大肠杆菌GalP-GlK途径功能分析  

Functional Analysis of GalP-GlK Pathway in Escherichia coli

作　　者：李文 王彩喆 牟林云 王正祥 LI Wen;WANG Caizhe;MOU Linyun;WANG Zhengxiang(College of Chemical Engineering and Materials Science,Tianjin University of Science&Technology,Tianjin 300457,China;Tianjin Key Laboratory of Industrial Microbiology,Tianjin 300457,China;College of Biotechnology,Tianjin University of Science&Technology,Tianjin 300457,China)

机构地区：[1]天津科技大学化工与材料学院,天津300457 [2]天津市工业微生物重点实验室,天津300457 [3]天津科技大学生物工程学院,天津300457

出　　处：《天津科技大学学报》2024年第4期39-45,72,共8页Journal of Tianjin University of Science & Technology

基　　金：天津市杰出人才计划项目(JC20200309)。

摘　　要：大肠杆菌(Escherichia coli)主要经磷酸转移酶系统(phosphotransferase system,PTS)完成葡萄糖的底物水平磷酸化,启动葡萄糖代谢。为了拓展大肠杆菌的葡萄糖转运代谢能力,在删除大肠杆菌PTS系统的基础上,通过对半乳糖转运蛋白(GalP)与葡萄糖激酶(GlK)的过量表达搭建不依赖PTS系统的GalP-GlK葡萄糖转运途径,研究并探索大肠杆菌葡萄糖代谢的启动新方式及其效率,并考察其生理代谢的变化。通过基因重组技术删除大肠杆菌B0013-025基因组中ptsHI-crr、ptsG及mlc基因,获得了突变株025P。此突变株在以葡萄糖为唯一碳源的培养基中几乎不生长,PTS系统被破坏后,葡萄糖转运能力出现了严重缺失。进一步利用启动子gap增强galP与glk的表达,所获得的重组菌株025PG恢复了经氧化磷酸化启动葡萄糖代谢的能力。强化GalP-GlK途径不仅可以启动葡萄糖代谢,还可以启动半乳糖与阿拉伯糖代谢,代谢强弱排序为葡萄糖(μ=0.31 h^(-1))>半乳糖(μ=0.27h^(-1))>阿拉伯糖(μ=0.21h^(-1))>果糖=木糖(μ=0.01h^(-1))。因此,不依赖PTS系统的大肠杆菌菌株025PG在强化GalP-GlK途径后能够重新代谢葡萄糖,此结果为后续相关工业菌株的遗传选育提供了理论基础。The metabolic pathway of glucose in Escherichia coli is mainly initialized through a phosphotransferase system(PTS)for substrate level phosphorylation of glucose.In order to expand the ability of glucose transport and metabolism in E.coli,a PTS-independent glucose transport pathway(GalP-GlK)was established through the overexpression of galactose transporter(GalP)and glucokinase(GlK)on the basis of deleting the PTS system of E.coli.The new initiation mode and efficiency of glucose metabolism of E.coli were investigated,and the changes of physiological metabolism were further explored.Mutant strain 025P was obtained by deleting ptsHI-crr,ptsG and mlc genes from E.coli B0013-025 genome by gene recombinant technology.The mutant strain 025P grew hardly in the medium with glucose as the only carbon source,indicating a significant loss of glucose transport capacity after the destruction of PTS system.The expression of galP and glk was further enhanced by promoter gap,and the ability to initiate glucose metabolism by oxidative phosphorylation of the recombinant strain 025PG was restored.Strengthening the GalP-GlK pathway could not only initiate glucose metabolism,but also galactose and arabinose metabolism.The order of metabolic strength was glucose(μ=0.31h^(-1))>galactose(μ=0.27h^(-1))>arabinose(μ=0.21h^(-1))>fructose=xylose(μ=0.01h^(-1)).In conclusion,the PTS-independent strain 025PG was able to re-metabolize glucose after overexpression of GalP-GlK pathway,which has provided a theoretical basis for the sub-sequent genetic selection of related industrial strains.

关 键 词：大肠杆菌 半乳糖转运蛋白 葡萄糖激酶 PTS系统 糖类转运代谢 

分 类 号：Q936[生物学—微生物学]