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.

High‐efficiency transformation of archaea by direct PCR products with its application to directed evolution of a thermostable enzyme

“…All values were averages of the results of three independent experiments. The DNA sequences of positive colonies identified by colony PCR were validated by DNA sequencing (GENEWIZ, Beijing, China) (Kanai et al ., 2015).…”

Extremely thermophilic microorganisms as metabolic engineering platforms for production of fuels and industrial chemicals

“…T. kodakarensis has been metabolically engineered for improved hydrogen production, resulting in a strain capable of producing several times more hydrogen than wild-type (Santangelo et al, 2011 ). Efforts to further improve hydrogen production are ongoing (Kanai et al, 2015 ), and T. kodakarensis shows promise as a bio-hydrogen production platform. Strains optimized for overexpression and secretion of enzymes have also been developed (Takemasa et al, 2011 ).…”

Plasmid Curing is a Promising Approach to Improve Thermophiles for Biotechnological Applications: Perspectives in Archaea

“…Moreover, thermophiles often have remarkable properties useful for bioprocesses. A good example is the hyperthermophilic archaeon Thermococcus kodakarensis KOD1, which can use protons as an electron accepter in catabolism to generate molecular hydrogen and has been studied as a hydrogen production tool [7].…”