Exacerbation of substrate toxicity by IPTG in Escherichia coli BL21(DE3) carrying a synthetic metabolic pathway

Microbial Cell Factories

Research

Miroslava Sedlackova¹  Radek Fedr²  Karel Soucek³  Pavel Dvorak⁴  Zbynek Prokop⁴  Lukas Chrast⁴  Radka Chaloupkova⁴  Jiri Damborsky⁴  Pablo I. Nikel⁵  Víctor de Lorenzo⁵ 

[1] Department of Histology and Embryology, Faculty of Medicine, Masaryk University, 625 00, Brno, Czech Republic;International Clinical Research Center, St. Anne’s University Hospital, Pekarska 53, 656 91, Brno, Czech Republic;Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i., Kralovopolska 135, 612 65, Brno, Czech Republic;International Clinical Research Center, St. Anne’s University Hospital, Pekarska 53, 656 91, Brno, Czech Republic;Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i., Kralovopolska 135, 612 65, Brno, Czech Republic;Department of Experimental Biology, Faculty of Science, Masaryk University, 625 00, Brno, Czech Republic;Loschmidt Laboratories, Department of Experimental Biology and Research Centre for Toxic Compounds in the Environment RECETOX, Faculty of Science, Masaryk University, Kamenice 5/A13, 625 00, Brno, Czech Republic;International Clinical Research Center, St. Anne’s University Hospital, Pekarska 53, 656 91, Brno, Czech Republic;Systems and Synthetic Biology Program, Centro Nacional de Biotecnología CNB-CSIC, Cantoblanco, 28049, Madrid, Spain;

关键词: Metabolic burden;    Substrate toxicity;    Escherichia coli;    Heterologous metabolic pathway;    Isopropyl β-D-1-thiogalactopyranoside;    Lactose;    1,2,3-trichloropropane;    Metabolic engineering;   

DOI  :  10.1186/s12934-015-0393-3

 received in 2015-07-25, accepted in 2015-12-05,  发布年份 2015

来源: Springer

【 摘 要 】

BackgroundHeterologous expression systems based on promoters inducible with isopropyl-β-D-1-thiogalactopyranoside (IPTG), e.g., Escherichia coli BL21(DE3) and cognate LacIQ/PlacUV5-T7 vectors, are commonly used for production of recombinant proteins and metabolic pathways. The applicability of such cell factories is limited by the complex physiological burden imposed by overexpression of the exogenous genes during a bioprocess. This burden originates from a combination of stresses that may include competition for the expression machinery, side-reactions due to the activity of the recombinant proteins, or the toxicity of their substrates, products and intermediates. However, the physiological impact of IPTG-induced conditional expression on the recombinant host under such harsh conditions is often overlooked.ResultsThe physiological responses to IPTG of the E. coli BL21(DE3) strain and three different recombinants carrying a synthetic metabolic pathway for biodegradation of the toxic anthropogenic pollutant 1,2,3-trichloropropane (TCP) were investigated using plating, flow cytometry, and electron microscopy. Collected data revealed unexpected negative synergistic effect of inducer of the expression system and toxic substrate resulting in pronounced physiological stress. Replacing IPTG with the natural sugar effector lactose greatly reduced such stress, demonstrating that the effect was due to the original inducer’s chemical properties.ConclusionsIPTG is not an innocuous inducer; instead, it exacerbates the toxicity of haloalkane substrate and causes appreciable damage to the E. coli BL21(DE3) host, which is already bearing a metabolic burden due to its content of plasmids carrying the genes of the synthetic metabolic pathway. The concentration of IPTG can be effectively tuned to mitigate this negative effect. Importantly, we show that induction with lactose, the natural inducer of Plac, dramatically lightens the burden without reducing the efficiency of the synthetic TCP degradation pathway. This suggests that lactose may be a better inducer than IPTG for the expression of heterologous pathways in E. coli BL21(DE3).

【 授权许可】

CC BY   
© Dvorak et al. 2015