A novel screening protocol for the isolation of hydrogen producing Chlamydomonas reinhardtii strains

<p>Abstract</p> <p>Background</p> <p>Sealed <it>Chlamydomonas reinhardtii </it>cultures evolve significant amounts of hydrogen gas under conditions of sulfur depletion. However, the eukaryotic green alga goes through drastic metabolic changes during this nutritional stress resulting in cell growth inhibition and eventually cell death. This study aimed at isolating <it>C. reinhardtii </it>transformants which produce hydrogen under normal growth conditions to allow a continuous hydrogen metabolism without the stressful impact of nutrient deprivation.</p> <p>Results</p> <p>To achieve a steady photobiological hydrogen production, a screening protocol was designed to identify <it>C. reinhardtii </it>DNA insertional mutagenesis transformants with an attenuated photosynthesis to respiration capacity ratio (P/R ratio). The screening protocol entails a new and fast method for mutant strain selection altered in their oxygen production/consumption balance. Out of 9000 transformants, four strains with P/R ratios varying from virtually zero to three were isolated. Strain <it>apr</it>1 was found to have a slightly higher respiration rate and a significantly lower photosynthesis rate than the wild type. Sealed cultures of <it>apr</it>1 became anaerobic in normal growth medium (TAP) under moderate light conditions and induced [FeFe]-hydrogenase activity, yet without significant hydrogen gas evolution. However, Calvin-Benson cycle inactivation of anaerobically adapted <it>apr</it>1 cells in the light led to a 2-3-fold higher <it>in vivo </it>hydrogen production than previously reported for the sulfur-deprived <it>C. reinhardtii </it>wild type.</p> <p>Conclusion</p> <p>Attenuated P/R capacity ratio in microalgal mutants constitutes a platform for achieving steady state photobiological hydrogen production. Using this platform, algal hydrogen metabolism can be analyzed without applying nutritional stress. Furthermore, these strains promise to be useful for biotechnological hydrogen generation, since high <it>in vivo </it>hydrogen production rates are achievable under normal growth conditions, when the photosynthesis to respiration capacity ratio is lowered in parallel to down regulated assimilative pathways.</p

Cyanide Binding to [FeFe]-Hydrogenase Stabilizes the Alternative Configuration of the Proton Transfer Pathway

Hydrogenases are H2 converting enzymes that harbor catalytic cofactors in which iron (Fe) ions are coordinated by biologically unusual carbon monoxide (CO) and cyanide (CN−) ligands. Extrinsic CO and CN−, however, inhibit hydrogenases. The mechanism by which CN− binds to [FeFe]-hydrogenases is not known. Here, we obtained crystal structures of the CN−-treated [FeFe]-hydrogenase CpI from Clostridium pasteurianum. The high resolution of 1.39 Å allowed us to distinguish intrinsic CN− and CO ligands and to show that extrinsic CN− binds to the open coordination site of the cofactor where CO is known to bind. In contrast to other inhibitors, CN− treated crystals show conformational changes of conserved residues within the proton transfer pathway which could allow a direct proton transfer between E279 and S319. This configuration has been proposed to be vital for efficient proton transfer, but has never been observed structurally

Die Bindung von Cyanid an [FeFe]‐Hydrogenasen stabilisiert die alternative Konfiguration des Protonentransferpfads

Hydrogenasen sind H2-umsetzende Metalloenzyme und enthalten katalytische Kofaktoren, deren Eisenionen durch biologisch ungewöhnliche Kohlenmonoxid- (CO) und Cyanid- (CN−) Liganden koordiniert sind. Externes CO und CN−hemmt Hydrogenasen jedoch. Der molekulare Mechanismus der Bindung von CN− an [FeFe]-Hydrogenasen ist unbekannt. In dieser Studie präsentieren wir Kristallstrukturen der mit CN− behandelten [FeFe]-Hydrogenase CpI aus Clostridium pasteurianum. Auf Grund der hohen Auflösung von 1.39 Å können wir die intrinsischen CO- und CN−-Liganden voneinander unterscheiden. Wir zeigen, dass externes CN− die offene Bindestelle des Kofaktors besetzt, an die auch externes CO bindet. Im Gegensatz zu anderen Inhibitoren zeigen die CN−-behandelten Kristalle Konformationsänderungen konservierter Reste des Protonentransferpfads, die einen direkten Austausch von Protonen zwischen den Aminosäuren E279 und S319 ermöglichen. Diese Konformation wurde als notwendig für einen effizienten Protonentransfer vorgeschlagen, doch wurde sie bisher nicht strukturell nachgewiesen

Phylogenetic and molecular analysis of hydrogen-producing green algae

A select set of microalgae are reported to be able to catalyse photobiological H2 production from water. Based on the model organism Chlamydomonas reinhardtii, a method was developed for the screening of naturally occurring H2-producing microalgae. By purging algal cultures with N2 in the dark and subsequent illumination, it is possible to rapidly induce photobiological H2 evolution. Using NMR spectroscopy for metabolic profiling in C. reinhardtii, acetate, formate, and ethanol were found to be key compounds contributing to metabolic variance during the assay. This procedure can be used to test algal species existing as axenic or mixed cultures for their ability to produce H2. Using this system, five algal isolates capable of H2 production were identified in various aquatic systems. A phylogenetic tree was constructed using ribosomal sequence data of green unicellular algae to determine if there were taxonomic patterns of H2 production. H2-producing algal species were seen to be dispersed amongst most clades, indicating an H2-producing capacity preceded evolution of the phylum Chlorophyta

Association of Ferredoxin:NADP+ oxidoreductase with the photosynthetic apparatus modulates electron transfer in Chlamydomonas reinhardtii

R.M. acknowledges support from the MEXT (Ministry of Education, Culture, Sports, Science and Technology, 15K21122). T.H. gratefully acknowledges support from the DFG (DIP project cooperation “Nanoengineered optoelectronics with biomaterials and bioinspired assemblies”) and the Volkswagen Foundation (LigH2t). G.K. acknowledges support from CREST, Japan Science and Technology Agency. M.H. acknowledges support from the DFG (Deutsche Forschungsgemeinschaft, HI 739/13-1)

The influence of exogenous organic carbon assimilation and photoperiod on the carbon and lipid metabolism of Chlamydomonas reinhardtii

Microalgae are a promising platform for the production of renewable fuels and oleochemicals. Despite significant research efforts to understand the mechanisms of algal lipid accumulation, the influence of commercially relevant growth conditions on the lipid metabolism is poorly understood. To characterise the impact of differing organic carbon availabilities and photoperiod on the response of the model alga Chlamydomonas reinhardtii to nitrogen stress, the expression of key genes involved in the central carbon metabolism were monitored over a time-course of nitrogen deprivation. In addition, the growth, PSII integrity, chlorophyll content, triacylglycerol (TAG) content, starch content, and fatty acid composition were characterised. Results indicate that both organic carbon availability and photoperiod regulate the lipid accumulation response of C. reinhardtii. Under mixotrophic conditions, organic carbon uptake is favoured over photosynthesis, transcript abundance of lipid synthesis genes rapidly increase and acetate is funnelled to TAG synthesis. In contrast, autotrophic cultures lacking organic carbon experienced a slower rate of photosynthetic degradation and funnelled the majority of sequestered carbon to starch synthesis. Dark periods induced catabolism of both starch and TAG in autotrophic cultures but TAG alone in mixotrophic cultures. Furthermore, diurnal light enhanced starch synthesis under mixotrophic conditions. Finally, transcript analysis indicated that PGD1, important for the routing of oleic acid to TAG, was reliant on organic carbon availability, resulting in reduced C18:1 fatty acid accumulation in autotrophic cultures

Analytical approaches to photobiological hydrogen production in unicellular green algae

Several species of unicellular green algae, such as the model green microalga Chlamydomonas reinhardtii, can operate under either aerobic photosynthesis or anaerobic metabolism conditions. A particularly interesting metabolic condition is that of “anaerobic oxygenic photosynthesis”, whereby photosynthetically generated oxygen is consumed by the cell’s own respiration, causing anaerobiosis in the culture in the light, and induction of the cellular “hydrogen metabolism” process. The latter entails an alternative photosynthetic electron transport pathway, through the oxygen-sensitive FeFe-hydrogenase, leading to the light-dependent generation of molecular hydrogen in the chloroplast. The FeFe-hydrogenase is coupled to the reducing site of photosystem-I via ferredoxin and is employed as an electron-pressure valve, through which electrons are dissipated, thus permitting a sustained electron transport in the thylakoid membrane of photosynthesis. This hydrogen gas generating process in the cells offers testimony to the unique photosynthetic metabolism that can be found in many species of green microalgae. Moreover, it has attracted interest by the biotechnology and bioenergy sectors, as it promises utilization of green microalgae and the process of photosynthesis in renewable energy production. This article provides an overview of the principles of photobiological hydrogen production in microalgae and addresses in detail the process of induction and analysis of the hydrogen metabolism in the cells. Furthermore, methods are discussed by which the interaction of photosynthesis, respiration, cellular metabolism, and H(2) production in Chlamydomonas can be monitored and regulated