis, which contributes a fine example for secondary metabolite studies of certain talented Streptomyces strain(s).

壮观链霉菌CPCC 200148已知是大观霉素和曲张链菌素产生菌。项目申请人对该菌株脂溶性次级代谢产物进行LC-MS分析，发现了灵菌红素、spectinabilin和新化合物线索等，基因组草图序列生物信息学分析显示了超乎寻常数量次级代谢产物合成基因簇；因此 CPCC 200148具有丰富的次级代谢产物合成能力。本项目：（1）通过诱变激活沉默基因、改变培养基组成和培养条件、固/液体发酵过程跟踪等，实现CPCC 200148次级代谢产物谱最大化，从中发现新化合物；（2）依据基因组测序和生物信息学分析结果，选择1-2个沉默基因簇，通过基因组挖掘技术获得新次级代谢产物，特别是聚酮/聚肽类新化合物。预期本项目发现新化合物3-5个，构建CPCC 200148次级代谢产物与生物合成基因簇之间的对应关系，为链霉菌单菌株次级代谢产物系统研究树立一个新标杆。

壮观链霉菌 CPCC 200148 具有丰富的次级代谢产物合成能力，并曾用于大观霉素生产。本项目对CPCC 200148产生的脂溶性次级代谢产物进行了系统研究，分析鉴定了曲张链菌素、灵菌红素和新金链菌素等10余个化合物，发现了1个具有NRPS-PKS杂合骨架的新分子（杭肽霉素，hangtaimycin），以及1个属于二酮哌嗪类的R构型TDD（tryptophan-dehydrobutyrine diketopiperazine）新分子。杭肽霉素具有弱的抗菌和细胞毒等生物活性。完成了CPCC 200148基因组测序，表明它具有1个9.6Mb线性染色体和1个817kb线性质粒。antiSMASH分析等表明它具有40余个编码次级代谢产物生物合成基因簇，其中包括大观霉素、曲张链菌素、灵菌红素、新金链菌素和杭肽霉素等主要次级代谢产物基因簇，以及若干编码萜类和NRPS类次级代谢产物的基因簇。上述研究结果，为今后对CPCC 200148进行深入的基础（杭肽霉素生物合成机制）与应用开发（理性提高大观霉素等生产水平）研究，奠定了良好工作基础。.采用类似CPCC 200148研究策略，对其他两株链霉菌产生的次级代谢产物进行了研究：从CPCC 204095中发现了具有环庚三烯酮结构单元的isatropolone新组分3个，并首次发现isatropolones具有自噬激活活性；从链霉菌CPCC 204980中发现氧杂蒽酮类抗生素cervinomycin新组分4个，具有显著的抗菌和肿瘤细胞毒活性。.对CPCC 200148等链霉菌琼脂平板发酵培养操作模式进行了优化，特别是接种时采用硅胶刷替代三角玻璃棒，显著提高了工作效率和质量。此外，设计了盖底一体式培养皿，可减少一次性培养皿制造原料——聚苯乙烯用量三分之一，且可简化制造工艺流程，申请并获得实用新型专利授权。