title: Overproduction of the membrane-bound [NiFe]-hydrogenase in Thermococcus kodakarensis and its effect on hydrogen production.
abstract: The hyperthermophilic archaeon Thermococcus kodakarensis can utilize sugars or pyruvate for growth. In the absence of elemental sulfur, the electrons via oxidation of these substrates are accepted by protons, generating molecular hydrogen (H2). The hydrogenase responsible for this reaction is a membrane-bound [NiFe]-hydrogenase (Mbh). In this study, we have examined several possibilities to increase the protein levels of Mbh in T. kodakarensis by genetic engineering. Highest levels of intracellular Mbh levels were achieved when the promoter of the entire mbh operon (TK2080-TK2093) was exchanged to a strong constitutive promoter from the glutamate dehydrogenase gene (TK1431) (strain MHG1). When MHG1 was cultivated under continuous culture conditions using pyruvate-based medium, a nearly 25% higher specific hydrogen production rate (SHPR) of 35.3 mmol H2 g-dcw(-1) h(-1) was observed at a dilution rate of 0.31 h(-1). We also combined mbh overexpression using an even stronger constitutive promoter from the cell surface glycoprotein gene (TK0895) with disruption of the genes encoding the cytosolic hydrogenase (Hyh) and an alanine aminotransferase (AlaAT), both of which are involved in hydrogen consumption (strain MAH1). At a dilution rate of 0.30 h(-1), the SHPR was 36.2 mmol H2 g-dcw(-1) h(-1), corresponding to a 28% increase compared to that of the host T. kodakarensis strain. Increasing the dilution rate to 0.83 h(-1) or 1.07 h(-1) resulted in a SHPR of 120 mmol H2 g-dcw(-1) h(-1), which is one of the highest production rates observed in microbial fermentation.
authors.affiliation: Laboratory of Biochemical Engineering, Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University Kyoto, Japan ; Japan Science and Technology Agency, Core Research of Evolutional Science and Technology Tokyo, Japan.
authors.forename: Tamotsu
authors.initials: T
authors.lastname: Kanai
authors.affiliation: Laboratory of Biochemical Engineering, Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University Kyoto, Japan ; Japan Science and Technology Agency, Core Research of Evolutional Science and Technology Tokyo, Japan.
authors.forename: Jan-Robert
authors.initials: JR
authors.lastname: Simons
authors.affiliation: Laboratory of Biochemical Engineering, Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University Kyoto, Japan.
authors.forename: Ryohei
authors.initials: R
authors.lastname: Tsukamoto
authors.affiliation: Taiyo Nippon Sanso Corporation Tokyo, Japan.
authors.forename: Akihito
authors.initials: A
authors.lastname: Nakajima
authors.affiliation: Taiyo Nippon Sanso Corporation Tokyo, Japan.
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Omori
authors.affiliation: Laboratory of Biochemical Engineering, Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University Kyoto, Japan.
authors.forename: Ryoji
authors.initials: R
authors.lastname: Matsuoka
authors.affiliation: Laboratory of Biochemical Engineering, Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University Kyoto, Japan.
authors.forename: Haruki
authors.initials: H
authors.lastname: Beppu
authors.affiliation: Japan Science and Technology Agency, Core Research of Evolutional Science and Technology Tokyo, Japan ; Research Organization of Science and Technology, Ritsumeikan University Kusatsu, Japan.
authors.forename: Tadayuki
authors.initials: T
authors.lastname: Imanaka
authors.affiliation: Laboratory of Biochemical Engineering, Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University Kyoto, Japan ; Japan Science and Technology Agency, Core Research of Evolutional Science and Technology Tokyo, Japan.
authors.forename: Haruyuki
authors.initials: H
authors.lastname: Atomi
keywords: Thermococcus; archaea; dark fermentation; genetic engineering; hydrogen; hydrogenase; hyperthermophile