大肠杆菌tyrR基因剔除及其对苯丙氨酸生物合成的影响  

Knockout of tyrR Gene in Escherichia coli and Its Effects on the Phenylalanine Biosynthesis

作　　者：商量[1] 范长胜[1] 金瑞良[1] 刘东信[1] 王建刚[1] 尹隽[1] 宋大新[1] 

机构地区：[1]复旦大学生命科学学院微生物学系,上海200433

出　　处：《生物化学与生物物理学报》2003年第8期728-733,共6页

基　　金：国家自然科学基金资助项目 (No .3 0 0 70 0 2 0 )~~

摘　　要：TyrR是大肠杆菌芳香族氨基酸生物合成和运输途径中的一种全局性调控蛋白质。采用双交换同源重组的方法定位突变大肠杆菌染色体tyrR基因 ,在该基因中插入带有卡那霉素抗性基因的DNA片段 ,使之失活 ,实现基因剔除。经PCR、DNA测序、lacZ报告基因等多种方法证实了基因剔除的可靠性。tyrR基因剔除后 ,大肠杆菌芳香族氨基酸生物合成中受TyrR蛋白调控的关键酶的酶活力有所提高 :3 脱氧 2 阿拉伯庚酮糖 7 磷酸合成酶(DAHPS ,由aroG编码 )酶活力提高了 1.0 8倍 ,转氨酶 (AT ,由tyrB编码 )酶活力提高了 2 .70倍 ;突变菌株发酵生产苯丙氨酸的能力提高了 1.5 9倍 ;同时 ,与芳香族氨基酸运输相关的通透酶基因aroP(P)的阻遏被解除 ,细胞运输芳香族氨基酸的能力提高了 70 .2 %。TyrR is a global regulation protein encoded by tyrR gene which controls several transcriptional units involving biosynthesis and transportation of aromatic amino acids in Escherichia coli. In this work, the tyrR gene was knocked out by a double cross homologous recombination. The tyrR - mutant was verified by structural identification by PCR and sequencing, and functional demonstration using lacZ reporter gene. In tyrR - mutant, the activities of two key enzymes in the phenylalanine biosynthesis pathway, whose expression was controlled by TyrR, DAHPS and AT, had been shown to elevate by a 1.08 fold and 2.70 fold compared with the parent strain, respectively. The yield of phenylalanine biosynthesis in the mutant was 1.59 times higher than that of wild type strain. The repression on the transcription of aroP, encoding an aromatic amino acid permease, was eliminated, resulting in a 70.2% increase of the aromatic amino acid transportation in tyrR - mutant strain.

关 键 词：基因剔除 苯丙氨酸生物合成 代谢调控 大肠杆菌 

分 类 号：Q75[生物学—分子生物学]