Abstract Microbes that generate copious amounts of hydrogen (H2) via dark fermentation are a promising means to evolve and improve renewable biofuels. Many anaerobic hyperthermophilic archaea, such as the fast-growing, genetically tractable, heterotroph Thermococcus kodakarensis, produce generous quantities of H2 and provide an idealized platform to further optimize naturally high levels of biohydrogen reduction. Precise genetic manipulations and modifications to growth conditions have already resulted in substantial increases to H2 output but additional improvements are desired. An unexamined and potentially valuable route towards increased H2 production is to tether select electron donor and acceptor proteins together to reroute and maximize the flow of electrons towards H2 production. Such strategies have shown promise in Bacteria and Eukarya but have not yet been investigated in thermophilic Archaea. Here, we generate and evaluate twelve novel T. kodakarensis strains wherein a proteinaceous electron carrier (a ferredoxin, Fd) is physically tethered to the membrane-bound-hydrogenase (MBH), the sole H2 producing enzyme, to direct electron flux towards biohydrogen generation. Growth assessments and H2 output measurements demonstrate that strains encoding protein-fusions evolve up to ~ 40% more H2 per cell than the host strain. Eliminating H2 consumption and alternative routes of electron sinks in concert with protein tethering further increased H2 output per cell for a maximum increase of ~ 66% over the host strain. Our results demonstrate that rerouting electron flux via protein tethering coupled with the elimination of reductant sinks is a promising means towards improved biohydrogen production in T. kodakarensis. Key points <UnorderedList Mark="Bullet"> <ItemContent> Protein tethering between redox proteins can reroute electron flux in vivo. </ItemContent> <ItemContent> Enforced protein proximity results in ~ 40% increases in H2 production per cell. </ItemContent> <ItemContent> Protein-tethering provides a generalizable framework to redirect redox metabolism. </ItemContent> </UnorderedList>

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

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2026年3月6日

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Sere A. Williams,
David A. Crosby,
Danielle M. Riley,
Teagan P. Rockwood,
Brett W. Burkhart,
Meghan E. Stettler,
Thomas J. Santangelo

摘要

Abstract

Microbes that generate copious amounts of hydrogen (H₂) via dark fermentation are a promising means to evolve and improve renewable biofuels. Many anaerobic hyperthermophilic archaea, such as the fast-growing, genetically tractable, heterotroph Thermococcus kodakarensis, produce generous quantities of H₂ and provide an idealized platform to further optimize naturally high levels of biohydrogen reduction. Precise genetic manipulations and modifications to growth conditions have already resulted in substantial increases to H₂ output but additional improvements are desired. An unexamined and potentially valuable route towards increased H₂ production is to tether select electron donor and acceptor proteins together to reroute and maximize the flow of electrons towards H₂ production. Such strategies have shown promise in Bacteria and Eukarya but have not yet been investigated in thermophilic Archaea. Here, we generate and evaluate twelve novel T. kodakarensis strains wherein a proteinaceous electron carrier (a ferredoxin, Fd) is physically tethered to the membrane-bound-hydrogenase (MBH), the sole H₂ producing enzyme, to direct electron flux towards biohydrogen generation. Growth assessments and H₂ output measurements demonstrate that strains encoding protein-fusions evolve up to ~ 40% more H₂ per cell than the host strain. Eliminating H₂ consumption and alternative routes of electron sinks in concert with protein tethering further increased H₂ output per cell for a maximum increase of ~ 66% over the host strain. Our results demonstrate that rerouting electron flux via protein tethering coupled with the elimination of reductant sinks is a promising means towards improved biohydrogen production in T. kodakarensis.

Key points

Protein tethering between redox proteins can reroute electron flux in vivo.

Enforced protein proximity results in ~ 40% increases in H₂ production per cell.

Protein-tethering provides a generalizable framework to redirect redox metabolism.