PT  - JOURNAL ARTICLE
AU  - Lemaire, Olivier N.
AU  - Belhamri, Mélissa
AU  - Shevchenko, Anna
AU  - Wagner, Tristan
TI  - Carbon monoxide-driven bioethanol production operates via a tungsten-dependent catalyst
AID  - 10.1101/2024.07.29.605569
DP  - 2024 Jan 01
TA  - bioRxiv
PG  - 2024.07.29.605569
4099  - http://biorxiv.org/content/early/2024/09/04/2024.07.29.605569.short
4100  - http://biorxiv.org/content/early/2024/09/04/2024.07.29.605569.full
AB  - 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.Competing Interest StatementThe authors have declared no competing interest.