通过DNA shuffling技术获得初步解除苯丙氨酸反馈抑制的3-脱氧-D-阿拉伯庚酮糖-7-磷酸合成酶(AroG)突变株  

Screening for 3-deoxy-D-arabino-hepeulosonate-7-Phosphate Synthase(AroG) Mutants Resistant to Phenylalanine Feedback-inhibition by Using DNA Shuffling Method

作　　者：郝瑞昕[1] 边英男[1] 李晓萍[1] 管栋印[1] 江培翃[1] 黄伟达[1] 

机构地区：[1]复旦大学生物化学系,上海200433

出　　处：《复旦学报（自然科学版）》2010年第3期287-292,共6页Journal of Fudan University：Natural Science

基　　金：教育部博士点基金资助项目(20040246019)

摘　　要：aroG基因编码的3-脱氧-D-阿拉伯庚酮糖-7-磷酸合成酶(3-deoxy-D-arabino-heptulosonate-7-phosphate synthase,AroG)是苯丙氨酸(phenylalanine,Phe)合成途径中的关键酶之一,受到苯丙氨酸的反馈抑制.为得到解除苯丙氨酸反馈抑制的突变AroG,分别以Escherichia coli(E.coli)野生型和突变型aroG基因以及Sal monella typhi mnrium(S.typhi mnrium)野生型aroG基因为亲本,通过DNA suffling技术对aroG进行改组,获得的改组分子与载体pET30a连接得到改组文库,转化到E.coliBL21(DE3)中,在含有Phe类似物对氟苯丙氨酸(p-FP)的培养基上筛选转化子,获得4个解除Phe反馈抑制的克隆.3-deoxy-D-arabino-heptulosonate-7-phosphate（DHAP） synthase AroG encoded by aroG gene catalyzes the first committed step in the phenylalanine biosynthesis pathway,which is feedback inhibited by phenylalanine.To get the AroG mutants resistant to the feedback inhibition of Phe,DNA shuffling were applied on three groups of aroG genes to generate a large,diverse library of random chimeras.The reassembled gene fragments were cloned into the vector pET-30a and transferred into E.coli BL21（DE3） to screen the mutants.Four mutants which relieved feedback inhibition on medium containing 10mg/mL of para-fluoro-phenylalanine（p-FP） were successfully obtained.

关 键 词：DNA SHUFFLING AROG 苯丙氨酸 反馈抑制 

分 类 号：Q789[生物学—分子生物学]