敲除ptsG基因及共表达透明颤菌血红蛋白提高大肠杆菌SHMT产量  

Knockout of ptsG and Co-Expression with Vitreoscilla Hemoglobin Enhance the Production of Serine Hydroxymethyltransferase in Escherichia coli

作　　者：韩琴 徐新星 王儒昕 李鑫 闫达中 吴菁 刘军 HAN Qin;XU Xinxing;WANG Ruxin;LI Xin;YAN Dazhong;WU Jing;LIU Jun(College of Biology and Pharmaceutical Engineering,Wuhan Polytechnic University,Wuhan 430023,China)

机构地区：[1]武汉轻工大学生物与制药工程学院

出　　处：《食品科学》2020年第2期119-125,共7页Food Science

基　　金：国家自然科学基金面上项目(31270112);湖北省自然科学基金项目(2014CFC1133)

摘　　要：利用Red同源重组技术敲除大肠杆菌BL21(DE3)的ptsG基因,得到ptsG基因缺失菌株BL21(DE3)ΔptsG。构建重组质粒,得到表达大肠杆菌丝氨酸羟甲基转移酶(serine hydroxymethyltransferase,SHMT)工程菌株BL21(DE3)/pET-glyA、BL21(DE3)ΔptsG/pET-glyA,及共表达SHMT和透明颤菌血红蛋白(Vitreoscilla hemoglobin,VHb)工程菌株BL21(DE3)ΔptsG/pET-SV。在LB培养基中,BL21(DE3)ΔptsG/pET-glyA菌株与BL21(DE3)/pET-glyA菌株生长情况没有明显差异,BL21(DE3)ΔptsG/pET-SV菌株稳定期的OD 600 nm值比BL21(DE3)ΔptsG/pET-glyA菌株提高了21.3%,在LBG培养基中,稳定期BL21(DE3)ΔptsG/pET-glyA菌株OD600 nm值比BL21(DE3)/pET-glyA菌株提高了19.4%,BL21(DE3)ΔptsG/pET-SV菌株OD600 nm值比BL21(DE3)ΔptsG/pET-glyA菌株提高了21.5%。在LBG培养基中,经过异丙基-β-D-硫代半乳糖苷诱导,与对照菌株BL21(DE3)/pET-28a相比较,两种单表达工程菌株产SHMT活力分别是其6.4倍和7.7倍,共表达工程菌株产SHMT活力是其9.6倍。实验结果表明,敲除ptsG基因能够增加大肠杆菌在含葡萄糖培养基中的生长量及SHMT表达量,共表达VHb能进一步提高菌株生长量和SHMT产量。The phosphotransferase system G(ptsG)-deleted strain BL21(DE3)ΔptsG was obtained by Red homologous recombination.Recombinant plasmids pET-glyA and pET-SV were constructed and transformed separately into the host strain.As a result,the engineered strains BL21(DE3)/pET-glyA and BL21(DE3)ΔptsG/pET-glyA for expression of serine hydroxymethyltransferase(SHMT)from Escherichia coli were obtained as well as BL21(DE3)ΔptsG/pET-SV for co-expression of E.coli SHMT and Vitreoscilla hemoglobin(VHb).In LB medium,there was no significant difference between the growth of BL21(DE3)/pET-glyA and that of BL21(DE3)ΔptsG/pET-glyA,but in the stationary phase,the OD600 nm of BL21(DE3)ΔptsG/pET-SV increased by 21.3%compared to that of BL21(DE3)ΔptsG/pET-glyA.In LBG medium,the OD 600 nm of BL21(DE3)ΔptsG/pET-glyA was 19.4%higher than that of BL21(DE3)/pET-glyA in the stationary phase,but was 21.5%lower than that of BL21(DE3)ΔptsG/pET-SV.In LBG medium,the SHMT activities produced by BL21(DE3)/pET-glyA,ΔptsG/pET-glyA andΔptsG/pET-SV were 6.4,7.7 and 9.6 times as high as that produced by the control strain,respectively.The results showed that knockout of the ptsG gene could increase the growth of E.coli in glucose-containing medium and SHMT expression,and this effect was enhanced by co-expression with VHb.

关 键 词：RED同源重组 丝氨酸羟甲基转移酶 透明颤菌血红蛋白 ptsG基因 共表达 

分 类 号：Q819[生物学—生物工程]