大肠杆菌BL21(DE3)膜组分相关基因的敲除对重组蛋白胞外分泌的影响  

Effects of knockout genes related to outer membrane on extracellular secretion of recombinant proteins in Escherichia coli BL21(DE3)

作　　者：朱芸[1] 周有治 储建林[1] 何冰芳[1,2] 

机构地区：[1]南京工业大学生物与制药工程学院,江苏南京211816 [2]南京工业大学药学院,江苏南京211816

出　　处：《微生物学报》2015年第12期1551-1559,共9页Acta Microbiologica Sinica

基　　金：国家自然科学基金(21376119);国家"973"项目(2011CBA00807)~~

摘　　要：【目的】探究Escherichia coli BL21(DE3)中膜组分相关的脂多糖合成基因waa F或msb B的敲除对重组蛋白胞外分泌的影响。【方法】运用Red重组技术将E.coli BL21(DE3)染色体上的基因waa F或msb B敲除,构建敲除菌株E.coli BL21(Δwaa F)、E.coli BL21(Δmsb B)。将本实验室保存的带有β-呋喃果糖苷酶(β-fructofuranosidase,β-FFase)、青霉素G酰化酶(penicillin G acylase,PGA)基因的重组质粒p ET-ffase、p ET-pga分别转入敲除菌株及出发菌株中,构建工程菌株E.coli BL21(Δmsb B)/p ET-ffase、E.coli BL21(Δwaa F)/p ET-ffase、E.coli BL21(DE3)/p ET-ffase、E.coli BL21(Δmsb B)/p ET-pga、E.coli BL21(Δwaa F)/p ET-pga、E.coli BL21(DE3)/p ET-pga。最后通过摇瓶发酵研究敲除菌株对β-FFase、PGA胞外分泌的影响。【结果】当诱导表达4 h,以出发菌株E.coli BL21(DE3)为宿主时,β-呋喃果糖苷酶β-FFase的胞外分泌量占总表达量的2.6%,以敲除菌株Δmsb B为宿主时,胞外分泌量达到19.7%,而以敲除菌株Δwaa F为宿主时,胞外分泌量达到50.9%。另外,当诱导表达24 h,以敲除菌株Δwaa F为宿主时,青霉素G酰化酶PGA的胞外酶活是出发菌株中的4.1倍,达到1708 U/L。【结论】本研究成功构建了敲除菌株Δmsb B和Δwaa F,Δmsb B能明显增强β-FFase的胞外分泌,而Δwaa F对β-FFase和PGA的胞外分泌均有显著的强化作用。[Objective] We knocked out the genes related to lipopolysaccharide in outer membrane of Escherichia coli BL21 （DE3） to study the effects on extracellular secretion of recombinant proteins. [ Methods] We generated waaF or msbB knockout mutants [ E. coli BL21 （ △waaF） or E. coli BL21 （ AmsbB ） ] of E. coli BL21 （DE3） by using lambda-Red recombination system. Then, we transformed recombinant plasmids pET-ffase or pET-pga into E. coli BL21 （AmsbB） , E. coil BL21（AwaaF） and E. coli BL21 （DE3） respectively, to generate the engineering strains E. coli BL21 （AmsbB）/ pET-ffase , E. coli BL21 （AwaaF）/pET-ffase, E. coli BL21 （ DE3 ） / pET-ffase, E. coli BL21 （△msbB）/pET-pga, E. coli BL21 （AwaaF）/pET-pga and E. coli BL21 （DE3） /pET-pga. Finally, we studied the effects of mutants on extracellular secretion of beta- fructofuranosidase （EC 3.2. 1.26, beta-FFase） and penicillin G acylase （EC 3.5.1. 11 ） in shaking flask fermentation. [ Results] After induced expression for 4 hours, up to 19.7% of the beta-FFase activity was found in the culture medium with the msbB deletion mutant, and 50.9% with the waaF deletion mutant, compared to the original 2.6%. Besides, after induced expression for 24 hours, up to 1708 U/L extracellular activity of penicillin G acylase was found in the culture medium with the waaF deletion mutant, which was 4.1 times of the original. [ Conclusion] Knockout mutants （△msbB and △waaF） had significantly higher excretion of beta-FFase and the waaF deletion mutant had higher excretion of penicillin G acylase.

关 键 词：大肠杆菌BL21(DE3) 膜脂 基因敲除 

分 类 号：Q786[生物学—分子生物学]