Controlling cell-free metabolism through physiochemical perturbations
Controlling cell-free metabolism through physiochemical perturbations
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DOI：
10.1016/j.ymben.2017.11.005
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发表时间：
2018-01-01
影响因子：
8.4
通讯作者：
Jewett, Michael C.
中科院分区：
文献类型：
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作者：
Karim, Ashty S.;Heggestad, Jacob T.;Jewett, Michael C.
由于细胞代谢的复杂性，在细胞中建立生物合成途径和工程代谢反应可能是耗时的。这些复杂性通常使定义最佳生物合成系统所需的生物合成途径设计的组合测试复杂化。为了简化生物合成系统的优化，我们最近报道了一种用于途径构建和测试的新的无细胞框架。在这个框架中，多个粗细胞提取物选择性地富含单个途径酶，然后将其混合以在一天的时间尺度上构建完整的生物合成途径。这种快速构建途径的方法通过提供独特的设计自由度来修改和控制基础和应用生物技术的生物系统，从而有助于研究代谢途径的性能。这项工作的目标是证明在我们的无细胞框架中，通过扰动生理化学条件，使用正丁醇合成作为模型来探测生物合成途径性能的能力。我们进行了三个独特的案例研究。首先，我们展示了我们的无细胞方法的力量，通过使用机器人液体处理器绘制理化景观来最大限度地提高生物合成产量。这使我们能够确定NAD和CoA是控制无细胞正丁醇代谢的最重要因素。其次，我们比较了从富集裂解物、异源表达和无细胞蛋白质合成构建途径的两种不同方法之间的代谢谱差异。我们发现PEP利用的磷酸盐，沿着其他理化试剂，在无细胞蛋白质合成偶联的粗裂解物代谢系统操作过程中抑制最佳的无细胞正丁醇代谢。第三，我们表明，非磷酸化的二次能源底物可用于燃料无细胞蛋白质合成和正丁醇生物合成。总之，我们的工作突出了使用无细胞系统探索理化扰动的容易性，并建议需要一个更可控的，多步骤的，分离的无细胞框架，用于未来的途径原型和酶的发现工作。
Building biosynthetic pathways and engineering metabolic reactions in cells can be time-consuming due to complexities in cellular metabolism. These complexities often convolute the combinatorial testing of biosynthetic pathway designs needed to define an optimal biosynthetic system. To simplify the optimization of biosynthetic systems, we recently reported a new cell-free framework for pathway construction and testing. In this framework, multiple crude-cell extracts are selectively enriched with individual pathway enzymes, which are then mixed to construct full biosynthetic pathways on the time scale of a day. This rapid approach to building pathways aids in the study of metabolic pathway performance by providing a unique freedom of design to modify and control biological systems for both fundamental and applied biotechnology. The goal of this work was to demonstrate the ability to probe biosynthetic pathway performance in our cell-free framework by perturbing physiochemical conditions, using n-butanol synthesis as a model. We carried out three unique case studies. First, we demonstrated the power of our cell-free approach to maximize biosynthesis yields by mapping physiochemical landscapes using a robotic liquid-handler. This allowed us to determine that NAD and CoA are the most important factors that govern cell-free n-butanol metabolism. Second, we compared metabolic profile differences between two different approaches for building pathways from enriched lysates, heterologous expression and cell-free protein synthesis. We discover that phosphate from PEP utilization, along with other physiochemical reagents, during cell-free protein synthesis-coupled, crude-lysate metabolic system operation inhibits optimal cell-free n-butanol metabolism. Third, we show that non-phosphorylated secondary energy substrates can be used to fuel cell-free protein synthesis and n-butanol biosynthesis. Taken together, our work highlights the ease of using cell-free systems to explore physiochemical perturbations and suggests the need for a more controllable, multi-step, separated cell-free framework for future pathway prototyping and enzyme discovery efforts.