Zhu Yun, Zhou Youzhi, Chu Jianlin, He Bingfang
Wei Sheng Wu Xue Bao. 2015 Dec 4;55(12):1551-9.
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.
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 (AmsbB) , 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.
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.
Knockout mutants (ΔmsbB and ΔwaaF) had significantly higher excretion of beta-FFase and the waaF deletion mutant had higher excretion of penicillin G acylase.
敲除大肠杆菌BL21(DE3)外膜中与脂多糖相关的基因，以研究其对重组蛋白胞外分泌的影响。
利用λ-Red重组系统构建大肠杆菌BL21(DE3)的 waaF或msbB基因敲除突变体[大肠杆菌BL21(ΔwaaF)或大肠杆菌BL21(ΔmsbB)]。然后，分别将重组质粒pET-ffase或pET-pga转化至大肠杆菌BL21(ΔmsbB)、大肠杆菌BL21(ΔwaaF)和大肠杆菌BL21(DE3)中，构建工程菌株大肠杆菌BL21(ΔmsbB)/pET-ffase、大肠杆菌BL21(ΔwaaF)/pET-ffase、大肠杆菌BL21(DE3)/pET-ffase、大肠杆菌BL21(ΔmsbB)/pET-pga、大肠杆菌BL21(ΔwaaF)/pET-pga和大肠杆菌BL21(DE3)/pET-pga。最后，通过摇瓶发酵研究突变体对β-呋喃果糖苷酶（EC 3.2.1.26，β-FFase）和青霉素G酰化酶（EC 3.5.1.11）胞外分泌的影响。
诱导表达4小时后，msbB基因缺失突变体的培养基中β-FFase活性最高可达19.7%，waaF基因缺失突变体的为50.9%，而原始菌株为2.6%。此外，诱导表达24小时后，waaF基因缺失突变体的培养基中青霉素G酰化酶的胞外活性最高可达1708 U/L，是原始菌株的4.1倍。
敲除突变体（ΔmsbB和ΔwaaF）的β-FFase分泌量显著更高，且waaF基因缺失突变体的青霉素G酰化酶分泌量更高。
