The Effect of Heating Rate on Escherichia coli Metabolism, Physiological Stress, Transcriptional Response, and Production of Temperature-induced Recombinant Protein: A Scale-Down Study
The Effect of Heating Rate on Escherichia coli Metabolism, Physiological Stress, Transcriptional Response, and Production of Temperature-induced Recombinant Protein: A Scale-Down Study
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DOI：
10.1002/bit.22084
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发表时间：
2009-02-01
影响因子：
3.8
通讯作者：
Ramirez, Octavio T.
中科院分区：
文献类型：
--
作者：
Caspeta, Luis;Flores, Noemi;Ramirez, Octavio T.
在实验室规模下，当在热诱导系统中表达异源蛋白时，可以容易地实现从30 ° C到42 ° C的突然步骤增加。然而，对于大规模培养物，由于热传递限制，仅缓慢的斜坡式温度升高是可能的，其中加热速率随着规模增加而降低。在这项工作中，转录和代谢的重组大肠杆菌菌株的温度诱导的合成前胰岛素原在高细胞密度培养物的反应进行了研究，在不同的加热速率。分别以6、1.7、0.8和0.4摄氏度/分钟的加热速率在模拟0.1、5、20和100米（3）发酵罐的缩小方法中进行了测试。最慢的加热速率获得最高的重组蛋白产量和浓度。随着加热速率的增加，产率和最大重组蛋白浓度降低，而更大部分的碳骨架以乙酸盐、乳酸盐和甲酸盐的形式损失。与30 ℃相比，当以6、1.7和0.8 ℃/min诱导培养物时，通过qRT-PCR定量的38和42 ℃下选定热休克基因的mR-NA水平增加2倍至超过42倍，但在0.4 ℃/min时没有观察到增加。对于以1.7和6 ℃/min诱导的培养物，在42 ℃下观察到胁迫基因spoT和relA的表达降低（1.5- 4倍），表明细胞在缓慢升温下能够适应应激。在6、1.7和0.8 ℃/min时，转录-翻译机制（tufB、rpoA和tig）基因的mRNA水平下降40%至80%，而在42 ℃时，0.4 ℃/min时出现短暂增加。编码前胰岛素原的基因的mRNA水平显示出与热休克基因转录物相似的特征，反映了可能的类似诱导机制。总之，所获得的结果表明，缓慢的加热速率，如那些可能发生在传统的大规模发酵罐，有利于异源蛋白质的合成，在本报告中使用的热诱导表达系统。了解加热速率对细菌生理学和产物形成的影响有助于合理设计规模缩小和规模LIP策略以及优化重组蛋白诱导方案。
At the laboratory scale, sudden step increases from 30 to 42 degrees C can be readily accomplished when expressing heterologous proteins in heat-inducible systems. However, for large scale-cultures only slow ramp-type increases in temperature are possible due to heat transfer limitations, where the heating rate decreases as the scale increases. In this work, the transcriptional and metabolic responses of a recombinant Escherichia coli strain to temperature-induced synthesis of pre-proinsulin in high cell density cultures were examined at different heating rates. Heating rates of 6, 1.7, 0.8, and 0.4 degrees C/min were tested in a scale-down approach to mimic fermentors of 0.1, 5, 20, and 100 m(3), respectively. The highest yield and concentration of recombinant protein was obtained for the slowest heating rate. As the heating rate increased, the yield and maximum recombinant protein concentration decreased, whereas a larger fraction of carbon skeletons was lost as acetate, lactate, and formate. Compared to 30 degrees C, the mR-NA levels of selected heat-shock genes at 38 and 42 degrees C, as quantified by qRT-PCR, increased between 2- to over 42-fold when cultures were induced at 6, 1.7, and 0.8 degrees C/min, but no increase was observed at 0.4 degrees C/min. Only small increases (between 1.5- and 4-fold) in the expression of the stress genes spoT and relA were observed at 42 degrees C for cultures induced at 1.7 and 6 degrees C/min, Suggesting that cells subjected to slow temperature 09 increases can adapt to stress. mRNA levels of genes from the transcription-translation machinery (tufB, rpoA, and tig) decreased between 40% and 80% at 6, 1.7 and 0.8 degrees C/min, whereas a transient increase occurred for 0.4 degrees C/min at 42 degrees C. mRNA levels of the gene coding for pre-proinsulin showed a similar profile to transcripts of heat-shock genes, reflecting a probable analogous induction mechanism. Altogether, the results obtained indicate that slow heating rates, such as those likely to occur in conventional large-scale fermentors, favored heterologous protein synthesis by the thermo-inducible expression system used in this report. Knowledge of the effect of heating rate on bacterial physiology and product formation is useful for the rational design of scale-down and scale-LIP strategies and optimum recombinant protein induction schemes.