鼠李糖脂减毒高产菌株的构建和盐霉素基因簇salBⅢ基因功能研究
发布时间：2018-10-30 15:58
【摘要】：鼠李糖脂是一种低毒性,易于生物降解的,具有良好选择性和专一性的糖脂类阴离子生物表面活性剂,在医药、食品、化妆品、环境保护等众多工业领域具有广泛的应用前景。铜绿假单胞菌(Pseudomas aeruginosa)是目前鼠李糖脂产量最高的菌株,但是其具有条件致病性,易感染免疫力低下的患者,临床感染率可以达到30%以上,直接用于工业生产存在很严重的安全隐患。5-烯醇丙酮酞莽草酸-3-磷酸合成酶基因aroA是细菌和植物莽草酸途径合成芳香族氨基酸的关键基因,而哺乳动物不具备这种途径。在前期研究中,本课题组利用在假单胞菌中建立的高效同源重组系统,成功敲除了铜绿假单胞菌PAO1(Pseudomas aeruginosa PAO1)的aroA基因,从而构建出了一株在添加有芳香族氨基酸的培养基中才能生长的减毒Pseudowas aeruginosa PAO1-△aroA。本课题在此基础上构建了一个鼠李糖脂表达质粒,该质粒包含鼠李糖脂合成相关基因鼠李糖脂转移酶复合体RhlAB、自转运酯酶基因EstA、庆大霉素抗性基因、接合转移起始位点oriT和一个广谱性复制子pBBRI。将该质粒电转化进入构建好的减毒菌株PAO1-AaroA中,多拷贝质粒可以增加鼠李糖脂合成相关基因在该菌中的拷贝数,且在发酵过程中不会丢失,通过鼠李糖脂甲苯乳化实验和测定鼠李糖脂发酵产量可确定该减毒菌株的鼠李糖脂产量可增加75%。最终,本课题成功构建了该高产鼠李糖脂的减毒PAO1-△aroA。盐霉素是由白色链霉菌DSM 41398产生的一种离子载体抗生素,它能够妨碍细胞内外Na+/Ca2+的传递,破坏膜内外离子平衡,造成细胞死亡,对革兰氏阳性菌(包括分枝杆菌和一些丝状真菌)及各种球虫有很强的抑制和杀灭作用。最近研究发现,盐霉素不仅能够杀死乳腺癌干细胞,还能抑制前列腺癌、肝癌和肺癌等多种肿瘤细胞的生长,具有比普通抗癌药物紫杉醇更好的药效。前期研究将盐霉素合成基因簇克隆至天蓝色链霉菌A3(2)中进行异源表达时发现并未表达出预期的盐霉素(m/= 733.6[M-H20+H]+),而是产生了一个盐霉素衍生物(m/z = 752.6[M+H]+)。本课题对盐霉素生物合成途径中的相关基因进行测序发现,salBⅢ基因发生突变,有六个核苷酸序列丢失,导致所编码蛋白连续缺少一个苏氨酸一个亮氨酸。之后对盐霉素基因簇上该基因进行遗传操作,研究清楚salBⅢ基因的功能。通过体内基因敲除和修复实验表明salBⅢ基因与盐霉素的第一个吡喃环的成环反应相关。
[Abstract]:Rhamnolipid is a kind of anionic biosurfactants with low toxicity, easy biodegradation, good selectivity and specificity. It has been widely used in many industrial fields such as medicine, food, cosmetics, environmental protection and so on. Pseudomonas aeruginosa (Pseudomas aeruginosa) is the strain with the highest yield of rhamnolipid, but it has a conditional pathogenicity and is susceptible to infection in patients with low immunity. The clinical infection rate can reach more than 30%. AroA gene is a key gene in the synthesis of aromatic amino acids by the pathway of shikimic acid in bacteria and plants. Mammals do not have this pathway. In previous studies, our team successfully knocked out the aroA gene of Pseudomonas aeruginosa (PAO1 (Pseudomas aeruginosa PAO1) by using the highly efficient homologous recombination system established in Pseudomonas aeruginosa (Pseudomonas aeruginosa). A attenuated Pseudowas aeruginosa PAO1- aroA. was constructed to grow in medium containing aromatic amino acids. In this study, a rhamnolipid expression plasmid was constructed, which contained the gene associated with rhamnolipid biosynthesis, RhlAB, self-transporter esterase gene EstA, and gentamicin resistance gene. The initiation site of conjugate transfer oriT and a broad-spectrum replicon pBBRI. The multicopy plasmid can increase the copy number of the genes associated with rhamnolipid biosynthesis in the strain PAO1-AaroA, and it will not be lost during fermentation. By emulsifying rhamnose glycolipid toluene and determining the fermentative yield of rhamnolipid, the rhamnolipid yield of the attenuated strain can be increased by 75%. Finally, the PAO1- aroA. with high yield of rhamnolipid was successfully constructed. Salinomycin is an ionic carrier antibiotic produced by Streptomyces albicans DSM 41398. It can block the transfer of Na / Ca2 in and out of cells, destroy the balance of ions inside and outside the membrane, and cause cell death. It can inhibit and kill Gram-positive bacteria (including Mycobacterium and some filamentous fungi) and various coccidia. Recent studies have found that salinomycin can not only kill breast cancer stem cells, but also inhibit the growth of many kinds of tumor cells, such as prostate cancer, liver cancer and lung cancer. In previous studies, when the salinomycin synthase gene cluster was cloned into Streptomyces aquilinum A3 (2) for heterologous expression, it was found that the expected salinomycin was not expressed (m / r = 733.6 [M-H20 H]). Instead, it produces a salinomycin derivative (m / z = 752.6 [M / M H]). In this study, we sequenced the related genes in the biosynthesis pathway of salinomycin and found that the salB 鈪，
本文编号：2300464
[Abstract]:Rhamnolipid is a kind of anionic biosurfactants with low toxicity, easy biodegradation, good selectivity and specificity. It has been widely used in many industrial fields such as medicine, food, cosmetics, environmental protection and so on. Pseudomonas aeruginosa (Pseudomas aeruginosa) is the strain with the highest yield of rhamnolipid, but it has a conditional pathogenicity and is susceptible to infection in patients with low immunity. The clinical infection rate can reach more than 30%. AroA gene is a key gene in the synthesis of aromatic amino acids by the pathway of shikimic acid in bacteria and plants. Mammals do not have this pathway. In previous studies, our team successfully knocked out the aroA gene of Pseudomonas aeruginosa (PAO1 (Pseudomas aeruginosa PAO1) by using the highly efficient homologous recombination system established in Pseudomonas aeruginosa (Pseudomonas aeruginosa). A attenuated Pseudowas aeruginosa PAO1- aroA. was constructed to grow in medium containing aromatic amino acids. In this study, a rhamnolipid expression plasmid was constructed, which contained the gene associated with rhamnolipid biosynthesis, RhlAB, self-transporter esterase gene EstA, and gentamicin resistance gene. The initiation site of conjugate transfer oriT and a broad-spectrum replicon pBBRI. The multicopy plasmid can increase the copy number of the genes associated with rhamnolipid biosynthesis in the strain PAO1-AaroA, and it will not be lost during fermentation. By emulsifying rhamnose glycolipid toluene and determining the fermentative yield of rhamnolipid, the rhamnolipid yield of the attenuated strain can be increased by 75%. Finally, the PAO1- aroA. with high yield of rhamnolipid was successfully constructed. Salinomycin is an ionic carrier antibiotic produced by Streptomyces albicans DSM 41398. It can block the transfer of Na / Ca2 in and out of cells, destroy the balance of ions inside and outside the membrane, and cause cell death. It can inhibit and kill Gram-positive bacteria (including Mycobacterium and some filamentous fungi) and various coccidia. Recent studies have found that salinomycin can not only kill breast cancer stem cells, but also inhibit the growth of many kinds of tumor cells, such as prostate cancer, liver cancer and lung cancer. In previous studies, when the salinomycin synthase gene cluster was cloned into Streptomyces aquilinum A3 (2) for heterologous expression, it was found that the expected salinomycin was not expressed (m / r = 733.6 [M-H20 H]). Instead, it produces a salinomycin derivative (m / z = 752.6 [M / M H]). In this study, we sequenced the related genes in the biosynthesis pathway of salinomycin and found that the salB 鈪，
本文编号：2300464
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