Faulkner Eilis, Barrett Mark, Okor Sola, Kieran Patricia, Casey Eoin, Paradisi Francesca, Engel Paul, Glennon Brian
School of Chemical and Bioprocess Engineering, Centre for Synthesis and Chemical Biology, University College Dublin (UCD), Dublin 4, Ireland.
Biotechnol Prog. 2006 May-Jun;22(3):889-97. doi: 10.1021/bp050327+.
A fed-batch process for the high cell density cultivation of E. coli TG1 and the production of the recombinant protein phenylalanine dehydrogenase (PheDH) was developed. A model based on Monod kinetics with overflow metabolism and incorporating acetate utilization kinetics was used to generate simulations that describe cell growth, acetate production and reconsumption, and glucose consumption during fed-batch cultivation. Using these simulations a predetermined feeding profile was elaborated that would maintain carbon-limited growth at a growth rate below the critical growth rate for acetate formation (mu < mu(crit)). Two starvation periods are incorporated into the feed profile in order to induce acetate utilization. Cell concentrations of 53 g dry cell weight (DCW)/L were obtained with a final intracellular product concentration of recombinant protein corresponding to approximately 38% of the total cell protein. The yield of PheDH was 129 U/mL with a specific activity of 1.2 U/mg DCW and a maximum product formation rate of 0.41 U/mg DCW x h. The concentration of aectate was maintained below growth inhibitory levels until 3 h before the end of the fermentation when the concentration reached a maximum of 10.7 g/L due to IPTG induction of the recombinant protein.
开发了一种用于大肠杆菌TG1高细胞密度培养和重组蛋白苯丙氨酸脱氢酶（PheDH）生产的补料分批培养工艺。使用基于莫诺德动力学并结合溢流代谢以及乙酸利用动力学的模型进行模拟，以描述补料分批培养过程中的细胞生长、乙酸产生和再消耗以及葡萄糖消耗。利用这些模拟结果制定了预定的补料曲线，该曲线将在低于乙酸形成临界生长速率（μ < μ(crit)）的生长速率下维持碳限制生长。在补料曲线中加入两个饥饿期以诱导乙酸利用。获得了53 g干细胞重量（DCW）/L的细胞浓度，重组蛋白的最终细胞内产物浓度约占总细胞蛋白的38%。PheDH的产量为129 U/mL，比活性为1.2 U/mg DCW，最大产物形成速率为0.41 U/mg DCW·h。在发酵结束前3 h之前，乙酸浓度一直维持在生长抑制水平以下，此时由于IPTG诱导重组蛋白，乙酸浓度达到最大值10.7 g/L。
