Engineering Synechocystis PCC6803 for hydrogen production: influence on the tolerance to oxidative and sugar stresses

Abstract

In the prospect of engineering cyanobacteria for the biological photoproduction of hydrogen, we have studied the hydrogen production machine in the model unicellular strain Synechocystis PCC6803 through gene deletion, and overexpression (constitutive or controlled by the growth temperature). We demonstrate that the hydrogenase-encoding hoxEFUYH operon is dispensable to standard photoautotrophic growth in absence of stress, and it operates in cell defense against oxidative (H₂O₂) and sugar (glucose and glycerol) stresses. Furthermore, we showed that the simultaneous over-production of the proteins HoxEFUYH and HypABCDE (assembly of hydrogenase), combined to an increase in nickel availability, led to an approximately 20-fold increase in the level of active hydrogenase. These novel results and mutants have major implications for those interested in hydrogenase, hydrogen production and redox metabolism, and their connections with environmental conditions.

Figure 1. Schematic representation of the Synechocystis wild-type and the mutant strains constructed in this study.

The Synechocystis spherical cells are represented by the green circles. The chromosome is shown as the black line form attached to the cell membrane, while the pTR-hypABCDEF and pCE-hypABCDEF replicating plasmids are represented by circles. The hoxEFUYH operon, the hypABCDEF genes and the antibiotic resistance markers are shown by large colored arrows, which indicate the direction of their transcription. The hatched (orange) arrow shows the λcI₈₅₇ gene encoding the temperature-sensitive repressor, which tightly controls the activity of the otherwise strong λp _R promoter (red triangle), depending on the growth temperature. The symbols are namely: Δ for deletion; CE for constitutive expression; TR for temperature-regulated expression; and WT for wild type.

Figure 2. Analysis of the Synechocystis TR-hoxEFUYH mutant for temperature-regulated high-level expression of the hoxEFUYH operon.

(A) Schematic representation of the hoxEFUYH operon locus in the wild-type strain (WT) and the TR-hoxEFUYH mutant (TR1). (B) Typical growth of the WT (squares) and TR1 cells (triangles) under standard light at 30°C (open symbols) or 39°C (black symbols). (C) Histogram representation of the ratio of the transcript abundance (measured by Real-time quantitative PCR) of each eight genes of the hoxEFUYH operon in the WT and TR1 cells grown at 30°C or 39°C. (D) Western blot analysis of the abundance of the HoxF and HoxH proteins in WT and TR1 cells grown at 30°C or 39°C. (E) Histograms representation of the hydrogenase activities of WT and TR1 cells grown at 30°C or 39°C in standard medium (MM) or MM* (MM +17 µM Fe) supplemented with 2.5 µM NiSO₄. All experiments were performed at least three times.

Figure 3. Analysis of the TR-hoxEFUYH-hypABCDEF mutant for temperature-regulated high-level expression of the hoxEFUYH and hypABCDEF genes.

All experiments were performed at least three times on cells grown under standard light, at 39°C to induce the expression of the hoxEFUYH operon and the hypABCDEF genes controlled by the λcI₈₅₇-λp _R system. (A) Typical growth of the WT (squares), TR-hoxEFUYH (TR1; white triangles) and TR-hoxEFUYH-hypABCDEF (TR2; grey triangles) strains. (B) Histogram plot representation of the transcript abundance (measured by Real-time quantitative PCR) of the eight genes of the hoxEFUYH operon (left part) and six hypABCDEF genes (right part) in WT (white bars), TR1 (grey bars) or TR2 (hatched bars) cells. (C) Western blot analysis of the abundance of the HoxF and HoxH proteins in WT, TR1 or TR2 cells. (D) Histograms representation of the hydrogenase activities of WT (light grey), TR1 (dark grey) or TR2 (hatched bars) growing in standard medium (MM) or MM* (MM+17 µM Fe) supplemented with 2.5 µM NiSO₄.

Figure 4. Analysis of the CE-hoxEFUYH and CE-hoxEFUYH-hypABCDEF mutants for strong constitutive expression of the hoxEFUYH alone or together with the hypABCDEF genes.

All experiments were performed at least three times on cells grown at 30°C under standard light. (A) Typical growth of the WT (squares), CE-hoxEFUYH (CE1; white triangles) and CE-hoxEFUYH-hypABCDEF (CE2; grey triangles) cells. (B) Histogram plot representation of the transcript abundance (measured by Real-time quantitative PCR) of the eight genes of the hoxEFUYH operon (left part) and the six hypABCDEF genes (right part) in the CE1 (grey bars) or CE2 (hatched bars) mutants, as compared to the WT strain (white bars). (C) Western blot analysis of the abundance of the HoxF and HoxH proteins in WT, CE1 or CE2 cells. (D) Histograms representation of the hydrogenase activities of WT (light grey), CE1 (dark grey) or CE2 cells (hatched bars) growing in standard medium (MM) or MM* (MM+17 µM Fe) supplemented with 2.5 µM NiSO₄.

Figure 5. Influence of the hoxEFUYH and hypABCDEF genes on the tolerance to H₂O₂, glucose or glycerol.

(A) Typical survival of the Synechocystis strains WT (open squares), ΔhoxEFUYH::Km^r (Δhox, open cercles) or CE-hoxEFUYH (CE1, open triangles) grown at 30°C and challenged for 1 hour with H₂O₂ under anaerobiose before plating under standard photoautotrophic conditions. (B) Typical survival to the anaerobic H₂O₂ stress of the WT strain (open squares), CE1 (open triangles), CE-hoxEFUYH-hypABCDEF (CE2, dark-grey triangles), CE-hypABCDEF (CE3, dark-grey squares). (C) Typical photoautotrophic growth of the wild-type (WT), DhoxEFUYH::Km^r (Δhox), CE-hoxEFUYH (CE1) and CE-hoxEFUYH-hypABCDEF (CE2) strains. Cells grown twice in standard (MM) liquid medium (up to mid-log phase) were inoculated in MM without or with glucose 10 mM or 300 µM glycerol, and incubated under light (3,000 luxes) during 7 days. All experiments were performed at least three times.