Manipulating Fermentation Pathways in the Hyperthermophilic Archaeon Pyrococcus furiosus for Ethanol Production up to 95 degrees C Driven by Carbon Monoxide Oxidation

Previously, the highest temperature for biological ethanol production was 85 degrees C. Here, we have engineered ethanol production at 95 degrees C by the hyperthermophilic archaeon Pyrococcus furiosus. Genetic engineering of hyperthermophilic organisms for the production of fuels and other useful chemicals is an emerging biotechnological opportunity. In particular, for volatile organic compounds such as ethanol, fermentation at high temperatures could allow for straightforward separation by direct distillation. Currently, the upper growth temperature limit for native ethanol producers is 72 degrees C in the bacterium Thermoanaerobacter ethanolicus JW200, and the highest temperature for heterologously-engineered bioethanol production was recently demonstrated at 85 degrees C in the archaeon Pyrococcus furiosus. Here, we describe an engineered strain of P. furiosus that synthesizes ethanol at 95 degrees C, utilizing a homologously-expressed native alcohol dehydrogenase, termed AdhF. Ethanol biosynthesis was compared at 75 degrees C and 95 degrees C with various engineered strains. At lower temperatures, the acetaldehyde substrate for AdhF is most likely produced from acetate by aldehyde ferredoxin oxidoreductase (AOR). At higher temperatures, the effect of AOR on ethanol production is negligible, suggesting that acetaldehyde is produced by pyruvate ferredoxin oxidoreductase (POR) via oxidative decarboxylation of pyruvate, a reaction known to occur only at higher temperatures. Heterologous expression of a carbon monoxide dehydrogenase complex in the AdhF overexpression strain enabled it to use CO as a source of energy, leading to increased ethanol production. A genome reconstruction model for P. furiosus was developed to guide metabolic engineering strategies and understand outcomes. This work opens the door to the potential for 'bioreactive distillation' since fermentation can be performed well above the normal boiling point of ethanol.IMPORTANCE Previously, the highest temperature for biological ethanol production was 85 degrees C. Here, we have engineered ethanol production at 95 degrees C by the hyperthermophilic archaeon Pyrococcus furiosus. Using mutant strains, we showed that ethanol production occurs by different pathways at 75 degrees C and 95 degrees C. In addition, by heterologous expression of a carbon monoxide dehydrogenase complex, ethanol production by this organism was driven by the oxidation of carbon monoxide. A genome reconstruction model for P. furiosus was developed to guide metabolic engineering strategies and understand outcomes.