【作者】 李周；

【导师】 赵圣印； 唐功利；

【作者基本信息】 东华大学 ， 有机化学， 2011， 硕士

【摘要】 链霉菌源的次级代谢物是天然产物药物的重要组成部分,所谓次级代谢是微生物在一定的生长时期(一般是稳定生长期),以初级代谢产物为前体,合成一些对微生物的生命活动没有明确功能的物质的过程。以初级代谢小分子产物为反应底物,通过一系列特定的酶蛋白催化反应,即多个不同功能的酶依次作用,最终得到微生物次级代谢产物。通过微生物全基因测序和生物信息学分析发现,微生物染色体中有许多小分子化合物的生物合成基因簇,而这些小分子化合物在微生物发酵过程中产量很低或者不产生,通过异源表达的方法可以发酵得到一些结构新奇的小分子化合物或者使化合物产量得到提高,这有利于我们发现更多的新化合物。本课题共分为三个部分：1)链霉菌Streptomyces rochei ATCC10739发酵、次级代谢产物Borrelidin分离纯化和结构确证；2)链霉菌Streptomyces TP-A2060菌株中几个小分子次级代谢产物基因簇的异源表达；3)链霉菌Streptomyces 371菌株中几个小分子次级代谢产物基因簇的异源表达。Borrelidin是从链霉菌(Streptomyces rochei ATCC 10739)中分离到的种18元大环内酯抗生素,其具有CDK(依赖于细胞周期的蛋白激酶)抑制活性和抑制血管生成活性。我们的工作主要是优化发酵条件并对化合物进行分离纯化,对其结构进行确证。在菌株发酵过程中对发酵条件进行优化,以稳定并提高化合物产量。最终确定了最适发酵条件：ISP-2培养基中添加1%甘油,起始pH值为6.0,培养温度30℃,相对湿度50%,发酵时间为7d。化合物经ESI-MS、HR-ESI-MS和1HNMR确证其结构与文献相符。Streptomyces TP-A2060是抗肿瘤抗生素Yatakemycin的产生菌,我们主要是对其中的几个小分子化合物基因进行异源表达。由全基因测序和生物信息学分析可知,化合物主要包括五个萜类化合物、一个芳香族聚酮化合物、一个无法预知结构类型的化合物。通过克隆质粒、异源表达质粒和突变株的构建,我们成功获得了全部基因簇的异源表达质粒、Tep-1和Tep-4的突变株,并对这两个突变株进行了小量发酵。希望通过原生质体转化得到其余突变株,最后发酵得到预期化合物。进一步的突变株构建和发酵工作还在进行中。Streptomyces 371是生物农药金核霉素的产生菌,我们所做的工作主要是希望通过异源表达获得可能的三个萜类化合物和一个芳香族聚酮化合物。目前已经成功得到了全部基因簇的异源表达质粒、Tep-7和Pkz的突变株,质谱结果显示两个突变株小量发酵得到了预期的萜类和聚酮化合物。进一步的研究工作还在进行中。更多还原

【Abstract】 Secondary metabolites producing by Streptomyces play important role in natural product drugs. Secondary metabolism is the process that microorganisms in certain period of growth (usually in stationary phase) synthesize a number of products with some uncertain functions from primary metabolic precursor. These process are carried out by a serial of enzymatic reactions in microorganisms using primary metabolites. Based on the genome sequencing of microbe and bioinformatics analysis, some gene clusters of other small molecules were elucidated in the microbial chromosomes. The production of these small molecules is usually very low or even can’t be detected during the fermentation. However, some structurally novel compounds were generated or the production was improved by heterologous expression of the biosynthetic gene clusters. It benefits us to discover-more novel compounds.Three parts are discussed in this dissertation, including 1) Fermentation of Streptomycete rochei ATCC10739, separation and isolation of secondary metabolite Borrelidin,2) Heterogenous expression of some small secondary metabolites’ gene clusters from Streptomyces TP-A2060, and 3) Heterogenous expression of some small secondary metabolites’gene clusters from Streptomyces 371.Borrelidin is a structurally distinct 18-membered macrolide antibiotic isolated from Streptomycete rochei ATCC10739, which was found to inhibit cyclindependent kinase (CDK) and show antiangiogenesis activity (IC50=0.4 ngmL-1). Our work is to optimize fermentation condition, separate and isolate this compound, and confirm its structure. We optimize the fermentation conditions in order to stabilize and increase borrelidin’s production. The optimal fermentation conditions were determined as follow:the ISP-2 medium with 1% glycerol; initial pH 6.0; culture temperature,30℃; relative humidity,50% and fermentation time,7 days. The structure of Borrelidin was confirmed by ESI-MS, HR-ESI-MS and 1HNMR in comparison with published rusults.Streptomyces TP-A2060 is the producing strain of antitumor antibiotic yatakemycin. In this part, senven gene clusters involved in biosynthesis of five terpenoids, a polyketide compound and an unknown compound from this strain were heterogenous expressioned. We cloned these gene clusters, inserted the DNA fragments into expressing vectors, and tried to transfer the expression plasmid into heterogenous host Streptomyces coelicolor CH999, now we obtained all expression plasmids and two mutant strains of Tep-1 and Tep-4. Two mutant strains of Tep-1 and Tep-4 were fermented. We hope we could get the rest of mutant strains through protoplast transformation soon. The expectant secondary metabolites are isolating through fermentation of mutant strains. The works need further exploration.Streptomyces 371 is the producing strain of biopesticide aureonucleomycin. We try to get three terpenoids and a polyketide compound by heterogenous expression strategy. Now, we had obtained all expression plasmids and mutant strains of Tep-7 and Pkz, and two secondary metabolites of an expectant terpenoid and a polyketide compound were detected by HPLC-MS from the fermentation broth. The isolation and structure eludication of these compounds are on the way.更多还原