深海宏基因组克隆子次生代谢产物的研究

发布时间：2018-04-21 12:29

本文选题：深海宏基因组 + 次生代谢产物　；参考：《厦门大学》2014年硕士论文

【摘要】：在微生物代谢产物及生合成途径的研究中,由于绝大多数微生物很难或不能通过分离培养方法获得纯培养,因而无法正确认识环境中微生物基因及其编码生合成代谢产物的多样性,无法全面认识自然界物种的多样性。因此,非可培养方法是获知绝大多数微生物的基因多样性及代谢多样性的主要方法。通过直接从环境样品中提取微生物的总DNA,并克隆到合适的可培养微生物宿主中,利用基因组学的研究策略研究环境样品所包含的全部微生物的遗传组成,已成为海洋等极端环境样品的重要分析方法。本文首先对源于深海沉积物样本的宏基因组文库中具有细胞毒活性的克隆子进行了化学筛选,通过与宿主大肠杆菌对比,发现克隆子10-1及25D7能够产生差异代谢产物。进而大规模发酵宿主大肠杆菌及10-1,对其代谢产物进行提取、分离与结构鉴定,得到18个化合物,为6个吲哚生物碱,5个氯霉素衍生物,7个环二肽。其中10-1产生的3-羟基-2-吲哚酮类化合物和靛玉红为差异成分,其生合成途径可能是加氧酶功能基因作用下氧化底物吲哚形成靛红,进而缩合成靛玉红。该生合成途径通过添加底物5-溴吲哚培养10-1得到证实。克隆子25D7的代谢产物的研究直接通过5-溴吲哚底物添加方式进行。发酵液在LC-MS指导下定向分离得到4个溴代产物。其中两个新的溴代双吲哚衍生物生合成途径是加氧酶基因和羟化酶基因表达产生2,3-吲哚-二酮和7-羟基吲哚,再缩合成双吲哚衍生物。通过上述研究,共从两株深海宏基因组克隆子和宿主发酵物中获得23个化合物,其中新化合物2个；验证了4个差异代谢物的生合成途径,为进一步开发和利用深海功能基因奠定了基础。
[Abstract]:In the study of microbial metabolites and biosynthesis pathway, the majority of microbes are difficult or unable to obtain pure culture by the method of isolation and culture. Therefore, it is impossible to correctly understand the diversity of microbial genes and their encoded metabolites in the environment, and to fully understand the diversity of natural species. Therefore, the non-culturable method is the main way to know the gene diversity and metabolic diversity of most microorganisms. By extracting the total DNA of microbes directly from environmental samples and cloning them into suitable culturable microorganism hosts, the genetic composition of all microbes contained in environmental samples was studied using genomics research strategies. It has become an important analytical method for extreme environmental samples such as the ocean. In this paper, the cytotoxic clones from macrogenomic library derived from deep-sea sediment samples were chemically screened. By comparison with host Escherichia coli, it was found that clone 10-1 and 25D7 could produce different metabolites. The metabolites of Escherichia coli and 10-1 were extracted, isolated and identified, and 18 compounds were obtained, including 6 indole alkaloids, 5 chloramphenicol derivatives and 7 cyclic dipeptides. The 3-hydroxy-2-indole compounds produced by 10-1 and indirubin were different components. The pathway of their synthesis may be the oxidation of indole to indirubin under the action of oxygenase function gene, and then the synthesis of indirubin. The biosynthesis pathway was confirmed by adding 5-bromoindole to culture 10-1. The metabolites of clone 25D7 were studied directly by adding 5-bromoindole substrate. Under the guidance of LC-MS, four brominated brominated products were obtained. Two new brominated diindole derivatives were synthesized by the expression of oxygenase gene and hydroxylase gene to produce 2-indole-3-diketone and 7-hydroxyindole, and then condensed to bis (indole) derivatives. Based on the above studies, 23 compounds were obtained from two deep-sea macrogenomic clones and host fermentations, 2 of which were new compounds, which verified the biosynthesis pathway of 4 differentially metabolites. It lays a foundation for the further development and utilization of deep-sea functional genes.
【学位授予单位】：厦门大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R915

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