Overproduction of the cyanobacterial hydrogenase and selection of a mutant thriving on urea, as a possible step towards the future production of hydrogen coupled with water treatment

Abstract

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 overproduce 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 exhibit 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).

Genomics of Urea Transport and Catabolism in Cyanobacteria: Biotechnological Implications

“…Many urease-endowed cyanobacteria can grow on urea as the sole nitrogen source (Collier et al, 1999) but a high-concentration of urea and/or prolonged cultivation on urea (≥10 mM) can be toxic to cyanobacteria. This finding was shown with Arthrospira PCC 8005 (edible cyanobacterium, Deschoenmaeker et al, 2017), Microcystis aeruginosa (fresh water cyanobacterium, Wu et al, 2015), Synechococcus PCC 7002 (costal cyanobacterium, Sakamoto et al, 1998) and Synechocystis PCC 6803 (euryhaline cyanobacterium, Veaudor et al, 2018). The cell death and color change (from blue-green to yellowish) triggered by the prolonged growth on urea could be due to lipid peroxidation, a phenomenon that increases in parallel with cell death and pigment oxidation (Sakamoto et al, 1998).…”

“…The cell death and color change (from blue-green to yellowish) triggered by the prolonged growth on urea could be due to lipid peroxidation, a phenomenon that increases in parallel with cell death and pigment oxidation (Sakamoto et al, 1998). By contrast, urease defective mutants of Synechococcus PCC7002 and Synechocystis PCC 6803 (inactivation of the ureC gene, see below) were not killed by prolonged incubation in the presence of a high urea concentration, demonstrating that urea-consumption driven by urease can become toxic (Sakamoto et al, 1998; Veaudor et al, 2018). Furthermore, the (marine) Synechococcus WH7803 strain and the (freshwater) Synechococcus PCC7942 strain cannot grow on urea and neither have urease activity (Collier et al, 1999).…”

A Genetic Toolbox for the New Model Cyanobacterium Cyanothece PCC 7425: A Case Study for the Photosynthetic Production of Limonene

“…After 7–10 days of cultivation on urea, Cyanothece PCC 7425 can turn yellowish, as previously observed in the phylogenetically distant cyanobacteria Anabaena cylindrica , Synechococcus PCC 7002 ( Sakamoto et al, 1998 ) and Synechocystis PCC 6803 growing on urea as the sole nitrogen source ( Veaudor et al, 2019 ). Again as observed in Synechocystis PCC 6803 ( Veaudor et al, 2018 ), once installed the chlorosis process decreased the cell viability measured by plating assays on standard growth medium (it contains nitrate, not urea). Interestingly, Cyanothece PCC 7425 grew up to increasing cell densities in response to increasing urea quantities, which needed to be supplied not all at once, but as small successive sub-doses along cell growth ( Supplementary Figure S1 ).…”

“…The ls nucleotide sequence was synthesized by the Eurofins company as a DNA segment flanked by Nde I and Eco RI restriction sites at its 5′- and 3′-ends, respectively. After cleavage with both Nde I and Eco RI, the ls gene was cloned downstream of the strong λ p R promoter of the pFC1-derivative pC plasmid for high-level constitutive gene expression ( Veaudor et al, 2018 ), which was opened with the same enzymes ( Supplementary Figures S6 , S7 ).…”

Mechanism and Application of the Catalytic Reaction of [NiFe] Hydrogenase: Recent Developments

“…Veaudor et al. overproduced [NiFe] H 2 ase in cyanobacterium Synechocystis PCC6803 using various types of genetic manipulations (e. g., promoter replacement and gene cloning in replicating plasmid expression vector) . The new strain overproduced the following twelve proteins: HoxEFUYH (H 2 production proteins), HoxW (maturation protein of the HoxH subunit protein), and HypABCDEF (assembly proteins of the Ni−Fe redox center).…”