A cell-free framework for rapid biosynthetic pathway prototyping and enzyme discovery
A cell-free framework for rapid biosynthetic pathway prototyping and enzyme discovery
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DOI：
10.1016/j.ymben.2016.03.002
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发表时间：
2016-07-01
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.;Jewett, Michael C.
加快设计-构建-测试（DBT）周期是生物化学工程面临的一个根本挑战。为了应对这一挑战，我们报告了一个新的无细胞蛋白质合成驱动的代谢工程（CFPS-ME）框架快速生物合成途径原型。在我们的框架中，用于合成靶小分子的无细胞混合物以混合匹配的方式从粗细胞裂解物组装，所述粗细胞裂解物含有来自异源过表达的选择性富集的途径酶或通过CEPS在裂解物中直接产生途径酶。作为模型，我们将我们的方法应用于正丁醇生物合成，表明大肠杆菌裂解物在体外支持高度活跃的17步CoA依赖性正丁醇途径。无细胞环境中灵活性的提高使我们能够操纵理化条件，进入酶节点，发现新的酶，并利用线性DNA模板建立酶组原型，以研究途径性能。我们预计CFPS-ME将有助于定义，操纵和理解加速DBT循环的代谢途径，而无需重新设计生物体。(C)2016国际代谢工程学会。爱思唯尔公司出版All rights reserved.
Speeding up design-build-test (DBT) cycles is a fundamental challenge facing biochemical engineering. To address this challenge, we report a new cell-free protein synthesis driven metabolic engineering (CFPS-ME) framework for rapid biosynthetic pathway prototyping. In our framework, cell-free cocktails for synthesizing target small molecules are assembled in a mix-and-match fashion from crude cell lysates either containing selectively enriched pathway enzymes from heterologous overexpression or directly producing pathway enzymes in lysates by CEPS. As a model, we apply our approach to n-butanol biosynthesis showing that Escherichia coli lysates support a highly active 17-step CoA-dependent n-butanol pathway in vitro. The elevated degree of flexibility in the cell-free environment allows us to manipulate physiochemical conditions, access enzymatic nodes, discover new enzymes, and prototype enzyme sets with linear DNA templates to study pathway performance. We anticipate that CFPS-ME will facilitate efforts to define, manipulate, and understand metabolic pathways for accelerated DBT cycles without the need to reengineer organisms. (C) 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.