Emmerling M, Bailey J E, Sauer U
Institute of Biotechnology, ETH Zürich, CH-8093 Zürich, Switzerland.
Biotechnol Bioeng. 2000 Mar 5;67(5):623-7. doi: 10.1002/(sici)1097-0290(20000305)67:5<623::aid-bit13>3.0.co;2-w.
Glycolytic fluxes in resting Escherichia coli were enhanced by overexpression of heterologous pyruvate kinases (Pyk) from Bacillus stearothermophilus and Zymomonas mobilis, but not homologous Pyk. Compared to the control, an increase of 10% in specific glucose consumption and of 15% in specific ethanol production rates was found in anaerobic resting cells, expressing the heterologous Pyks, that were harvested from exponentially growing aerobic cultures. A further increase in glycolytic flux was achieved by simultaneous overexpression of E. coli phosphofructokinase (Pfk) and Pyk with specific glucose consumption and ethanol production rates of 25% and 35% greater, respectively, than the control. Fluxes to lactate were not significantly affected, contrary to previous observations with resting cells harvested from anaerobically growing cultures. To correlate the physiology of resting cells with the physiology of cells prior to harvest, we determined the relevant growth parameters from aerobic and anaerobic precultures. We conclude that glycolytic fluxes in E. coli with submaximal (aerobic) metabolic activity can be increased by overexpression of pyruvate kinases which do not require allosteric activation or co-overexpression with Pfk. However, such improvements require more extensive engineering in E. coli with near maximal (anaerobic) metabolic activity.
通过过量表达嗜热脂肪芽孢杆菌和运动发酵单胞菌的异源丙酮酸激酶（Pyk），而非同源Pyk，可增强静止状态大肠杆菌中的糖酵解通量。与对照相比，从指数生长的需氧培养物中收获的表达异源Pyk的厌氧静止细胞，其比葡萄糖消耗增加了10%，比乙醇产生速率增加了15%。通过同时过量表达大肠杆菌磷酸果糖激酶（Pfk）和Pyk，糖酵解通量进一步增加，其比葡萄糖消耗和乙醇产生速率分别比对照高25%和35%。与之前从厌氧生长培养物中收获的静止细胞的观察结果相反，流向乳酸的通量没有受到显著影响。为了将静止细胞的生理学与收获前细胞的生理学联系起来，我们从需氧和厌氧预培养物中确定了相关的生长参数。我们得出结论，通过过量表达不需要变构激活或与Pfk共过量表达的丙酮酸激酶，可以增加具有次最大（需氧）代谢活性的大肠杆菌中的糖酵解通量。然而，对于具有接近最大（厌氧）代谢活性的大肠杆菌，这种改进需要更广泛的工程改造。
