Recombinant E. Coli High-density Fermentation Process For Production Of Human Collagen Optimization Study

Recombinant Escherichia coli BL21 was used to express human-like collagen in fed-batch culture. The fermentation control strategy such as the nitrogen-feeding modes (fast cycle with 0.5 min intervals, and slow cycle with 4 min intervals), oxygen-enrichment methods (improving fennenter's pressure, supplying oxygen-enriched air, and glucose feedback control) and the variation of inducement strength and specific growth rates before and after induction were studied, furthermore, through metabolic flux analyze and draw kinetics models, the optimization of feeding were obtained.1. The optimum fermentation operation model was in the way of the fast feeding nitrogen substrates cycle at the rate of 3.34⁵.06*10^-3 L/min, to sustain dissolved oxygen around 20% by improving fennenter's pressure properly (0.3-0.8* 10⁵ Pa), keep at 42 ℃ for 2³ h and 39 ℃ for 5⁶ h cultivation for induction, and the final cell density and human-likecollagen concentration could reach 68.94 g/L (DCW) and 13.16 g/L respectively.2. The specific growth rate affected the cell growth and human-like collagen expressionbefore induction, and the optimum specific growth rate before induction was 0.15-0.20 h^-1 in order to yield higher cell and human-like collagen; different specific growth rates after induction also had great influence on the cell growth and human-like collagen expression after induction, the intracellular human-like collagen production would be inhibited at lower or higher specific growth rate, and the optimum specific growth rate after induction was 0.04-0.05 h^-1. At the optimum specific growth rate, the final cell density and human-like collagen concentration could reach 69.5 g/L (DCW) and 13.8 g/L respectively.3. â‘  The specific growth rate before induction influenced not only cell growth at Phase III but also human-like collagen synthesis at Phase IV. At phase III, with specific growth rate increasing, the carbon flux of Pentose Phosphate Pathway (PP), which synthesizing enough precursors and NADPH to satisfy the need of cell growth, increased to it's peak value, then decreased, and flux was the largest at 0.15 h^-1. While at Phase IV, with specific growth rate increasing, the carbon flux of PP decreased gradually, and the carbon flux of Tricarboxylic Acid Cycle (TCA), which providing enough precursors and energy to synthesize human-like collagen, decreased slightly with specific productivity of human-like collagen increasing. Themetabolic flux analysis testified that the optimum specific growth rate was 0.15⁰.20 h"1 before induction. (2) The specific growth rate after induction had influence on the cell growth and human-like collagen synthesis at Phase IV. After induction, with the specific growth rate increasing, the carbon flux of PP increased to apply enough precursors and NADPH to satisfy the need of the cell growth, and the carbon flux of TCA decreased to the lowest at 0.04⁰.05 h"1 resulting from the consumption of the precursors of human-like collagen, and then increased greatly which shown that the energy was wasted more at higher specific growth rate. Metabolic flux analysis testified the optimum specific growth rate after induction was 0.04-0.05 h"1. (3) With fermentation going, the carbon flux of PP decreased greatly and that from the PP pathway feedback to Embden-Meyerhof-Parnas (EMP) pathway also deceased greatly. At the same time, the carbon flux of TCA increased markedly to produce enough energy to sustain cell activity and synthesize human-like collagen.4. The methods of lsqnonlin and Runge-kutta (ode45) in MATLAB were used to get the optimization parameters of the kinetics, and the experiment data agreed well to calculated data by the kinetics.At batch culture, the kinetics were0.7275 M 0.674 + 5'ds 0.7275*dt 0.352(0.674 + 5)'At fed-batch culture (before induction), the kinetics were0.7275 M 0.674 + 5'At the phase of induction, the kinetics were 0.7275M 0.674 + 5'n+ 0.054+ â– 0.321 0.0647r = -2.160 ju + 0.489—-5. On the base of Pontryagin's minimum principle, with the aim function of the highestyield of human-like collagen, feeding rate (Fs) was solved from the kinetics above by the method of fmincon in MATLAB. And at single control phase, Fs didn't have any relation to the cooperation variable (X.) and its formula was same to that in the kinetics. In the fed-batch culture, in order to yield higher human-like collagen, the glucose feeding rate would close to the exponential feeding rate at the optimum specific growth rate.