Nejvíce citovaný článek - PubMed ID 24973068
A Pseudomonas putida strain genetically engineered for 1,2,3-trichloropropane bioremediation
BACKGROUND: Heterologous expression systems based on promoters inducible with isopropyl-β-D-1-thiogalactopyranoside (IPTG), e.g., Escherichia coli BL21(DE3) and cognate LacI(Q)/P(lacUV5)-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. RESULTS: The 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. CONCLUSIONS: IPTG 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 P lac , 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).
