In vitro ketone production continues to be a challenge due to the biochemical features of the enzymes involved-even when some of them have been extensively characterized (e.g. thiolase from Clostridium acetobutylicum), the assembly of synthetic enzyme cascades still face significant limitations (including issues with protein aggregation and multimerization). Here, we designed and assembled a self-sustaining enzyme cascade with acetone yields close to the theoretical maximum using acetate as the only carbon input. The efficiency of this system was further boosted by coupling the enzymatic sequence to a two-step ATP-regeneration system that enables continuous, cost-effective acetone biosynthesis. Furthermore, simple methods were implemented for purifying the enzymes necessary for this synthetic metabolism, including a first-case example on the isolation of a heterotetrameric acetate:coenzyme A transferase by affinity chromatography.
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