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

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.