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

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. An order of enzymes, hold the cells: For chemical and fuel production from biomass, chemical methods are often supplemented by biological means, such as microbial systems. It is demonstrated here that microbial reaction cascades can be redesigned and simplified to an extent that cell‐free bulk production of chemicals and fuels from biomass becomes feasible. Specifically, glucose is converted to ethanol and to isobutanol by using only six and eight enzymes respectively and one cofactor. Larger alcohols such as isobutanol are known to poison microbial systems.