Crucial Role of the Chaperonin GroES/EL for Heterologous Production of the Soluble Methane Monooxygenase from Methylomonas methanica MC09.

Methane is a widespread energy source and can serve as an attractive C1 building block for a future bioeconomy. The soluble methane monooxygenase (sMMO) is able to break the strong C-H bond of methane and convert it to methanol. The high structural complexity, multiplex cofactors, and unfamiliar folding or maturation procedures of sMMO have hampered the heterologous production and thus biotechnological applications. Here, we demonstrate the heterologous production of active sMMO from the marine Methylomonas methanica MC09 in Escherichia coli by co-synthesizing the GroES/EL chaperonin. Iron determination, electron paramagnetic resonance spectroscopy, and native gel immunoblots revealed the incorporation of the non-heme diiron centre and homodimer formation of active sMMO. The production of recombinant sMMO will enable the expansion of the possibilities of detailed studies, allowing for a variety of novel biotechnological applications.

Catalytic and Spectroscopic Properties of the Halotolerant Soluble Methane Monooxygenase Reductase from Methylomonas methanica MC09.

The soluble methane monooxygenase receives electrons from NADH via its reductase MmoC for oxidation of methane, which is itself an attractive C1 building block for a future bioeconomy. Herein, we present biochemical and spectroscopic insights into the reductase from the marine methanotroph Methylomonas methanica MC09. The presence of a flavin adenine dinucleotide (FAD) and [2Fe2S] cluster as its prosthetic group were revealed by reconstitution experiments, iron determination and electron paramagnetic resonance spectroscopy. As a true halotolerant enzyme, MmoC still showed 50 % of its specific activity at 2 M NaCl. We show that MmoC produces only trace amounts of superoxide, but mainly hydrogen peroxide during uncoupled turnover reactions. The characterization of a highly active reductase is an important step for future biotechnological applications of a halotolerant sMMO.

From inert gas to fertilizer, fuel and fine chemicals: N2 reduction and fixation.

The 100th anniversary of a leading nitrogen fixation technology developer like CASALE SA is a reason to reflect over the 20th century successful solution of the problem of world food supply, and to look out for solutions for sustainable developments with respect to ammonia production. We review the role of nitrogen as essential chemical constituent in photosynthesis and biology, and component of ammonia as it is used as fertilizer for primary production by photosynthesis for farming and food supply and its future role as energy carrier. While novel synthesis methods and very advanced synchrotron based x-ray analytical techniques are being developed, we feel it is important to refer to the historical and economical context of nitrogen. The breaking of the N≡N triple bond remains a fundamental chemical and energetic problem in this context. We review the electrochemical ammonia synthesis as an energetically and environmentally benign method. Two relatively novel X-ray spectroscopy methods, which are relevant for the molecular understanding of the catalysts and biocatalysts, i.e. soft X-ray absorption spectroscopy and nuclear resonant vibration spectroscopy are presented. We illustrate the perceived reality in fertilizer usage on the field, and fertilizer production in the factory complex with photos and thus provide a contrast to the academic view of the molecular process of ammonia function and production.

Rewiring cyanobacterial photosynthesis by the implementation of an oxygen-tolerant hydrogenase.

Molecular hydrogen (H2) is considered as an ideal energy carrier to replace fossil fuels in future. Biotechnological H2 production driven by oxygenic photosynthesis appears highly promising, as biocatalyst and H2 syntheses rely mainly on light, water, and CO2 and not on rare metals. This biological process requires coupling of the photosynthetic water oxidizing apparatus to a H2-producing hydrogenase. However, this strategy is impeded by the simultaneous release of oxygen (O2) which is a strong inhibitor of most hydrogenases. Here, we addressed this challenge, by the introduction of an O2-tolerant hydrogenase into phototrophic bacteria, namely the cyanobacterial model strain Synechocystis sp. PCC 6803. To this end, the gene cluster encoding the soluble, O2-tolerant, and NAD(H)-dependent hydrogenase from Ralstonia eutropha (ReSH) was functionally transferred to a Synechocystis strain featuring a knockout of the native O2 sensitive hydrogenase. Intriguingly, photosynthetically active cells produced the O2 tolerant ReSH, and activity was confirmed in vitro and in vivo. Further, ReSH enabled the constructed strain Syn_ReSH+ to utilize H2 as sole electron source to fix CO2. Syn_ReSH+ also was able to produce H2 under dark fermentative conditions as well as in presence of light, under conditions fostering intracellular NADH excess. These findings highlight a high level of interconnection between ReSH and cyanobacterial redox metabolism. This study lays a foundation for further engineering, e.g., of electron transfer to ReSH via NADPH or ferredoxin, to finally enable photosynthesis-driven H2 production.

Insights into electron transfer and bifurcation of the Synechocystis sp. PCC6803 hydrogenase reductase module.

The NAD+-reducing soluble [NiFe] hydrogenase (SH) is the key enzyme for production and consumption of molecular hydrogen (H2) in Synechocystis sp. PCC6803. In this study, we focused on the reductase module of the SynSH and investigated the structural and functional aspects of its subunits, particularly the so far elusive role of HoxE. We demonstrated the importance of HoxE for enzyme functionality, suggesting a regulatory role in maintaining enzyme activity and electron supply. Spectroscopic analysis confirmed that HoxE and HoxF each contain one [2Fe2S] cluster with an almost identical electronic structure. Structure predictions, alongside experimental evidence for ferredoxin interactions, revealed a remarkable similarity between SynSH and bifurcating hydrogenases, suggesting a related functional mechanism. Our study unveiled the subunit arrangement and cofactor composition essential for biological electron transfer. These findings enhance our understanding of NAD+-reducing [NiFe] hydrogenases in terms of their physiological function and structural requirements for biotechnologically relevant modifications.