Overproduction of the membrane-bound [NiFe]-hydrogenase in Thermococcus kodakarensis and its effect on hydrogen production.

Tamotsu Kanai,Yoshiyuki Omori,Ryohei Tsukamoto,Jan-Robert Simons,Haruyuki Atomi,Haruki Beppu,Ryoji Matsuoka,Tadayuki Imanaka,Akihito Nakajima

doi:10.3389/fmicb.2015.00847

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.

Highlights

In view of the high demand for renewable energy resources, biological hydrogen (H2) produced by photosynthetic and anaerobic fermentative microorganisms is a promising biofuel that has attracted research activities during the last decades (Hallenbeck, 2009; Oh et al, 2011; Rittmann et al, 2015)
Construction of T. kodakarensis Strains that Overexpress the membrane-bound [NiFe]-hydrogenase (Mbh) Genes In T. kodakarensis, the membrane-bound hydrogenase, Mbh, is the key enzyme that is responsible for the evolution of H2 (Kanai et al, 2011)
The mbh operon can be divided into two regions; the former region containing genes presumed to encode Na+/H+ antiporter subunits (Na/H region; mbh structural genes (mbhA)-I; TK2080-TK2088), FIGURE 4 | Expression levels of the [NiFe]-hydrogenase large subunit (MbhL) in the constructed T. kodakarensis strains

Summary

Introduction

In view of the high demand for renewable energy resources, biological hydrogen (H2) produced by photosynthetic and anaerobic fermentative microorganisms is a promising biofuel that has attracted research activities during the last decades (Hallenbeck, 2009; Oh et al, 2011; Rittmann et al, 2015). The hyperthermophilic archaeon T. kodakarensis grows on media with pyruvate or carbohydrates (such as soluble starch or maltodextrin) (Morikawa et al, 1994; Atomi et al, 2004). It displays one of the highest cell-specific H2 production rates when grown in a continuous culture (up to 60 mmol g-dcw−1 h−1) with pyruvate (Kanai et al, 2005). Bacteria typically exhibit maximum cell-specific H2 production rates below 40 mmol g-dcw−1 h−1 (Rittmann and Herwig, 2012), but have the advantage to reach higher cell densities

Methods

Results

Conclusion