| The project was primarily focused on recombinant Escherichia coli BL 21 strain in high cell density cultivation expressing recombinant human-like collagen (RHLC). Several factors that influenced the cell growth and RHLC production were investigated, including:fermentor pressure shifting strategy, kinetics models, induction conditions (induction temperature, pH, and C/N), metabolic flux analysis, and plasmid stability, further more, the glucose starvation induced cross-protection was studied as a pre-induction operation.a) The results demonstrated that increasing the fermentor pressure was an effective way to avoid the oxygen transfer capacity limitation, and the increase in dissolved CO2 content did not affect the growth of the recombinant E. coli BL21 strain. At the end of the fermentation process in a 12.8 L fermentor, the cell density (represented by dry cell weight, DCW) reached 77.3 g·L-1, and the concentration of RHLC reached 14.1 g·L-1. In addition, the oxygen transfer capacity(KlaC*) value decreased drastically at about 5 hours after induction. This was probably because of the increased concentration of extracellular DNA owing to cell lysis, indicating that the cells had to be harvested. The kinetics models of cell growth were obtained:(a) Batch phase,(b) Fed-batch phase,(c) Induction phase, b) Production of RHLC by thermo-induction was investigated in a 30 L bioreactor. The effects of induction temperature (T), pH, and carbon to nitrogen molar ratio of the nutrient medium (C/N) were examined. The optimal thermo-induction protocol for RHLC production were determined by using a model coupling genetic algorithm (GA) and artificial neural networks (ANN). The optimal operating conditions were as follows: maintenance of induction temperature at 42℃for 3 h and then at 39.4℃till the end, induction pH at 7.03, and C/N at 4.8 (mol/mol). The theoretical maximum concentration of RHLC was 12.5 g/L, while the experimental value was 12.1 g/L under the optimal induction conditions.c) Metabolic flux analysis was studied supposing the mid metabolic reaction at stable state.The results demonstrated that:(a) At fed-batch stage, the carbon metabolic flux has some differences between glucose feedback and fermentor shifting strategy. The carbon flux of Pentose Phosphate Pathway (PP) was more in cultivation controlled by glucose feedback than fermentor shifting strategy. While the carbon flux of Tricarboxylic Acid Cycle (TCA) was more in cultivation controlled by fermentor shifting strategy.(b) At induction stage, the carbon flux of PP pathway was the lowest one in cultivation induced at 42℃till the end, which had the strongest induction level preferring synthesis of product precursors. While the most one was the cultivation induced at 42℃for 3 h then reduced to 37℃, which had the weakest induction level preferring synthesis of cell growth precursors. The cultivation induced at 42℃for 3 h then reduced to 39℃had a proper induction level.(c) The carbon metabolic flux had some differences among three different induction pH conditions. The carbon flux of PP pathway was the most one at pH 6.8, and the cell growth was better at pH 6.8 condition. The carbon flux of TCA cycle was higher at pH 7.0 and 7.2, indicating that alkalescence environment might more suitable for protein product synthesis.(d) The carbon metabolic flux had some differences among three different induction C/N conditions. The carbon flux of TCA cycle was lowest at C/N=6, whose acetic acid accumulation was highest and bad for synthesis of foreign protein. When C/N=5, the carbon flux of PP pathway was higher than that at C/N=4, and the specific product synthesis rate was highest, and increasing nitrogen source ratio properly would be beneficial for foreign protein production.d) Affect of glucose starvation and yeast extract powder starvation on cell growth were compared in high cell density cultivation. The results demonstrated that glucose starvation was more suitable as pre-induction operation. But the starvation time could not be too long, otherwise would influence the foreign protein production and lead to cell lysis. Glucose starvation for 0.5 h was proper for cell growth and production.e) Plasmid stability of recombinant E. coli was studied by different cultivation control strategies in a 30 L fermentor. The result showed that the higher the specific growth rate was, the lower the plasmid stability was. The cultivation temperature had the same regulation as the specific growth rate. The plasmid stability in glucose feedback strategy was higher than that in fermentor shifting strategy. The plasmid loss was significant at induction stage, and the plasmid stability could be enhanced by reducing induction temperature.
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