大肠杆菌BL21(DE3)氨基葡萄糖代谢调控相关基因的敲除及glmS在其中的表达研究  

A Preliminary Study of Key Regulated Enzymes Forglucosamine Metabolism in Escherichia coli BL21(DE3)

作　　者：涂宇鹏[1] 何京桦[1] 乐科易[1] 严维耀[1] 

机构地区：[1]复旦大学生命科学学院微生物学与微生物工程系,上海200433

出　　处：《复旦学报（自然科学版）》2012年第6期796-802,共7页Journal of Fudan University：Natural Science

摘　　要：氨基葡萄糖(Glucosamine,GlcN)是一类广泛使用的保健品,对于人类软骨再生及关节炎的临床治疗都具有良好的疗效.为构建以代谢工程为基础的氨基葡萄糖生产菌株,采用λRed同源重组系统将大肠杆菌BL21(DE3)中甘露糖—磷酸转移酶系统操纵子(manXYZ)和乙酰氨基葡萄糖(GlcNAc)转运和代谢特异性系统操纵子(nagBACD-nagE)进行双剔除.随后构建含新型氨基葡萄糖合成酶基因(glmS)的表达载体pETG,并将它转化该双操纵子剔除菌株.结果表明,改造后的菌株不能以GlcN或GlcNAc作为碳源进行代谢生长.表达GlmS菌株的上清液经Ni-NTA亲和层析纯化,其酶活性达8.63U/mg,表明该菌株已具备发酵生产GlcN的初步特性.As a class of widely used healthcare products in the world, glucosamine （GlcN） provides good regeneration of human joint cartilage and palliation the pain or disability of osteoarthritis （OA）. For the purposes to create a new method of GlcN production based on metabolic engineering, operon manXYZ（mannose-specific PTS enzymes） and operon nagBACD-nagE （include acetyl glucosamine-specific transporter, deacetylase, deaminase and its regulatory proteins） of Escherichia coli BL21 （DE3） were deleted using ）,Red homology recombination system. A new expression vector pETG was constructed in order to highly express the GlmS protein. Compared to the wild type Escherichia coli BL21 （DE3）, the engineered strain failed to use glucosamine or acetyl-glucosamine as carbon source. By the modified Morgan-Elson method, an 8. 63 U/mg activity of glmS protein was identified after purification by Ni-NTA affinity chromatography. These suggest that the result bacterial has initial characteristics of the ideal GlcN production strain.

关 键 词：大肠杆菌BL21(DE3) 氨基葡萄糖 λRed同源重组 氨基葡萄糖合成酶 代谢工程 

分 类 号：Q786[生物学—分子生物学]