| Summary: | Natural products with significant biological activities continuously act as rich sources for drug discovery and development. To harness the potential of these valuable compounds, robust methods need to be developed for their rapid and sustainable production. Cell-free biosynthesis of pharmaceutical natural products by in vitro reconstruction of the entire biosynthetic pathways represents one such solution. In this review, we focus on in vitro biosynthesis of two important classes of natural products, polyketides (PKs) and nonribosomal peptides (NRPs). First, we summarize purified enzyme-based systems for the biosynthesis of PKs, NRPs, and PK/NRP hybrids. Then, we introduce the cell-free protein synthesis (CFPS)-based technology for natural product production. With that, we discuss challenges and opportunities of cell-free synthetic biology for in vitro biosynthesis of natural products. Keywords: Cell-free synthetic biology, Purified enzymes, Cell-free protein synthesis, Polyketides, Nonribosomal peptides, In vitro biosynthesis, Natural products
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