Recent advances in applying cell-free systems for high-value and complex natural product biosynthesis

• Cell-free systems enable total biosynthesis of complex natural products. • Cell-free systems are robust to express long biosynthetic gene clusters (>10 kb). • Cell-free reactions allow for easy optimization and enhanced production. • Cell-free platforms are powerful for rapidly screening and generating new natural products and analogs. Natural products have complex chemical structures and exhibit diverse bioactivities. To harness these high-value natural compounds, robust approaches are required for their rapid and sustainable production. Recently, cell-free systems as one promising solution have been applied to produce natural products by reconstitution of their entire biosynthetic pathways in vitro . Here, we highlight recent advances in cell-free biosynthesis of natural products and discuss future challenges and opportunities. We envision that cell-free technology holds tremendous potential for natural product research such as high-throughput screening of peptide-based natural product analogs/variants, expression of toxic metabolic pathways, characterization of enzyme functions, and activation of silent biosynthetic gene clusters.