Rerouting reductant flux via protein tethering enhances biohydrogen production in Thermococcus kodakarensis

The efficiency of hydrogen (H 2 ) production via dark fermentation is dictated, in large part, by how electrons are distributed among competing reductant usage pathways and ultimately, the share of electrons that get delivered to hydrogenases for H₂ evolution (Thauer et al. 1977 ; Maier 1996 ; Amend and Shock 2001 ; Lewalter and Müller 2006 ; McNeely et al. 2010 ; Martin and Thauer 2017 ). While studies focused on mesophilic derived dark fermentation have produced significant gains (Agapakis et al. 2010 ; Eilenberg et al. 2016 ; Koo et al. 2016 ; Mazzoli et al. 2024 ; Vauthier et al. 2026 ), anaerobic hyperthermophiles offer an attractive alternative due to their thermostable enzymes, high metabolic rates, and capacity to maintain efficient reduction–oxidation (redox) balance under extreme conditions, providing idealized systems for probing and engineering electron flux for biohydrogen generation (Rittmann and Herwig 2012 ; Lipscomb et al. 2014 ; Bae et al. 2015 ; Aslam et al. 2017 ; Simons et al. 2020 ). Energy conservation in anaerobic microbes depends on the orchestration of reductant flux via redox proteins such as ferredoxins (Fds), hydrogenases, and oxidoreductases which maintain redox balance and generate reduced cofactors in the absence of oxygen (Santangelo et al. 2011 ). Hyperthermophilic archaea must also maintain protein integrity while controlling electron transfer at elevated temperatures—near the limits of life—thus their redox-responsive proteins must direct efficient and selective electron transfers at high temperature. In response, electron flux in hyperthermophiles is mediated by small, specialized redox proteins that function near thermodynamic limits (Schäfer et al. 1999 ; Page et al. 1999 ; Mayer and Müller 2014 ; Nguyen et al. 2017 ). The specialized electron transfer pathways of hyperthermophilic archaea are thus an appealing model for understanding fundamental bioenergetics, for developing high-temperature biotechnologies, and for enhancing the production of biohydrogen.