Recombinant Human Collagen Expression System For Process Optimization

Recombinant Escherichia coli BL21 was used to express human-like collagen in fed-batch cultivation. The fermentation control strategy such as the inducement opportunity and the variation of inducement strength, oxygen-enrichment methods (improving fermentor's pressure, supplying oxygen-enriched air, and glucose feedback control), specific growth rates before and after induction and the nitrogen-feeding modes (grading nitrogen-feeding and continuous nitrogen-feeding)were studied, furthermore, through kinetics models, the optimization of feeding were obtained:1. The optimum inducement time was near the end of logarithmic phase, when cultivation at 42 ℃ for 2-3 h and 39 ℃ for 5-6 h for the formation of target protein, the final biomass and human-like collagen 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 collagenexpression before induction, and the optimal specific growth rate before induction was 0.15-0.20 h-1 in order to yield higher biomass 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's produced 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 biomass and human-like collagen concentration could reach 69.5 g/L (DCW) and 13.8 g/L respectively.3. The optimal fermentation operation model was the grading nitrogen-feeding, to sustain dissolved oxygen around 20%, fermentor's pressure was properly increased(0.3~0.8*10~5 Pa), the final biomass and human-like collagen concentrationcould reach 76.01 g/L (DCW) and 16.75 g/L respectively.4. The methods of lsqnonlin and Runge-kutta (ode45) in MATLAB were used toget the optimization parameters of the kinetics, and the experiment data agreed well to calculated data by the kinetics.At batch cultvation, the kinetics were0.7275fi 0.674 + s ' ds 0.727s*- 0.054*;dt 0.352(0.674At fed-batch cultivation (before induction), the kinetics were 0.72750.674 + 5 dx 0.7275* 1 r 0.7275*r dt 0.674 + 5 sf -s 0.179(0.674 + 5)+ 0.054*1dv v 0.7275*— =-------f------------------+ 0.054*1;dt 5,-5 0.179(0.674 + 5)At the phase of induction, the kinetics were0.7275μ =-----------,0.674 + 50 002dx 0.7275* * . 0.7275* nnCA 1 . 1 _o n__ 0.002,,— =---------------------[--------------------+ 0.054* +-------(-1.238m + 0.511---------)*]dt 0.674 + 5 5^5 0.097(0.674 + 5) 0.064 μ;ih238 + 0.51U[ +0.054, + (1.238^ + 0.511dt 0.674 + 5 sf -s 0.097(0.674 + 5) 0.064 μ_i_ Mm0064dt sf -5 0.097(0.674 + 5) 0.064...