Metabolic footprint analysis of recombinant Escherichia coli strains during fed-batch fermentations
Metabolic footprint analysis of recombinant Escherichia coli strains during fed-batch fermentations
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DOI：
10.1039/c0mb00143k
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发表时间：
2011-01-01
影响因子：
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通讯作者：
Rocha, Isabel
中科院分区：
文献类型：
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作者：
Carneiro, Sonia;Villas-Boas, Silas G.;Rocha, Isabel
代谢足迹已经成为表征表型和区分由环境和/或遗传改变引起的特定代谢状态的一种有价值的分析方法。大肠杆菌细胞中外源蛋白生产的代谢影响特别令人感兴趣，因为在这一过程中触发了许多细胞应激，限制了总体生产力，例如严格的反应。由于对重组生物过程中代谢反应的了解仍然很少，代谢足迹可以提供内在代谢调节的相关信息。因此，我们测量并解释了大肠杆菌W3110和Delta relA突变菌株在不同实验条件下重组分批补料发酵过程中产生的代谢足迹。iptg诱导的异源蛋白表达导致glyoxylate shunt抑制剂在培养液中迅速积累，这表明清除了这一复变途径，以补充为异源蛋白的额外形成而退出的TCA中间体。营养变化在重组细胞代谢中也很关键，这表明细胞采用不同的策略来抵消细胞代谢中的不平衡，包括某些代谢物的分泌，这些代谢物很可能被用作生存过程的代谢缓解。
Metabolic footprinting has become a valuable analytical approach for the characterization of phenotypes and the distinction of specific metabolic states resulting from environmental and/or genetic alterations. The metabolic impact of heterologous protein production in Escherichia coli cells is of particular interest, since there are numerous cellular stresses triggered during this process that limit the overall productivity, e. g. the stringent response. Because the knowledge on the metabolic responses in recombinant bioprocesses is still scarce, metabolic footprinting can provide relevant information on the intrinsic metabolic adjustments. Thus, the metabolic footprints generated by E. coli W3110 and the Delta relA mutant strain during recombinant fed-batch fermentations at different experimental conditions were measured and interpreted. The IPTG-induction of the heterologous protein expression resulted in the rapid accumulation of inhibitors of the glyoxylate shunt in the culture broth, suggesting the clearance of this anaplerotic route to replenish the TCA intermediaries withdrawn for the additional formation of the heterologous protein. Nutritional shifts were also critical in the recombinant cellular metabolism, indicating that cells employ diverse strategies to counteract imbalances in the cellular metabolism, including the secretion of certain metabolites that are, most likely, used as a metabolic relief to survival processes.