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.

Conclusion: 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).

Figure 1

Western blot probed with Strep tactin AP conjugate of StrepII-tagged CrHydA1 in S. oneidensis AS52A1N and AS52A1C strains. Lanes 1 and 3: cell-debris pellet, lanes 2 and 4: cell-free extracts. M: molecular weight marker (kDa).

Figure 2

Gel-electrophoretic analysis of purified, StrepII-tagged CrHydA1. (A): Coomassie blue staining. (B): Immunoblot detection with Strep tactin AP conjugate. Lanes 1: HydA1 extracted after growth under DMSO-respiring conditions (~1 μg); lanes 2: HydA1 extracted after growth under TMAO-respiring conditions (~1 μg); lanes 3: HydA1 extracted after growth under thiosulfate-respiring conditions (~1 μg). M: molecular weight marker (kDa).