Cell‐Free Metabolic Engineering: Production of Chemicals by Minimized Reaction Cascades

Abstract The limited supply of fossil resources demands the development of renewable alternatives to petroleum‐based products. Here, biobased higher alcohols such as isobutanol are versatile platform molecules for the synthesis of chemical commodities and fuels. Currently, their fermentation‐based production is limited by the low tolerance of microbial production systems to the end products and also by the low substrate flux into cell metabolism. We developed an innovative cell‐free approach, utilizing an artificial minimized glycolytic reaction cascade that only requires one single coenzyme. Using this toolbox the cell‐free production of ethanol and isobutanol from glucose was achieved. We also confirmed that these streamlined cascades functioned under conditions at which microbial production would have ceased. Our system can be extended to an array of industrially‐relevant molecules. Application of solvent‐tolerant biocatalysts potentially allows for high product yields, which significantly simplifies downstream product recovery.