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

Kateryna Sybirna,Hervé Bottin,Vincent Méjean,Pia Lindberg,Vincent Fourmond,Tatiana Antoine,Marc Rousset

doi:10.1186/1472-6750-8-73

Abstract

BackgroundThe eukaryotic green alga, Chlamydomonas reinhardtii, produces H2 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.ResultsBased on codon usage bias and hydrogenase maturation ability, the bacterium S. oneidensis, which possesses putative [Fe-Fe] and [Ni-Fe] hydrogenase operons, was selected as the best potential host for C. reinhardtii [Fe-Fe] hydrogenase expression. Hydrogen formation by S. oneidensis strain AS52 (ΔhydAΔhyaB) transformed with a plasmid bearing CrHydA1 and grown in the presence of six different substrates for anaerobic respiration was determined. A significant increase in hydrogen evolution was observed for cells grown in the presence of trimethylamine oxide, dimethylsulfoxide and disodium thiosulfate, showing that the system of S. oneidensis is efficient for heterologous expression of algal [Fe-Fe] hydrogenase.ConclusionIn the present work a new efficient system for heterologous expression and maturation of C. reinhardtii hydrogenase has been developed. HydA1 of C. reinhardtii was purified and shown to contain 6 Fe atoms/molecule of protein, as expected. Using DMSO, TMAO or thiosulfate as substrates for anaerobic respiration during the cell growth, 0.4 – 0.5 mg l-1(OD600 = 1) of catalytically active HydA1 was obtained with hydrogen evolution rate of ~700 μmol H2 mg-1 min-1.

Highlights

The eukaryotic green alga, Chlamydomonas reinhardtii, produces H2 under anaerobic conditions, in a reaction catalysed by a [Fe-Fe] hydrogenase HydA1
The opposite situation appeared with the E. coli expression system which resulted in a higher amount of enzyme (0.8 – 1 mg of HydA1 per liter of culture) but with hydrogen evolution rates of 150 μmol H2 min-1 mg-1 [8]
In contrast to the transcriptome analysis by Beliaev et al [12], we observed a significant increase of hydrogen formation due to the presence of the S. oneidensis [Fe-Fe] hydrogenase (HydA) with three substrates (trimethylamine oxide (TMAO), dimethylsulfoxide (DMSO) and disodium thiosulfate), compared to anaerobic conditions in the presence of other added respiratory substrates

Summary

Introduction

The eukaryotic green alga, Chlamydomonas reinhardtii, produces H2 under anaerobic conditions, in a reaction catalysed by a [Fe-Fe] hydrogenase HydA1. C. reinhardtii is a eukaryotic green alga which contains two monomeric [Fe-Fe] hydrogenases: HydA1 and HydA2 [3] Both proteins are encoded by nuclear genes. Heterologous expression of HydA1 has been previously performed in Clostridium acetobutylicum using endogenous maturation enzymes [7] and in Escherichia coli using a heterologously expressed maturation system [6,8] The former experiments using C. acetobutylicum resulted in the purification of 0.1 mg of HydA1 per liter of culture with hydrogen evolution rates of 760 μmol H2 min-1 mg-1 [7]. The opposite situation appeared with the E. coli expression system which resulted in a higher amount of enzyme (0.8 – 1 mg of HydA1 per liter of culture) but with hydrogen evolution rates of 150 μmol H2 min-1 mg-1 [8]

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Discussion

Conclusion