Weber Jan, Hoffmann Frank, Rinas Ursula
Biochemical Engineering Division, GBF German Research Center for Biotechnology, Mascheroder Weg 1, 38124 Braunschweig, Germany.
Biotechnol Bioeng. 2002 Nov 5;80(3):320-30. doi: 10.1002/bit.10380.
The impact of temperature-induced synthesis of human basic fibroblast growth factor (hFGF-2) in high-cell-density cultures of recombinant Escherichia coli was studied by estimating metabolic flux variations. Metabolic flux distributions in E. coli were calculated by means of a stoichiometric network and linear programming. After the temperature upshift, a substantially elevated energy demand for synthesis of hFGF-2 and heat shock proteins resulted in a redirection of metabolic fluxes. Catabolic pathways like the Embden-Meyerhof-Parnas pathway and the tricarboxylic acid (TCA) cycle showed significantly enhanced activities, leading to reduced flux to growth-associated pathways like the pentose phosphate pathway and other anabolic pathways. Upon temperature upshift, an excess of NADPH was produced in the TCA cycle by isocitrate dehydrogenase. The metabolic model predicted the involvement of a transhydrogenase generating additional NADH from NADPH, thereby increasing ATP regeneration in the respiratory chain. The influence of the temperature upshift on the host's metabolism was investigated by means of a control strain harboring the "empty" parental expression vector. The metabolic fluxes after the temperature upshift were redirected similarly to the production strain; the effects, however, were observed to a lesser extent and with different time profiles.
通过估算代谢通量变化，研究了温度诱导人碱性成纤维细胞生长因子（hFGF - 2）在重组大肠杆菌高细胞密度培养物中的合成所产生的影响。利用化学计量网络和线性规划计算大肠杆菌中的代谢通量分布。温度上调后，hFGF - 2和热休克蛋白合成所需的能量需求大幅增加，导致代谢通量重新分配。糖酵解途径和三羧酸（TCA）循环等分解代谢途径的活性显著增强，导致流向与生长相关途径（如磷酸戊糖途径和其他合成代谢途径）的通量减少。温度上调时，异柠檬酸脱氢酶在TCA循环中产生过量的NADPH。代谢模型预测，一种转氢酶可利用NADPH生成额外的NADH，从而增加呼吸链中的ATP再生。通过携带“空”亲本表达载体的对照菌株，研究了温度上调对宿主代谢的影响。温度上调后的代谢通量与生产菌株类似地重新分配；然而，观察到的影响程度较小且时间分布不同。
