Shewanella oneidensis: a new and efficient System for Expression and Maturation of heterologous [Fe-Fe] Hydrogenase from Chlamydomonas reinhardtii

Abstract

Background: The eukaryotic green alga, Chlamydomonas reinhardtii, produces H 2 under anaerobic conditions, in a reaction catalysed by a [Fe-Fe] hydrogenase HydA1. For further biochemical and biophysical studies a suitable expression system of this enzyme should be found to overcome its weak expression in the host organism. Two heterologous expression systems used up to now have several advantages. However they are not free from some drawbacks. In this work we use bacterium Shewanella oneidensis as a new and efficient system for expression and maturation of HydA1 from Chlamydomonas reinhardtii.

Purification and Characterization of the [NiFe]-Hydrogenase of Shewanella oneidensis MR-1

“…The data collected were compared to the data from Tc(V)-citrate (provided by Nancy Hess) and Tc(IV)O 2 ⅐ nH 2 O standards measured previously at the same beamline under similar conditions (43). (26,39), which provided a clean background for testing whether expressed MR-1 [NiFe]-H 2 ase was functional in vivo. We transformed an MR-1 mutant in which both hyaB and hydA genes were deleted (i.e., ⌬hyaB-⌬hydA) with pLS189 that contained the genes encoding MR-1 [NiFe]-H 2 ase (i.e., hyaA and hyaB).…”

“…Following verification by sequencing, pLS189 was introduced into an MR-1 mutant in which both hyaB and hydA (locus tag SO3920; the gene encoding the large subunit of [FeFe]-H 2 ase) genes were deleted to create MR-1 strain LS498 (24). pBBR-hydA1N was introduced into the same mutant to create LS484 (39). The MR-1 strains, plasmids, and primers used in this study are listed in Table 1.…”

“…To purify MR-1 [NiFe]-H 2 ase, it was expressed in the ⌬hyaB-⌬hydA mutant when 10 mM DMSO was used as the terminal electron acceptor (39). Following cell lysis and ultracentrifugation, the MR-1 [NiFe]-H 2 ase was isolated from the supernatant by using immobilized metal ion affinity chromatography.…”

The AbrB2 Autorepressor, Expressed from an Atypical Promoter, Represses the Hydrogenase Operon To Regulate Hydrogen Production in Synechocystis Strain PCC6803

“…Hydrogenase activities were measured by two standard methods. First, hydrogen evolution was measured with a modified Clark-type electrode (Hansatech, United Kingdom) (48) as described previously by some of us (46). A total of 2.5 ϫ 10 7 cells (1-ml culture at an optical density at 580 nm of 1) were harvested by a 5-min centrifugation at 5,000 rpm, resuspended in 25 l of 50 mM Tris-HCl (pH 7.5) buffer, and introduced into 500 l of a solution containing 50 mM Tris-HCl (pH 7.5), 20 mM Na-dithionite, and 5 mM methylviologen as the electron donor to hydrogenase.…”

Solar-to-bioH2 production enhanced by homologous overexpression of hydrogenase in green alga Chlorella sp. DT

“…There have been several reports that heterologous expression of HydA from C. reinhardtii was demonstrated in some bacterial hosts, for instance, Clostridium acetobutylicum [21], Escherichia coli [22], and Shewanella oneidensis [23]. However, the resulting expressed HydA did not produce H 2 at a high rate, simply due to the hosts lacking a maturation system.…”