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

Abstract: 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 OD600 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 and ΔptsG/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.

Key words: Red homologous recombination, serine hydroxymethyltransferase, Vitreoscilla hemoglobin, ptsG gene, co-expression