Enhancing thermo-induced recombinant protein production in Escherichia coli by temperature oscillations and post-induction nutrient feeding strategies

• A novel thermal induction strategy for recombinant protein production by E. coli was developed.
• Temperature oscillations between conditions adequate for optimal growth and full heterologous gene induction were applied.
• The strategy allows, for the first time, the simultaneous expression of recombinant protein while maintaining cell growth.
• A several-fold increase in biomass and recombinant protein concentration was achieved.
• A decrease in the concentration of toxic organic acids was achieved.

Traditional strategies for production of thermo-induced recombinant protein in Escherichia coli consist of a two-phase culture, with an initial growth stage at low temperature (commonly 30 °C) followed by a production stage where temperature is increased stepwise (commonly up to 42 °C). A disadvantage of such strategies is that growth is inhibited upon temperature increase, limiting the duration of the production stage and consequently limiting recombinant protein production. In this work, a novel oscillatory thermo-induction strategy, consisting on temperature fluctuations between 37 and 42 °C or 30 and 42 °C, was tested for improving recombinant protein production. In addition, the induction schemes were combined with one of three different nutrient feeding strategies: two exponential and one linear. Recombinant human preproinsulin (HPPI), produced under control of the λPL-cI857 system in the E. coli BL21 strain, was used as the model protein. Compared to the conventional induction scheme at constant temperature (42 °C), longer productive times were attained under oscillatory induction, which resulted in a 1.3- to 1.7-fold increase in maximum HPPI concentration. Temperature oscillations led to a 2.3- to 4.0-fold increase in biomass accumulation and a decrease of 48–62% in the concentration of organic acids, compared to conventional induction. Under constant induction, growth ceased upon temperature increase and the maximum concentration of HPPI was 3.9 g/L, regardless of the post-induction feeding strategy used. In comparison, the combination of temperature oscillations and a high nutrient-feeding rate allowed sustained growth after induction and reaching up to 5.8 g/L of HPPI.