The dual effect of a ferredoxin-hydrogenase fusion protein in vivo: successful divergence of the photosynthetic electron flux towards hydrogen production and elevated oxygen tolerance

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.

Photosynthetic fuel for heterologous enzymes: the role of electron carrier proteins

“…4b). This yielded 2.5-fold higher hydrogen productivities in the presence of competing FNR and NADP + in vitro (Yacoby et al 2011) and also showed 4.5-fold increased hydrogenase activity in vivo (Eilenberg et al 2016). In a similar manner, fusing ferredoxin with the first cytochrome P450 of the dhurrin biosynthetic pathway, CYP79A1 (Fig.…”

“…These hydrogenases fall into two classes, containing [Fe-Fe] (algal) or [Ni-Fe] (cyanobacterial) redox centers. Different strategies have been employed to address O 2 inhibition of hydrogenase activity and improve their competitiveness towards reduced ferredoxin, ranging from gene fusion with ferredoxin to reconfigure the iron-sulfur clusters, to conversion of a hydrogen-consuming uptake hydrogenase into a hydrogen-generating one (Yacoby et al 2011;Dubini and Ghirardi 2014;Eilenberg et al 2016;Raleiras et al 2016). …”

“…The importance of electron carriers in biotechnological applications of photosynthetic organisms is apparent from several recent examples where successful redox tuning increased the productivity of novel metabolic routes. Thus, there is much unexplored potential for redirecting photosynthetic reducing power, and engineering of photosynthetic electron transfer chains to accommodate heterologous enzymes could be an important or the hydrogenase HydA (Yacoby et al 2011;Eilenberg et al 2016) improves their ability to sequester electrons in the presence of competing FNR, demonstrating the viability of this strategy for both soluble and thylakoid membrane-localized enzymes. Black wavy lines indicate flexible Gly-Ser-rich linkers, which were used in both studies.…”

O2 sensitivity and H2 production activity of hydrogenases—A review

“…To make effective O 2 ‐tolerant hydrogenases, operations on the O 2 diffusion channels to the active site may be considered (Liebgott et al, ). Alternatively, other engineering strategies, such as chemical treatment (Rodríguez‐Maciá et al, ), silicification‐induced cell aggregation (Xiong et al, ), hydrogel protection (Plumeré et al, ), and protein fusion (Eilenberg et al, ) have been successfully adopted to protect hydrogenases from O 2 ‐driven damage. Both site‐directed and random mutagenesis studies have been done to improve O 2 tolerance of the enzyme (Bingham, Smith, & Swartz, ; Ghirardi, ).…”

“…Both site‐directed and random mutagenesis studies have been done to improve O 2 tolerance of the enzyme (Bingham, Smith, & Swartz, ; Ghirardi, ). By the development of high‐throughput screening methods (Wecker & Ghirardi, ; Wecker, Meuser, Posewitz, & Ghirardi, ) including those based on the cell‐free technology (Koo et al, ; Koo, Schnabel, Liong, Evitt, & Swartz, ), [FeFe] hydrogenase variants with improved O 2 tolerance have been achieved (Bingham et al, ; Eilenberg et al, ; Koo & Swartz, ). Even so, this limited success is still not sufficient for practical applications, inviting future efforts towards further improving O 2 tolerance of the enzyme.…”