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Abstract:
Microbial alcohol production from waste gasses is a game changer for sustainable carbon cycling and remediation. While the biotechnological process employing Clostridium autoethanogenum to transform syngas (H2/CO2/CO) is blooming, the reactions involved in ethanol biosynthesis remain to be demonstrated. Here, we experimentally showed that ethanol production initiates via a tungsten-dependent aldehyde:ferredoxin oxidoreductase (AFOR), which reduces acetate to acetaldehyde. Such an unfavourable reaction has often been considered unsuitable for a biological process. To answer this riddle, we demonstrated that the thermodynamic pull of CO-oxidation and ethanol synthesis is crucial for triggering acetate reduction. The experimental setup performed with native CO-dehydrogenase and AFOR highlighted the key role of ferredoxin in stimulating the activity of both metalloenzymes and electron shuttling. The crystal structure of holo AFOR refined to 1.59-Å resolution, together with its biochemical characterisation, provides new insights into the reaction mechanism and the specificities of this enzyme fundamental to sustainable biofuel production.
