含苏氨酸操纵子重组质粒的构建及其对大肠杆菌L-苏氨酸积累的影响  

Construction of recombinant plasmids containing threonine operon and their effects on L-threonine accumulation

作　　者：张雪[1,2] 闫继爱[1,2] 于雷[2] 张国强[2] 张芸[2] 陈宁[1] 温廷益[2] 

机构地区：[1]天津科技大学生物工程学院,天津300457 [2]中国科学院微生物研究所,北京100101

出　　处：《微生物学报》2009年第5期591-596,共6页Acta Microbiologica Sinica

基　　金：国家“863计划”(2006AA02Z216)~~

摘　　要：【目的】通过分子生物学手段构建重组质粒,将其转入野生型大肠杆菌W3110,分析含苏氨酸操纵子基因的质粒及质粒定点突变解除反馈抑制时,对L-苏氨酸积累的影响。【方法】以W3110染色体DNA为模板,PCR扩增苏氨酸操纵子基因,即启动子THrLp、编码前导肽基因thrL以及thrA、thrB、thrC基因,通过重叠延伸PCR的方法对thrA基因定点突变,解除苏氨酸对它的反馈抑制,构建出重组表达质粒pWYE112和pWYE134,5L发酵实验测定L-苏氨酸的产量。【结果】经5L发酵罐发酵产酸实验,W3110的L-苏氨酸产量为0.036±0.004g/L,携带含苏氨酸操纵子质粒的W3110菌株L-苏氨酸产量为2.590±0.115g/L,质粒上thrA解除反馈抑制后,L-苏氨酸的产量增加到9.223±1.279g/L。【结论】过表达苏氨酸操纵子基因可以使L-苏氨酸积累,进一步解除thrA基因的反馈抑制,可以增强L-苏氨酸积累的效果,为L-苏氨酸工程菌改造的进一步研究奠定了基础。[ Objective ] We reconstructed two recombinant plasmids and studied their effects on L-threonine accumulation of Escherichia coli W3110. [ Methods] We amplified the threonine operon containing ThrLp promoter, lead peptide thrL, thrA, thrB and thrC genes by PCR from E. coli W3110 chromosome and ligated it into the pMD19 T-vector. Site-directed mutation were carried out by gene splicing by overlap extension PC1/ to release the feedback inhibition of aspartokinase I （ thrA ）. Two recombinant plasmids pWYEll2 and pWYE134 were transformed into E. coli W3110 by eleetroporation. Fed-batch eultures of E. coli W3110 were carried out in 5-Liter fermentors and the L-threonine concentration was measured by HPLC. [Results] Fed-batch fermentation results showed that E. coli W3110 could accumulate little L-threonine （0.036 ±0.004 g/L） but recombinant E. coli W3110 harboring the plasmid pWYEll2 containing a threonine operon exhibited a L-threonine production of 2.590 ±0. 115 g/L. Furthermore, L-threonine production reached 9.223± 1.279 g/L when the feedback inhibition of thrA was released. [ Conclusion] Overexpression of threonine operon can lead to the accumulation of L-threonine. Further release of feedback inhibition of aspartokinase I can enhance its accumulation.

关 键 词：L-苏氨酸 重叠延伸PCR 大肠杆菌 发酵 定点突变 

分 类 号：Q78[生物学—分子生物学]