【作者】 张旭东；

【导师】 陈华； 王鹏；

【作者基本信息】 合肥工业大学 ， 制药工程（专业学位）， 2021， 硕士

【摘要】 玫瑰孢链霉菌发酵得到的次级代谢产物达托霉素是一种具有全新杀菌机制的环脂肽类抗生素。提高达托霉素的产量满足耐药菌株感染用药成为了研究人员的奋斗目标。近年来,我国达托霉素的市场逐年递增,但目前发酵生产达托霉素的菌株效价低,无法满足人们的临床用药需求。随着分子生物学、合成生物学等技术的应用,在产物生物合成途径上改造菌株、研究分析代谢产物相关基因的功能、提高微生物天然产物的产量已经成为新的趋势。本论文以玫瑰孢链霉菌为实验对象,对达托霉素生物合成途径中GntR家族转录调控子进行了研究,主要内容如下:首先,将本实验室保藏的玫瑰孢链霉菌HF-1801,从前体癸酸的添加时间、癸酸的单次添加量和接种量三方面进行了发酵工艺优化。确定癸酸的补料时间为发酵开始后48h,单次补料体积为0.15%,接种量为4%,经过优化后的达托霉素产量最高达到226mg/L。其次,为了在基因水平上改造玫瑰孢链霉菌,建立了遗传操作体系:该菌株对安普霉素等四种常用抗生素均敏感;分子克隆PCR反应体系中退火温度62℃,DMSO添加量5%时,PCR反应有较好的结果;建立了玫瑰孢链霉菌-大肠杆菌接合转移体系。最后,选取GntR家族转录调控子SSIG＿RS21680基因进行研究,构建敲除菌株和过表达菌株,通过不同的固体培养基培养,发现该基因对菌体生长形态分化没有较大影响,通过相同条件下的发酵培养,发现该基因对达托霉素的产量有一定的负调控作用。更多还原

【Abstract】 Daptomycin,a secondary metabolite of Streptomyces roseosporus fermentation,is a cyclic lipopeptide antibiotic with a completely new bactericidal mechanism.Improving the yield of daptomycin to meet the drug-resistant strain infection drug use has become a struggling goal of researchers.In recent years,the market of daptomycin in China has increased year by year,but at present,the strain producing daptomycin by fermentation has a low titer,which can not meet people’s clinical medication needs.With the application of molecular biology,synthetic biology and other technologies,it has become a new trend to engineer strains on the pathway of product biosynthesis,study and analyze the function of genes related to metabolites,and improve the production of microbial natural products.n this paper,we used Streptomyces roseosporus as an experimental agent to study the transcriptional regulators of the GntR family in the daptomycin biosynthetic pathway,as follows:First,the fermentation process was optimized using Streptomyces roseosporus HF-1801 stored in our laboratory in terms of the addition time of precursor capric acid,single addition of capric acid and inoculum size.The feeding time of capric acid was determined to be 48 h after the start of fermentation with a single feed volume of0.15 % and an inoculum size of 4%,and the optimized daptomycin yield reached a maximum of 226 mg/L.Second,to engineer S.roseosporus at the genetic level,a genetic manipulation system was established: the strain was sensitive to all four commonly used antibiotics including Apramycin,a molecular cloning PCR reaction system with annealing temperature of 62 ℃and DMSO addition of 5% gave better results,and a S.roseosporus-Escherichia coli conjugative transfer system was established.Finally,SSIG＿RS21680,a transcriptional regulator of the GntR family,was selected to study in order to construct the knockout and overexpression strains,which were cultured in different solid media and found that this gene did not have a large effect on the morphological differentiation of the bacteriophage growth,while it was found to negatively regulate the production of daptomycin by fermentation and Cultivation under the same conditions.更多还原