Abstract
BackgroundHydrogen photo-production in green algae, catalyzed by the enzyme [FeFe]-hydrogenase (HydA), is considered a promising source of renewable clean energy. Yet, a significant increase in hydrogen production efficiency is necessary for industrial scale-up. We have previously shown that a major challenge to be resolved is the inferior competitiveness of HydA with NADPH production, catalyzed by ferredoxin-NADP+-reductase (FNR). In this work, we explored the in vivo hydrogen production efficiency of Fd-HydA, where the electron donor ferredoxin (Fd) is fused to HydA and expressed in the model organism Chlamydomonas reinhardtii.ResultsWe show that once the Fd-HydA fusion gene is expressed in micro-algal cells of C. reinhardtii, the fusion enzyme is able to intercept photosynthetic electrons and use them for efficient hydrogen production, thus supporting the previous observations made in vitro. We found that Fd-HydA has a ~4.5-fold greater photosynthetic hydrogen production rate standardized for hydrogenase amount (PHPRH) than that of the native HydA in vivo. Furthermore, we provide evidence suggesting that the fusion protein is more resistant to oxygen than the native HydA.ConclusionsThe in vivo photosynthetic activity of the Fd-HydA enzyme surpasses that of the native HydA and shows higher oxygen tolerance. Therefore, our results provide a solid platform for further engineering efforts towards efficient hydrogen production in microalgae through the expression of synthetic enzymes.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0601-3) contains supplementary material, which is available to authorized users.
Highlights
We have shown that more than 85 % of the electron flux is naturally directed to NADPH production [9]
In our previous work [9], we showed that by substituting HydA for ferredoxin [GlyX4-Ser]X3 hydrogenase (Fd-HydA) fusion protein in in vitro reconstituted reactions, we diverted 60 % of the electron flux towards hydrogen production, while limiting NADPH production
Isolation of transgenic H2 producing algal strains To study the in vivo H2 production rate of the Fd-HydA fusion, we first obtained a C. reinhardtii strain with negligible hydrogen production background, i.e., hydA1-1 hydA2-1 double mutant that was derived from the parental strain D66 [15]
Summary
Hydrogen gas production occurs in certain microorganisms that catalyze its production through two enzyme families: nitrogenases that fix atmospheric nitrogen into ammonia while emitting gaseous hydrogen as a byproduct, and hydrogenases that either produce or oxidize hydrogen directly. Algal [FeFe]-hydrogenase (hereafter HydA) is of great industrial interest due to its extremely high turnover rate. HydA is extremely sensitive to oxygen, which is a direct product of photosynthesis. The enzyme’s typical half-life under aerobic conditions ranges from a few seconds for algal HydA to several minutes for the Clostridia [FeFe]-hydrogenase, which contains additional iron sulfur clusters [3, 4]. Oxygen sensitivity of HydA constitutes a primary technological barrier to overcome in order to make commercial production of hydrogen feasible
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