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.

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