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.

The sustainable capture and conversion of carbon dioxide (CO2) is key to achieving a circular carbon economy. Bioelectrocatalysis, which aims at using renewable energies to power the highly specific, direct transformation of CO2 into value added products, holds promise to achieve this goal. However, the functional integration of CO2‐fixing enzymes onto electrode materials for the electrosynthesis of stereochemically complex molecules remains to be demonstrated. Here, we show the electricity‐driven regio‐ and stereoselective incorporation of CO2 into crotonyl‐CoA by an NADPH‐dependent enzymatic reductive carboxylation. Co‐immobilization of a ferredoxin NADP+ reductase and crotonyl‐CoA carboxylase/reductase within a 2,2′‐viologen‐modified hydrogel enabled iterative NADPH recycling and stereoselective formation of (2S)‐ethylmalonyl‐CoA, a prospective intermediate towards multi‐carbon products from CO2, with 92±6 % faradaic efficiency and at a rate of 1.6±0.4 μmol cm−2 h−1. This approach paves the way for realizing even more complex bioelectrocatalyic cascades in the future.

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