Production of biomass and recombinant human-like collagen in Escherichia coli processes with different CO2 pulses.

The evolution of CO(2) in a fed-batch culture of recombinant Escherichia coli containing human-like collagen (HLC) cDNA was determined with an O(2)-enriched air supply (40%, v/v) in a 12.8 l fermentor; a maximum CO(2) concentration of 12.7% in the effluent gas was detected. The CO(2) pulse injection experiments showed that: (1) a 20% CO(2) pulse introduced in the batch cultivation phases inhibited cell growth but if introduced in the fed-batch cultivation phases slightly stimulated growth; and (2) CO(2) inhibited HLC expression only in the expression phase, where the final HLC concentration decreased by 34% under a 3 h 20% CO(2) pulse. The higher the CO(2) concentration and/or the longer the duration of the CO(2) pulse, the stronger the stimulatory or inhibitory effects.

Analysis of metabolic flux in Escherichia coli expressing human-like collagen in fed-batch culture.

Metabolic flux distributions of recombinant Escherichia coli BL21 expressing human-like collagen were determined by means of a stoichiometric network and metabolic balancing. At the batch growth stage, the fluxes of the pentose phosphate pathway were higher than the fluxes of the fed-batch growth phase and the production stage. After the temperature was increased, there was a substantially elevated energy demand for synthesizing human-like collagen and heat-shock proteins, which resulted in changes in metabolic fluxes. The activities of the Embden-Meyerhof-Parnas pathway and the tricarboxylic acid cycle were significantly enhanced, leading to a reduction in the fluxes of the pentose phosphate pathway and other anabolic pathways. The temperature upshift also caused an increase in NADPH production by isocitrate dehydrogenase in the tricarboxylic acid cycle. The metabolic model predicted the involvement of a transhydrogenase that generates additional NADH from NADPH, thereby increasing ATP regeneration in the respiratory chain. These data indicated that the maintenance energy for cellular activity increased with the increase in biomass in fed-batch culture, and that cell growth and synthesis of human-like collagen could clearly represent the changes in metabolic fluxes. At the production stage, more NADPH was used to synthesize human-like collagen than for maintaining cellular activity, cell growth, and cell propagation.

Characteristics of fed-batch cultures of recombinant Escherichia coli containing human-like collagen cDNA at different specific growth rates.

Fed-batch cultures of recombinant Escherichia coli BL21 for producing human-like collagen were performed at different specific growth rates (0.1 approximately 0.25 h(-1)) before induction and at a constant value of 0.05 h(-1) after induction by the method of pseudo-exponential feeding. Although the final biomass (around 69 g l(-1)) was almost the same in all fed-batch cultures, the highest product concentration (13.6 g l(-1)) was achieved at the specific growth rate of 0.15 h(-1) and the lowest (9.6 g l(-1)) at 0.25 h(-1). The mean productivity of human-like collagen was the highest at 0.15 h(-1) (0.57 g l(-1)h(-1)) and the lowest at 0.1 h(-1) (0.35 g l(-1 )h(-1)). In the phase before induction, the cell yield coefficient (Y(X/S)) decreased when the specific growth rate increased, while the formation of acetic acid increased upto 2.5 g l(-1) at 0.25 h(-1). The mean product yield coefficient (Y(P/S)) also decreased with specific growth rate increasing. The respiration quotient (RQ) increased slightly with specific growth rate increasing before induction, and the mean value of RQ was around 72%. The optimum growth rate for human-like collagen production was 0.15 approximately 0.2 h(-1).