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

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

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 H2 production in Chlamydomonas can be monitored and regulated

Das anaerobe Leben der photosynthetischen Alge Chlamydomonas reinhardtii\textit {Chlamydomonas reinhardtii}

Die einzellige Grünalge $\textit {Chlamydomonas reinhardtii}$ ist ein pflanzlicher Organismus, der seinen jüngeren Verwandten, den höheren Pflanzen, in vielerlei Hinsicht ähnelt. Jedoch besitzt er Charakteristika, die für einen eukaryontischen photosynthetischen Organismus recht ungewöhnlich sind. Unter Schwefelmangel bilden belichtete $\textit {C. reinhardtii}$-Zellen einen lang anhaltenden anaeroben Stoffwechsel aus, während dessen Wasserstoff durch eine an die Photosynthese gekoppelte Hydrogenase und Formiat durch das bakterien-typische Enzym Pyruvat-Formiat-Lyase gebildet werden. Diese Arbeit gelangt zu verschiedenen neuen Erkenntnissen bezüglich des komplexen Metabolismus unter Schwefelmangel mit Schwerpunkten auf die physiologischen Anpassungen, die in der ersten Phase der Adaptation erfolgen, der Elektronenquelle für die H2-Produktion und die vitale Rolle der H2-Bildung für die Algen. Weiterhin wurde das außergewöhnlich vielfältige Gärungssystem von $\textit {C. reinhardtii}$ genetisch und biochemisch analysiert.The unicellular green alga $\textit {Chlamydomonas reinhardtii}$ is a plant organism that shares many features with its younger relatives, the higher plants. This alga, however, also possesses characteristics that are quite unusual for a eukaryotic photosynthetic organism. It has a complex fermentative metabolism that is marked by the production of hydrogen and formate, catalysed by an iron-hydrogenase coupled to photosynthesis and by the bacterial-type pyruvate formate-lyase, respectively. A sustained anaerobic metabolism is established when $\textit {C. reinhardtii}$ cells are deprived of sulphur. This study provides deeper insights into this complex metabolism. It arrives at several new conclusions regarding metabolic adjustments that occur in the initial phase of sulphur deprivation, the electron source for hydrogen production and the vital role of the hydrogenase pathway. Furthermore, the remarkable multifarious fermentative system of $\textit {C. reinhardtii}$ was characterised in genetic and biochemical details

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 H2 production in Chlamydomonas can be monitored and regulated