title: 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 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.
  
  
  RESULTS
  Based 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 (Delta hydA Delta hyaB) 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.
  
  
  CONCLUSIONS
  In 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 approximately 700 micromol H2 mg(-1) min(-1).
authors.affiliation: CEA, DSV, iBiTec-S, SB2SM, LPB (URA CNRS 2096), 91191 Gif-sur-Yvette cedex, France. kateryna.sybirna@cea.fr
authors.forename: Kateryna
authors.initials: K
authors.lastname: Sybirna
authors.forename: Tatiana
authors.initials: T
authors.lastname: Antoine
authors.forename: Pia
authors.initials: P
authors.lastname: Lindberg
authors.forename: Vincent
authors.initials: V
authors.lastname: Fourmond
authors.forename: Marc
authors.initials: M
authors.lastname: Rousset
authors.forename: Vincent
authors.initials: V
authors.lastname: Méjean
authors.forename: Hervé
authors.initials: H
authors.lastname: Bottin
mesh_terms: D000818:Animals; D016825:Chlamydomonas reinhardtii / Q000201:enzymology* / Q000235:genetics*; D004789:Enzyme Activation; D004795:Enzyme Stability; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D006864:Hydrogenase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D020592:Shewanella / Q000201:enzymology* / Q000235:genetics*
chemical_list: D011994:Recombinant Proteins; D006864:Hydrogenase
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