Effects of high-level expression of A₁-ATPase on H₂ production in Thermococcus kodakarensis

The hyperthermophilic archaeon Thermococcus kodakarensis can grow on pyruvate or maltooligosaccharides through H₂ fermentation. H₂ production levels of members of the Thermococcales are high, and studies to improve their production potential have been reported. Although H₂ production is primary metabolism, here we aimed to partially uncouple cell growth and H₂ production of T. kodakarensis. Additional A₁-type ATPase genes were introduced into T. kodakarensis KU216 under the control of two promoters; the strong constitutive cell surface glycoprotein promoter, P_csg, and the sugar-inducible fructose-1,6-bisphosphate aldolase promoter, P_fba. Whereas cells with the A₁-type ATPase genes under the control of P_csg displayed only trace levels of growth, cells with P_fba (strain KUA-PF) displayed growth sufficient for further analysis. Increased levels of A₁-type ATPase protein were detected in KUA-PF cells grown on pyruvate or maltodextrin, when compared to the levels in the host strain KU216. The growth and H₂ production levels of strain KUA-PF with pyruvate or maltodextrin as a carbon and electron source were analyzed and compared to those of the host strain KU216. Compared to a small decrease in total H₂ production, significantly larger decreases in cell growth were observed, resulting in an increase in cell-specific H₂ production. Quantification of the substrate also revealed that ATPase overexpression led to increased cell-specific pyruvate and maltodextrin consumptions. The results clearly indicate that ATPase production results in partial uncoupling of cell growth and H₂ production in T. kodakarensis.