The hyperthermophilic archaeon Thermococcus kodakarensis can grow on pyruvate or maltooligosaccharides through H-2 fermentation. H-2 production levels of members of the Thermococcales are high, and studies to improve their production potential have been reported. Although H-2 production is primary metabolism, here we aimed to partially uncouple cell growth and H-2 production of T. kodakarensis. Additional A(1)-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 A(1)-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 H-2 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 H-2 production, significantly larger decreases in cell growth were observed, resulting in an increase in cell-specific H-2 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 H-2 production in T. kodakarensis. (C) 2020, The Society for Biotechnology, Japan. All rights reserved.