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)

摘要:

摘要:【目的】探究Escherichia coli BL21(DE3)中膜组分相关的脂多糖合成基因waaF或msbB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waaF或msbB敲除，构建敲除菌株E.coli BL21(ΔwaaF)、E.coli BL21(ΔmsbB)。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pET-ffase、pET-pga分别转入敲除菌株及出发菌株中，构建工程菌株E.coli BL21(ΔmsbB)/pET-ffase、E.coli BL21(ΔwaaF)/pET-ffase、E.coli BL21(DE3)/pET-ffase、E.coli BL21(ΔmsbB)/pET-pga、E.coli BL21(ΔwaaF)/pET-pga、E.coli BL21(DE3)/pET-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ΔmsbB为宿主时，胞外分泌量达到19.7%，而以敲除菌株ΔwaaF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ΔwaaF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ΔmsbB和ΔwaaF，ΔmsbB能明显增强β-FFase的胞外分泌，而ΔwaaF对β-FFase和PGA的胞外分泌均有显著的强化作用。

Abstract:

Abstract:［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 (ΔwaaF) or E.coli BL21(ΔmsbB)］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 (ΔmsbB)，E.coli BL21(ΔwaaF) and E.coli BL21 (DE3) respectively，to generate the engineering strains E.coli BL21 (ΔmsbB) /pET-ffase，E.coli BL21 (ΔwaaF)/pET-ffase，E.coli BL21 (DE3)/pET-ffase，E.coli BL21 (ΔmsbB)/pET-pga，E.coli BL21 (ΔwaaF)/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 (ΔmsbB and ΔwaaF) had significantly higher excretion of beta-FFase and the waaF deletion mutant had higher excretion of penicillin G acylase.