一株产macrolactins芽孢杆菌PKS基因簇启动子的研究
发布时间：2018-07-29 19:54
【摘要】：Macrolactins是一类新型大环内酯类化合物,具有抗菌、免疫抑制、抗肿瘤等多种生物学活性,在医药领域具有极大的应用前景。目前野生菌发酵产macrolactins的产量较低,限制了对此类化合物的研究与应用,因此改造相关生物合成基因簇以提高macrolactins的产量具有十分重要的意义。本文以实验室前期筛选获得的一株能产macrolactins的野生菌ZJU-2011为研究对象,通过测序确定其为一株新型的解淀粉芽孢杆菌,将其PKS基因簇中高保守的AT结构域及可能的启动子区域与参考基因簇进行比对,确定了该基因簇的存在并发现其启动子元件位于第一模块前1Kb左右的位置。以绿色荧光蛋白为报告基因构建启动子检测载体,与P43启动子比较确定该基因簇原始启动子为弱启动子,进一步研究发现原始启动子是一个受葡萄糖调节的启动子,最适作用温度为37℃。以P43启动子为基础通过饱和突变构建启动子突变文库。优化确定聚合酶环形延伸克隆(CPEC)最适的退火温度为61.6℃,最佳的PCR循环数为25个。以LacZ作为报告基因通过CPEC克隆构建启动子突变文库,以β-半乳糖苷酶的酶活表征启动子的强度,筛选得到11个正向突变株,其中标号为“7”的突变株β-半乳糖苷酶酶活为P43启动子控制下的167.7%。对采用同源重组法在ZJU-2011中进行基因敲除的可行性进行研究。确定解淀粉芽孢杆菌ZJU-2011的最适转化条件为甘氨酸添加量为1%,菌体OD600为0.9左右,质粒进行去甲基化处理,电场强度为21KV/cm,电阻为200Ω。构建以mazF毒蛋白为反筛标记的整合载体pIEFMLN,通过同源重组成功筛选到了单交换阳性转化子,但经实验验证双交换重组的概率低于1/1500,因此初步判断同源重组法不适用于解淀粉芽孢杆菌ZJU-2011的基因敲除。尝试构建解淀粉芽孢杆菌中的CRISPR-Cas基因编辑系统。成功构建CRISPR-Cas9相关的敲除载体pHYpCasMLN,经验证该质粒已成功转入ZJU-2011 中。
[Abstract]:Macrolactins is a new class of macrolides with many biological activities, such as antibacterial, immunosuppressive, antitumor and so on. At present, the yield of fermentative macrolactins from wild bacteria is relatively low, which limits the research and application of these compounds. Therefore, it is of great significance to modify the related biosynthetic gene clusters to increase the yield of macrolactins. In this paper, a new strain of Bacillus amylolyticus, ZJU-2011, which can produce macrolactins, was selected as a new strain of Bacillus amylolyticus by sequencing. The highly conserved AT domain and possible promoter region in the PKS gene cluster were compared with the reference gene cluster. The existence of the gene cluster was determined and the position of the promoter element located about 1Kb in front of the first module was found. Green fluorescent protein (GFP) was used as a reporter gene to construct promoter detection vector. Compared with P43 promoter, the primordial promoter of the gene cluster was identified as weak promoter, and the primordial promoter was found to be a glucose regulated promoter. The optimum action temperature is 37 鈩，
本文编号：2153816
[Abstract]:Macrolactins is a new class of macrolides with many biological activities, such as antibacterial, immunosuppressive, antitumor and so on. At present, the yield of fermentative macrolactins from wild bacteria is relatively low, which limits the research and application of these compounds. Therefore, it is of great significance to modify the related biosynthetic gene clusters to increase the yield of macrolactins. In this paper, a new strain of Bacillus amylolyticus, ZJU-2011, which can produce macrolactins, was selected as a new strain of Bacillus amylolyticus by sequencing. The highly conserved AT domain and possible promoter region in the PKS gene cluster were compared with the reference gene cluster. The existence of the gene cluster was determined and the position of the promoter element located about 1Kb in front of the first module was found. Green fluorescent protein (GFP) was used as a reporter gene to construct promoter detection vector. Compared with P43 promoter, the primordial promoter of the gene cluster was identified as weak promoter, and the primordial promoter was found to be a glucose regulated promoter. The optimum action temperature is 37 鈩，
本文编号：2153816
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