The modification of intracellular metabolic pathways by metabolic engineering has generated many engineered strains with relatively high yields of various target products in the past few decades. However, the unpredictable accumulation of toxic products, the cell membrane barrier, and competition between the carbon flux of cell growth and product synthesis have severely retarded progress toward the industrial-scale production of many essential chemicals. On the basis of an in-depth understanding of intracellular metabolic pathways, scientists intend to explore more sustainable methods and construct a cell-free biosynthesis system in vitro. In this review, the synthesis and application of pyruvate as a platform compound is used as an example to introduce cell-free biosynthesis systems. We systematically summarize a proposed methodology workflow of cell-free biosynthesis systems, including pathway design, enzyme mining, enzyme modification, multienzyme assembly, and pathway optimization. Some new methods, such as machine learning, are also mentioned in this review.

中文翻译：

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无细胞生物合成系统：丙酮酸生物合成和转化体外高效平台的方法论和展望

在过去的几十年中，通过代谢工程对细胞内代谢途径的修饰已经产生了许多具有相对较高的各种目标产物产量的工程菌株。然而，不可预测的有毒产物积累、细胞膜屏障以及细胞生长的碳通量与产物合成之间的竞争严重阻碍了许多必需化学品的工业规模生产。在深入了解细胞内代谢途径的基础上，科学家们打算探索更可持续的方法，并在体外构建无细胞生物合成系统。本文以丙酮酸作为平台化合物的合成和应用为例，介绍无细胞生物合成系统。我们系统地总结了提出的无细胞生物合成系统的方法学工作流程，包括途径设计、酶挖掘、酶修饰、多酶组装和途径优化。这篇评论中还提到了一些新方法，例如机器学习。