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

TLDR

The limited fossil resource supply drives the need for renewable alternatives, and biobased higher alcohols such as isobutanol are promising platform molecules, yet fermentation‑based production is constrained by low microbial tolerance and substrate flux. The study aims to develop a cell‑free system using a minimized glycolytic cascade that requires only one coenzyme. The authors constructed a cell‑free minimized glycolytic cascade that uses a single coenzyme and can be expanded to produce various industrial molecules. The cell‑free system produced ethanol and isobutanol from glucose, operated under conditions lethal to microbes, and with solvent‑tolerant biocatalysts can achieve high yields and simplify downstream recovery.

Abstract

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.