Department of Biological and Chemical Engineering, Aarhus University, Aarhus, Denmark.
Biochemical Engineering (AVT.BioVT), RWTH Aachen University, Aachen, Germany.
Biotechnol Bioeng. 2024 Jun;121(6):1789-1802. doi: 10.1002/bit.28697. Epub 2024 Mar 12.
Decoupling cell formation from recombinant protein synthesis is a potent strategy to intensify bioprocesses. Escherichia coli strains with mutations in the glucose uptake components lack catabolite repression, display low growth rate, no overflow metabolism, and high recombinant protein yields. Fast growth rates were promoted by the simultaneous consumption of glucose and glycerol, and this was followed by a phase of slow growth, when only glucose remained in the medium. A glycerol-repressible genetic circuit was designed to autonomously induce recombinant protein expression. The engineered strain bearing the genetic circuit was cultured in 3.9 g L glycerol + 18 g L glucose in microbioreactors with online oxygen transfer rate monitoring. The growth was fast during the simultaneous consumption of both carbon sources (C-sources), while expression of the recombinant protein was low. When glycerol was depleted, the growth rate decreased, and the specific fluorescence reached values 17% higher than those obtained with a strong constitutive promoter. Despite the relatively high amount of C-source used, no oxygen limitation was observed. The proposed approach eliminates the need for the substrate feeding or inducers addition and is set as a simple batch culture while mimicking fed-batch performance.
从重组蛋白合成中解耦细胞形成是强化生物过程的一种有效策略。在葡萄糖摄取成分中发生突变的大肠杆菌菌株缺乏分解代谢物阻遏，表现出低生长速率、无溢出代谢和高重组蛋白产量。通过同时消耗葡萄糖和甘油来促进快速生长速率，然后进入仅在培养基中存在葡萄糖的缓慢生长阶段。设计了一个甘油可诱导的遗传回路来自动诱导重组蛋白表达。带有遗传回路的工程菌株在微型生物反应器中以在线氧传递速率监测的方式，在 3.9 g/L 甘油+18 g/L 葡萄糖中进行培养。在同时消耗两种碳源（C 源）时，生长速度很快，而重组蛋白的表达水平较低。当甘油耗尽时，生长速率下降，比使用强组成型启动子获得的特定荧光值高 17%。尽管使用了相对较高量的 C 源，但没有观察到氧气限制。所提出的方法消除了对底物进料或诱导剂添加的需求，并设定为简单的分批培养，同时模拟了补料分批的性能。
