【作者】 陈艳；

【导师】 张佑红； 谌颉；

【作者基本信息】 武汉工程大学 ， 生物化工， 2015， 硕士

【摘要】 粘质沙雷氏菌是一种广泛存在于自然界中的革兰氏阴性菌,该菌是包括人类在内的哺乳动物、腔肠动物、昆虫、线虫、植物等的条件致病菌。该菌的次级代谢产物灵菌红素具备多种生物活性,其中最引人注意的是灵菌红素引起癌细胞凋亡的作用机制完全不同于当前的临床抗癌药物,是很有开发潜力的抗癌药物。现阶段对细胞周期的研究主要停留在真核细胞上,本文创新性地将流式细胞术应用到原核细胞中研究粘质沙雷氏菌的生长代谢规律,可为临床解决该菌引起的感染和灵菌红素产量提高提供理论指导,也为原核生物细胞周期规律的研究提供重要参考价值。本论文主要研究了碳源对粘质沙雷氏菌次级代谢调控的影响,首先在以蔗糖为碳源的培养基中添加环磷酸腺苷(cyclic adenosine monophosphate,cAMP),研究cAMP在碳源代谢过程中的作用,然后用牛肉膏、蔗糖、甘油、葡萄糖分别发酵生产灵菌红素,比较不同碳源对灵菌红素合成的影响。论文研究工作如下:(1)细胞内cAMP的浓度升高时,碳源利用效率变高,细胞分裂及DNA复制速度随之加快,细胞形态结构发生改变,灵菌红素的生物合成过程被阻断,表明cAMP影响细菌碳源代谢过程。(2)用不同碳源发酵生产灵菌红素,菌体干重比较:牛肉膏>蔗糖>甘油>葡萄糖,灵菌红素浓度比较:蔗糖>葡萄糖>甘油>牛肉膏。结合流式细胞仪检测分析得:碳源种类不同时,碳源代谢途径随之改变,导致细菌分裂与遗传物质复制速率、细胞形态结构、灵菌红素生产能力的改变。实验表明碳源对粘质沙雷氏菌次级代谢有重要的调控作用,在四种碳源,牛肉膏、蔗糖、甘油、葡萄糖中,蔗糖是最佳碳源。(3)细菌在正常代谢下,细胞分裂生长,遗传物质复制分配过程,次级代谢状态与碳源代谢是一个协调的过程。本文还对粘质沙雷氏菌在5 L发酵罐中的碳源、磷酸盐、培养基配方、溶氧、补料过程进行了初步探讨。结果为:发酵中保持溶氧30%以上,限制加入磷酸盐,初始培养基配方:蔗糖0.25%,蛋白胨1.5%,氯化钠0.5%,无水氯化镁0.25%,吐温-80 0.5%;发酵至50 h时进行补料,补料配方:蔗糖0.5%,吐温0.5%。更多还原

【Abstract】 Serratia marcescens(S. marcescens) is a Gram-negative bacteria, which widely exists in environment. It is reported as an opportunistic pathogen of coelenterates, insects,nematodes, plants, and mammals including humans. The nature red pigment prodigiosin(PG) is a kind secondary metabolite of S. marcescens, it has been certified possessing multiple biological activities. It is noticeable because the mechanism of cancer cell apoptosis resulted from PG is quite different from the current clinical anti-cancer drugs.It is promising to be new anti-cancer drugs. Research on the cell cycle is mainly in the eukaryotic cell currently, however, this article innovatively applied flow cytometry to study the pattern of growth and metabolism of prokaryotic cells in S. marcescens. The present work may provide a theoretical basis for the clinical study of infections caused by this Gram-negative bacteria and improving PG production, and but also is an important reference for studying prokaryotic cell cycle.In order to find out the regulation of carbon source on secondary metabolic in S.marcescens, first the cyclic adenosine monophosphate(cAMP) was added in the medium,sucrose as the carbon source, to study the influence of cAMP on carbon metabolism in bacteria. Then we use different carbon sources such as beef extract, sucrose, glycerol and glucose to compare the synthesis of the secondary metabolites PG. The main work was as follows: 1. As the intracellular cAMP level elevated, carbon source utilization was more efficiency, and cells division,DNA replication accelerated, and the morphology of the cells changed, and at last the biosynthesis of PG was blocked. Those results show that cAMP plays an important role in the carbon metabolism. 2. Fermentation with different carbon sources, the dry cell weight was compared: beef extract >sucrose> glycerol>glucose, and PG level was compared: sucrose> glucose> glycerol >beef extract.Combined with flow cytometry analysis, we obtained the following results: using different carbon sources, it causes a series of changes in the metabolic pathway, cell division and growth speed, replication rate of genes, morphological structure, and PG production. To sum up, we know that carbon sources enormously influenced thesecondary metabolites PG biosynthesis in S. marcescens. Of the four carbons, beef extract, sucrose, glycerol and glucose, sucrose is more suitable. 3 The results also confirmed that cell division and growth, gene duplication and the genome distribution,secondary metabolic states are normally coordinated with nutrient substances.We investigated the fermentation process of S. marcescens in five liters fermenter,including the effects of carbon, phosphate, media formulations, dissolved oxygen and feed supplement on PG production. The preliminary results are as flollows: to maintain more than 30% dissolved oxygen, and limit phosphate; initial media formulations: 0.25%sucrose, 1.5% peptone, 0.5% NaCl, 0.25% anhydrous magnesium chloride, 0.5%Tween-80; feeding supplements after 60 hours and its formulations: 0.5% sucrose and 0.5 % Tween-80.更多还原