The field of metabolic engineering has yielded remarkable accomplishments in using cells to produce valuable molecules, and cell-free expression (CFE) systems have the potential to push the field even further. However, CFE systems still face some outstanding challenges, including endogenous metabolic activity that is poorly understood yet has a significant impact on CFE productivity. Here, we use metabolomics to characterize the temporal metabolic changes in CFE systems and their constituent components, including significant metabolic activity in central carbon and amino acid metabolism. We find that while changing the reaction starting state via lysate preincubation impacts protein production, it has a comparatively small impact on metabolic state. We also demonstrate that changes to lysate preparation have a larger effect on protein yield and temporal metabolic profiles, though general metabolic trends are conserved. Finally, while we improve protein production through targeted supplementation of metabolic enzymes, we show that the endogenous metabolic activity is fairly resilient to these enzymatic perturbations. Overall, this work highlights the robust nature of CFE reaction metabolism as well as the importance of understanding the complex interdependence of metabolites and proteins in CFE systems to guide optimization efforts.

中文翻译：

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基于大肠杆菌的无细胞系统中的代谢动力学

代谢工程领域在利用细胞生产有价值的分子方面取得了显著成就，而无细胞表达 (CFE) 系统有可能进一步推动该领域的发展。然而，CFE 系统仍然面临一些突出的挑战，包括对 CFE 生产率有重大影响但了解甚少的内源性代谢活动。在这里，我们使用代谢组学来表征 CFE 系统及其组成成分的时间代谢变化，包括中心碳和氨基酸代谢中的显着代谢活动。我们发现，在通过以下方式改变反应起始状态时裂解物预孵育影响蛋白质的产生，它对代谢状态的影响相对较小。我们还证明，裂解物制备的变化对蛋白质产量和时间代谢曲线有更大的影响，尽管一般代谢趋势是保守的。最后，虽然我们通过有针对性地补充代谢酶来提高蛋白质产量，但我们表明内源性代谢活动对这些酶扰动具有相当的弹性。总的来说，这项工作强调了 CFE 反应代谢的稳健性，以及理解 CFE 系统中代谢物和蛋白质复杂的相互依赖性以指导优化工作的重要性。