Rewiring hydrogenase-dependent redox circuits in cyanobacteria

Abstract

Hydrogenases catalyze the reversible reaction 2H(+) + 2e(-) ↔ H(2) with an equilibrium constant that is dependent on the reducing potential of electrons carried by their redox partner. To examine the possibility of increasing the photobiological production of hydrogen within cyanobacterial cultures, we expressed the [FeFe] hydrogenase, HydA, from Clostridium acetobutylicum in the non-nitrogen-fixing cyanobacterium Synechococcus elongatus sp. 7942. We demonstrate that the heterologously expressed hydrogenase is functional in vitro and in vivo, and that the in vivo hydrogenase activity is connected to the light-dependent reactions of the electron transport chain. Under anoxic conditions, HydA activity is capable of supporting light-dependent hydrogen evolution at a rate > 500-fold greater than that supported by the endogenous [NiFe] hydrogenase. Furthermore, HydA can support limited growth solely using H(2) and light as the source of reducing equivalents under conditions where Photosystem II is inactivated. Finally, we demonstrate that the addition of exogenous ferredoxins can modulate redox flux in the hydrogenase-expressing strain, allowing for greater hydrogen yields and for dark fermentation of internal energy stores into hydrogen gas.

Fig. 1.

Expression of HydA in Synechococcus elongatus sp. 7942. (A) Schematic of integrated genes. Maturation factors (HydEF, G) are expressed under the constitutive psba1 promoter. HydA is expressed under the IPTG-inducible PLac. (B) Western blot analysis for His-tagged HydA in wild-type or HydA-expressing strains under varied IPTG concentrations (predicted molecular mass = 65.6 kDa). (C) Representative, same day trial of evolved hydrogen gas from wild-type and HydA-expressing Synechococcus lysates 60 min following methyl viologen treatment under anaerobic conditions.

Fig. 2.

In vivo hydrogen production. (A) In vivo hydrogen production under anaerobic conditions (2.5% CO₂ in N₂) with DCMU treatment. (B) In vivo hydrogen production is dependent on light and electron transport from plastoquinone.

Fig. 3.

Hydrogenase-dependent chemoautotrophic growth. (A) HydA activity supports cell growth when coexpressed with hydrogenase maturation factors under anaerobic conditions in the presence of hydrogen gas. (B) Block of electron transfer from plastoquinone (with DBMIB) prevents hydrogenase-mediated growth. (C) Representative FACS analysis of cell viability in wild-type and HydA-expressing cells as measured by the vital dye Sytox blue. Data shown for cells after 7 d of incubation with DCMU and under a CO₂/H₂ atmosphere. (D) Reiterative rounds of growth under hydrogen atmosphere (4 d; DCMU/CO₂/H₂) followed by release of selective pressure (1 d; no DCMU air) enriches for HydA-expressing cells relative to wild type.

Fig. 4.

Ferredoxin incorporation alters electron flow to/from hydrogenase. (A) In vivo hydrogen production under anaerobic conditions in the presence of exogenous ferredoxins [Spinacea oleracea (S. Fd) and Clostridium acetobutylicum (C. Fd)]. (B) Incorporation of C. Fd allows dark fermentation to generate hydrogen gas. (C) Hydrogenase-bearing strains containing Clostridial ferredoxin are unable to support growth with hydrogen as the sole source of reducing equivalents.

Fig. 5.

Model of ferredoxin-mediated electron transfer to/from hydrogenase. Endogenous S. elongtaus ferredoxins (Syne. Fd) are capable of transferring electrons from PSI to exogenous HydA for the production of hydrogen. When exogenous Clostridial ferredoxin (C. Fd) is expressed, it is the preferred interacting partner for HydA (bold arrows). Although C. Fd can increase the flux of reducing equivalents to HydA under conditions favoring hydrogen evolution, exogenous ferredoxins are inefficient for electron transfer (faded arrows) to the plastoquinone (PQ) pool to allow for hydrogen-mediated growth.