利用DNA shuffling构建部分解除对氟苯丙氨酸反馈抑制的aroG  

Using DNA Shuffling to Construct the aroG Mutant Relieved the Feedback-Inhibition of para-fluoro-phenylalanine

作　　者：李晓萍[1] 边英男[1] 郝瑞昕[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5期568-574,共7页Journal of Fudan University：Natural Science

摘　　要：3-脱氧-D-阿拉伯庚酮糖-7-磷酸合成酶(DAHPs)是芳香族氨基酸生物合成途径中关键的限速酶,在大肠杆菌中有3种同工酶,分别由aroG(Phe)、aroH(Trp)和aroF(Tyr)基因编码,这3种酶分别受苯丙氨酸、色氨酸和酪氨酸反馈抑制.为得到解除苯丙氨酸反馈抑制的突变AroG,以Escherichia coli野生型aroG、aroH和aroF基因为亲本,通过DNAshuffling技术对aroG基因进行改组,将获得的改组aroG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2个能耐受10 mg/mLp-FP的克隆.这2个克隆的测序结果显示,其中一个发生了3个点突变:C129T,A531G,A1032G;另一个发生了3个点突变T77A,G883A,A1032G和4个碱基的插入突变即922位插入A,943位插入CG,971位插入A.2个改组AroG的最适pH和最适温度与野生型AroG相比无明显变化.Using the aroG,aroH and aroF of E.coli as the parental gene,the aroG mutant was constructed by DNA shuffling.To screen these mutants resistant to para-fluoro-phenylalanine（p-FP）,the reassembled gene was inserted into pET-30a and cloned into E.coli BL21（DE3）.Two mutants which relieved feedback inhibition on medium containing 10mg/mL p-FP were obtained.Sequence analysis results showed that： one had three point mutations： C129T,A531G,A1032G and the other one had three point mutations T77A,G883A,A1032G and four insertion mutations,including 922A,943CG,971A.The difference of temperature and pH between the wild type aroG and the recombinant aroG is not distinct.

关 键 词：DAHP合成酶 AROG aroF aroH DNA SHUFFLING Phe反馈抑制 

分 类 号：Q789[生物学—分子生物学]