Date Published: June 7, 2018
Publisher: Public Library of Science
Author(s): Théo Veaudor, Marcia Ortega-Ramos, Thichakorn Jittawuttipoka, Hervé Bottin, Corinne Cassier-Chauvat, Franck Chauvat, Wolfgang R. Hess.
Using a combination of various types of genetic manipulations (promoter replacement and gene cloning in replicating plasmid expression vector), we have overproduced the complex hydrogenase enzyme in the model cyanobacterium Synechocystis PCC6803. This new strain overproduces all twelve following proteins: HoxEFUYH (hydrogen production), HoxW (maturation of the HoxH subunit of hydrogenase) and HypABCDEF (assembly of the [NiFe] redox center of HoxHY hydrogenase). This strain when grown in the presence of a suitable quantities of nickel and iron used here exhibits a strong (25-fold) increase in hydrogenase activity, as compared to the WT strain growing in the standard medium. Hence, this strain can be very useful for future analyses of the cyanobacterial [NiFe] hydrogenase to determine its structure and, in turn, improve its tolerance to oxygen with the future goal of increasing hydrogen production. We also report the counterintuitive notion that lowering the activity of the Synechocystis urease can increase the photoproduction of biomass from urea-polluted waters, without decreasing hydrogenase activity. Such cyanobacterial factories with high hydrogenase activity and a healthy growth on urea constitute an important step towards the future development of an economical industrial processes coupling H2 production from solar energy and CO2, with wastewater treatment (urea depollution).
In response to the constant increase in energy consumption and resulting pollution by the growing and developping world population, it is important to develop new energy sources that are plentiful, renewable and environmentally friendly. Thus, the solar driven production of hydrogen (H2) is of special interest for many reasons. The annual solar flux received by Earth is in vast excess of the total energy used by human societies, and the burning of hydrogen liberates a high amount of energy (142 MJ/kg for H2 vs. 44.2 MJ/kg for oil) while producing only water (H2O) as a by-product. As a consequence, there is a growing interest in cyanobacteria, the photosynthetic microorganisms that can use solar energy, water, CO2, mineral and salts for the production of H2, while saving arable soils, fertilizers and fresh waters for agriculture (for recent reviews see [1–3]). Furthermore, several model cyanobacteria have a small sequenced genome easily manipulable, such as the presently studied unicellular strain Synechocystis PCC6803 (hereafter designated as Synechocystis). Its powerful genetics is necessary to attempt to increase the naturally low hydrogen production of cyanobacteria so as to reach levels that are of industrial interest.
We showed here that the simultaneous overproduction of all HoxEFUYHW and HypABCDEF proteins involved in the synthesis, maturation and assembly of the [NiFe] hydrogenase complex, combined with media improvement, led to a strong (about 25-fold) increase in the level of active hydrogenase. We also showed the counterintuitive notion that lowering the activity of the Synechocystis urease can improve the photoproduction of biomass, and in turn H2, from urea-polluted waters. The sophisticated strains constructed during this work displayed a higher increase in expression of the hoxEFUYH and hypABCDEF genes than that of hydrogenase activity. This finding indicates that limiting post-transcriptional factors need to be dealt with in order to engineer a powerful H2 producer of industrial importance. We think that the presently described strain with a healthy growth and an increased abundance of active hydrogenase is a suitable starting point for this important objective. Indeed, this hydrogenase overproducing strain should facilitate the purification and structural analysis of hydrogenase (it structure is as yet unknown), which, in turn, should facilitate the design of meaningfull strategies to increase its (low) tolerance to O2 that is massively produced by photosynthesis. Furthermore, recent in vitro data suggested that the hydrogenase enzyme could receive electrons not only from NAD(P)H but also the well conserved electron transfer proteins  ferredoxins . Thus, it will be interesting to try to further increase the hydrogenase activity of our high-hydrogenase level strain by overproducing each of the nine Synechocystis ferredoxins  along with its HoxEFUYHW and HypABCDEF proteins. It will also be important to examine the redox state of the cysteine amino-acids of the HoxH and HoxF subunits, since it has been shown that they can be oxidized [33,34], a finding that should stimulate the analysis of crosstalks between hydrogen production and redox (oxidative) stress .
The presently reported cyanobacterial factories with a large hydrogenase activity and healthy growth on urea will be very useful for the purification of large hydrogenase quantities for biochemical and structural analyses, in order to better understand this important enzyme and improve its (weak) tolerance to O2. Such work has interesting implications for future economically viable industrial production of H2 from solar energy, CO2 and urea-polluted waters.