Production of recombinant protein by a novel oxygen-induced system in Escherichia coli

Abstract

BackgroundThe SoxRS regulon of E. coli is activated in response to elevated dissolved oxygen concentration likely to protect the bacteria from possible oxygen damage. The soxS expression can be increased up to 16 fold, making it a possible candidate for recombinant protein expression. Compared with the existing induction approaches, oxygen induction is advantageous because it does not involve addition or depletion of growth factors or nutrients, addition of chemical inducers or temperature changes that can affect growth and metabolism of the producing bacteria. It also does not affect the composition of the growth medium simplifying the recovery and purification processes.ResultsThe soxS promoter was cloned into the commercial pGFPmut3.1 plasmid creating pAB49, an expression vector that can be induced by increasing oxygen concentration. The efficiency and the regulatory properties of the soxS promoter were characterized by measuring the GFP expression when the culture dissolved oxygen concentration was increased from 30% to 300% air saturation. The expression level of recombinant GFP was proportional to the oxygen concentration, demonstrating that pAB49 is a controllable expression vector. A possible harmful effect of elevated oxygen concentration on the recombinant product was found to be negligible by determining the protein-carbonyl content and its specific fluorescence.By performing high density growth in modified LB medium, the cells were induced by increasing the oxygen concentration. After 3 hours at 300% air saturation, GFP fluorescence reached 109000 FU (494 mg of GFP/L), representing 3.4% of total protein, and the cell concentration reached 29.1 g/L (DW).ConclusionsInduction of recombinant protein expression by increasing the dissolved oxygen concentration was found to be a simple and efficient alternative expression strategy that excludes the use of chemical, nutrient or thermal inducers that have a potential negative effect on cell growth or the product recovery.

Characterization of Endogenous and Reduced Promoters for Oxygen-Limited Processes Using Escherichia coli

“…Successful development of expression systems rely on the availability of well characterized promoters with suitable properties. For industrial applications, it is desirable that promoters can be induced by mechanisms that do not rely on the addition of a chemical agent, , shifts of pH or temperature . Oxygen limitation is an attractive means of inducing the expression of genes of interest.…”

Tracking of Dissolved Oxygen Distribution and Consumption Pattern in a Bespoke Bacterial Growth System

“…On the contrary, an increased concentration of oxygen would stress the microbial cells due to generation of toxic products, such as superoxide (), and produced in increased levels under elevated oxygen concentrations 6. These toxic products are more reactive than the molecular oxygen 26 and can provoke series of metabolic changes in the microbial cells such as oxidation of important macromolecules like protein and DNA oxidation 34. Therefore, a sufficient oxygen supply is crucial to microbial cells growth and to avoid detrimental effects of oxygen concentrations above and below the optimal concentration.…”

“…As the oxygen level is above the critical concentration, the specific uptake rate is constant and microbial cells are not starved. Generally, 5–10 % of the air saturation is considered the value of critical oxygen level under the average working conditions and it can be varied depending on the microbial species 26. In shaking flasks, the rate of oxygen transfer ( OTR ) is calculated from Eq.…”

Microbial stress: From molecules to systems (Sitges, November 2015)

“…Joseph Shiloach (National Institute of Diabetes Digestive and Kidney Disease/NIH, USA) reported an example of how oxidative stress faced by cells during growth can be exploited for the production of heterologous proteins. He showed that in response to high oxygen concentrations, soxS expression increased 16-fold in E. coli, making its promoter a possible candidate for inducing recombinant protein expression under these conditions [11]. Induction of recombinant protein expression by increasing the dissolved oxygen concentration was found to be a simple and efficient alternative expression strategy that excludes the use of chemical, nutrient or thermal inducers that have a potential negative effect on cell growth or on product recovery.…”