地中海拟无枝酸菌分支酸合酶基因失活的研究-生物化工专业论文.docx

万方数据 万方数据 Dissertation Submitted to Hebei University of Technology for The Master Degree of Biochemical Engineering STUDY ON GENE INACTIVATION OF CHORISMATE SYNTHASE IN AMYCOLATOPSIS MEDITERRANEI by Fan Fei Supervisor: Associate Prof. Jin Hong-xing May 2015 河北工业大学硕士学位论文 摘 要 利福霉素是在抗结核、麻风和艾滋病病发症中应用广泛的重要抗生素，基因调控 策略作为提高利福霉素生物合成的有效方法之一。为此，本论文以利福霉素工业生产 菌—地中海拟无枝酸菌为研究对象，探究了基因失活菌株的构建方法，为构建工业高 产菌株提供可行性依据。 由于地中海拟无枝酸菌属于革兰氏阳性菌，外源基因导入比较困难，且外源基因 导入率的大小直接影响同源重组的几率，所以本论文首先优化了地中海拟无枝酸菌电 击转化条件。以大肠杆菌—拟无枝酸菌穿梭质粒 pULVK1-Am 为载体，地中海拟无枝 酸菌为受体菌，考察了收集菌体时期、细胞壁弱化方式、电击缓冲液和电击参数等因 素对转化率的影响。结果表明以 0.1 μg/mL 氨苄青霉素处理细胞，OD600 为 0.7-0.8 时 收集菌体，用溶菌酶-LiAc-DTT 溶液于 37 oC 孵育 30 min，去离子水作电击缓冲液， 7.5 KV/cm、25 μF、750 ?下进行电击，转化率最高可达到 9.23×104 CFU/μg 质粒 DNA。 由于基因工程改造的工业生产菌株不能带有抗性标记，所以本论文通过重叠延伸 PCR 技术构建了没有抗性标记的分支酸合酶同源失活载体。用已经优化好的电击转化 法将其导入地中海拟无枝酸菌中，经过基因同源重组，得到分支酸合酶失活的菌株。 关键词：地中海拟无枝酸菌 分支酸合酶 基因失活 同源重组 I 地中海拟无枝酸菌分支酸合酶基因失活的研究 II 河北工业大学硕士学位论文 ABSTRACT The ansamycins antibiotic rifamycin produced by Amycolatopsis mediterranei is one of the important secondary polyketide metabolites. Currently, regulatory gene manipulation has been developed as one of the effective ways to improve antibiotic biosynthesis. This thesis studied the construction method of gene inactivation strain using A. mediterranei as the research object, in order to providing a feasible basis of building industrial high yield strain. Exogenous genes into the cells is more difficult, because A. mediterranei is Gram-positive bacterium. First this thesis optimized electroporation conditions of A. mediterranei since the electrotransformation efficiency directly affected the probability of homologous recombination. Some related factors such as collection period of mycelia, treatment of cell wall, electroporation buffer, and electroporation parameters are determined by using A. mediterranei as recipient bacterium and E. coli -Amycolatopsis shuttle plamid pULVK1-Am as the vector. Using 0.1 μg/mL ampicillin to treate A. mediterranei till OD600 up to 0.7-0.8, lysozy