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

Pavel Dvorak,Pavel Dvorak,Karel Soucek,Radek Fedr,Radek Fedr,Radek Fedr,Radek Fedr,Radka Chaloupkova,Radka Chaloupkova,Radka Chaloupkova,Radka Chaloupkova,Zbynek Prokop,Zbynek Prokop,Víctor De Lorenzo,Lukas Chrast,Miroslava Sedlackova,Jiri Damborsky,Lukas Chrast,Karel Soucek,Jiri Damborsky,Karel Soucek,Pablo I Nikel

doi:10.1186/s12934-015-0393-3

Abstract

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).Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0393-3) contains supplementary material, which is available to authorized users.

Highlights

Heterologous expression systems based on promoters inducible with isopropyl-β-D-1thiogalactopyranoside (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 E. coli BL21(DE3) and LacIQ/PlacUV5-T7 expression system suffers from certain drawbacks that mainly stem from the rapid and strong overexpression of recombinant proteins triggered by exposure to the synthetic inducer IPTG
The combined effects of multiple simultaneous stresses on the hosts and their engineered induction systems have not been examined in depth. To address this knowledge gap, we examined a recombinant strain of E. coli BL21(DE3) under conditions that provide an extreme combination of exogenous and endogenous stresses

Summary

Introduction

Heterologous expression systems based on promoters inducible with isopropyl-β-D-1thiogalactopyranoside (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. E. coli strain BL21(DE3) carries an inducible T7 RNA polymerase-dependent expression system that allows for the simple manipulation and tuning of protein production levels, and it has become a laboratory workhorse [1,2,3,4] This strain carrying commercial pET vectors or their derivatives has been the host of choice in numerous studies on recombinant protein expression [5, 6]. Fitness costs associated with the activities of the foreign proteins, which may cross-talk with the host’s extant metabolic network [11] and burdens linked to the components of the expression system, such as the IPTG inducer and its import into the cell, are frequently discussed [16, 17]

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Results

Conclusion