The emerging impact of cell-free chemical biosynthesis

[Display omitted] •Cell-free chemical biosynthesis (CFCB) adds flexibility and control to manufacturing.•CFCB enables modular assembly, flexible conditions, and direct access to reactions.•Key challenges are cost-effective cascade synthesis, stabilization, and operation.•Cascade and enzyme engineering, immobilization, and encapsulation can enhance CFCB. Biomanufacturing has emerged as a promising alternative to chemocatalysis for green, renewable, complex synthesis of biofuels, medicines, and fine chemicals. Cell-free chemical biosynthesis offers additional advantages over in vivo production, enabling plug-and-play assembly of separately produced enzymes into an optimal cascade, versatile reaction conditions, and direct access to the reaction environment. In order for these advantages to be realized on the larger scale of industry, strategies are needed to reduce costs of biocatalyst generation, improve biocatalyst stability, and enable economically sustainable continuous cascade operation. Here we overview the advantages and remaining challenges of applying cell-free chemical biosynthesis for commodity production, and discuss recent advances in cascade engineering, enzyme immobilization, and enzyme encapsulation which constitute important steps towards addressing these challenges.