敲除ptsG基因及共表达透明颤菌血红蛋白提高大肠杆菌产SHMT

食品科学 ›› 0, Vol. ›› Issue (): 0-0.

• 生物工程 • 下一篇

敲除ptsG基因及共表达透明颤菌血红蛋白提高大肠杆菌产SHMT

韩琴¹,徐新星¹,王儒昕¹,李鑫¹,闫达中²,吴菁¹,刘军¹

1. 武汉轻工大学生物与制药工程学院
2. 武汉轻工大学生物与制药工程学院

收稿日期:2018-11-14 修回日期:2019-11-15 出版日期:2020-01-25 发布日期:2020-01-19
通讯作者: 刘军 E-mail:junliu85@163.com
基金资助:
假单胞菌NyZ12 降解环己胺的分子机理研究

Knockout of ptsG and co-expression of Vitreoscilla hemoglobin enhance the production of SHMT in Escherichia coli

Qin HAN¹,Xing XinXU¹, ¹, ¹, ¹,Jing WU¹,Jun LIU

Received:2018-11-14 Revised:2019-11-15 Online:2020-01-25 Published:2020-01-19
Contact: Jun LIU E-mail:junliu85@163.com

摘要/Abstract

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，敲除ptsG基因的菌株与对照菌株生长情况没有明显差异，ΔptsG/pET-SV菌株稳定期的OD600 nm值比BL21（DE3）ΔptsG/pET-glyA菌株提高了21.3%，在LBG培养基中，稳定期敲除ptsG基因的菌株的OD600 nm值比对照菌株提高了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（Isopropyl-beta-D-thiogalactopyranoside，IPTG）诱导，与对照菌株BL21（DE3）/pET-glyA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。

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 ptsG-deleted strain BL21(DE3)ΔptsG was obtained by Red homologous recombination. Recombinant plasmids pET-glyA and pET-SV were constructed and transformed into host strains to obtain engineered strains BL21(DE3)/pET-glyA, BL21(DE3)ΔptsG/pET-glyA and BL21(DE3)ΔptsG/pET-SV, in which serine hydroxymethyltransferase (SHMT) of Escherichia coli was expressed, or E. coli SHMT and Vitreoscilla hemoglobin (VHb) were co-expressed, respectively. The growth of the above strains was studied. In LB medium, there was no significant difference in the growth between BL21(DE3)/pET-glyA and BL21(DE3)ΔptsG/pET-glyA, but in stable phase, the OD600 nm of BL21(DE3)ΔptsG/pET-SV increased by 21.3%, compared to that of BL21(DE3)ΔptsG/pET-glyA, respectively. In LBG medium, the OD600 nm of BL21(DE3)ΔptsG/pET-glyA was 19.4% higher than that of BL21(DE3)/pET-glyA in stable phase. However, BL21(DE3)ΔptsG/pET-SV biomass yield was more than that of BL21(DE3)ΔptsG/pET-glyA in stable phase, with the increment of OD600 nm by 21.5%. In LBG medium, for the two strains of single-expressing SHMT, BL21(DE3)/pET-glyA and BL21(DE3)ΔptsG/pET-glyA, the SHMT activities were 6.4 times and 7.7 times that of the control strain, respectively. The SHMT activity in the strain of co-expressing SHMT and VHb was 9.6 times that of the control strain. The results showed that the knockout of ptsG gene could increase the growth of E. coli in glucose-containing medium and the strain expression of SHMT, and co-expression of VHb could further enhance the strain growth and SHMT production in engineered strain.

Key words: Red homologous recombination, Serine hydroxymethyltransferase, Vitreoscilla hemoglobin, ptsG gene, Co-expression

中图分类号:

Q819

韩琴徐新星王儒昕李鑫闫达中吴菁刘军. 敲除ptsG基因及共表达透明颤菌血红蛋白提高大肠杆菌产SHMT[J]. 食品科学, 0, (): 0-0.

Qin HAN Xing XinXU Jing WU Jun LIU. Knockout of ptsG and co-expression of Vitreoscilla hemoglobin enhance the production of SHMT in Escherichia coli[J]. FOOD SCIENCE, 0, (): 0-0.

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敲除ptsG基因及共表达透明颤菌血红蛋白提高大肠杆菌产SHMT

Knockout of ptsG and co-expression of Vitreoscilla hemoglobin enhance the production of SHMT in Escherichia coli

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