Oxygen sensitivity of algal H2- production

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140

The green alga Chlamydomonas reinhardtii has a network of fermentation pathways that become active when cells acclimate to anoxia. Hydrogenase activity is an important component of this metabolism, and we have compared metabolic and regulatory responses that accompany anaerobiosis in wild-type C. reinhardtii cells and a null mutant strain for the HYDEF gene (hydEF-1 mutant), which encodes an [FeFe] hydrogenase maturation protein. This mutant has no hydrogenase activity and exhibits elevated accumulation of succinate and diminished production of CO 2 relative to the parental strain during dark, anaerobic metabolism. In the absence of hydrogenase activity, increased succinate accumulation suggests that the cells activate alternative pathways for pyruvate metabolism, which contribute to NAD(P)H reoxidation, and continued glycolysis and fermentation in the absence of O 2 . Fermentative succinate production potentially proceeds via the formation of malate, and increases in the abundance of mRNAs encoding two malateforming enzymes, pyruvate carboxylase and malic enzyme, are observed in the mutant relative to the parental strain following transfer of cells from oxic to anoxic conditions. Although C. reinhardtii has a single gene encoding pyruvate carboxylase, it has six genes encoding putative malic enzymes. Only one of the malic enzyme genes, MME4, shows a dramatic increase in expression (mRNA abundance) in the hydEF-1 mutant during anaerobiosis. Furthermore, there are marked increases in transcripts encoding fumarase and fumarate reductase, enzymes putatively required to convert malate to succinate. These results illustrate the marked metabolic flexibility of C. reinhardtii and contribute to the development of an informed model of anaerobic metabolism in this and potentially other algae.Chlamydomonas reinhardtii is a unicellular, soil-dwelling, photosynthetic green alga that has a diversity of fermentation pathways, inferred from the full genome sequence (1, 2). It uses these pathways for ATP production during anoxia, catabolizing starch and other intracellular carbon substrates into the predominant fermentation products formate, acetate, ethanol, CO 2 , and molecular hydrogen (H 2 ) in what is classified as heterofermentation (2-7). These metabolic pathways would be active primarily at night when high rates of respiration and the absence of photosynthetic O 2 evolution cause the rapid establishment of anoxia (8), especially in soil environments with high concentrations of microbes. Moreover, C. reinhardtii can balance the use of the tricarboxylic acid cycle with fermentation when the rate of respiratory O 2 consumption exceeds the rate of photosynthetic O 2 evolution (3, 4, 9). The catabolism of intracellular carbon stores during anoxic acclimation is a key component of C. reinhardtii metabolism as this alga does not appear to effectively assimilate extracellular sugars. Acquiring a better understanding of cellular metabolisms in algae such as C. reinhardtii under various conditions will facilitate the development o...

Section: Methodsmentioning

confidence: 99%

Flexibility in Anaerobic Metabolism as Revealed in a Mutant of Chlamydomonas reinhardtii Lacking Hydrogenase Activity

Dubini

Mus

Seibert

et al. 2009

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140

“…Hydrogenase activity was induced anaerobically in the dark, and H 2 photoproduction was monitored the following day. Rates of photosynthesis, respiration, and H 2 photoproduction in liquid cultures were determined as described previously (27).…”

Section: Methodsmentioning

confidence: 99%

“…Anaerobic Induction of Liquid Cell Suspensions-C. reinhardtii cultures were grown on Tris acetate-phosphate medium to ϳ20 g/ml total chlorophyll, centrifuged at 2500 ϫ g for 5 min, and resuspended in one-tenth volume of AIB induction buffer containing 50 mM potassium phosphate, pH 7, and 3 mM MgCl 2 (27). Samples were placed in vials that were wrapped with aluminum foil to exclude light, sealed with a rubber septum, flushed with argon for 15 min, and then incubated anaerobically in the dark at room temperature.…”

Section: Methodsmentioning

confidence: 99%

Discovery of Two Novel Radical S-Adenosylmethionine Proteins Required for the Assembly of an Active [Fe] Hydrogenase

Posewitz

King

Smolinski

et al. 2004

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378

385

To identify genes necessary for the photoproduction of H 2 in Chlamydomonas reinhardtii, random insertional mutants were screened for clones unable to produce H 2 . One of the identified mutants, denoted hydEF-1, is incapable of assembling an active [Fe] hydrogenase. Although the hydEF-1 mutant transcribes both hydrogenase genes and accumulates full-length hydrogenase protein, H 2 production activity is not observed. The HydEF protein contains two unique domains that are homologous to two distinct prokaryotic proteins, HydE and HydF, which are found exclusively in organisms containing [Fe] hydrogenase. In the C. reinhardtii genome, the HydEF gene is adjacent to another hydrogenase-related gene, HydG. Hydrogen has enormous potential to serve as a non-polluting fuel, alleviating the environmental and political concerns associated with fossil energy utilization. Among the most efficient H 2 -generating catalysts known are the [Fe] hydrogenase enzymes, which are found in numerous microorganisms, including the photosynthetic green alga Chlamydomonas reinhardtii (1, 2). Our research, aimed at identifying accessory proteins required for H 2 production in C. reinhardtii, has resulted in the discovery of two [Fe] hydrogenase assembly genes. These genes are essential for the formation of an active [Fe] hydrogenase and are conserved among sequenced organisms containing [Fe] hydrogenase enzymes.Algal H 2 production was first reported by Hans Gaffron and co-workers in seminal experiments performed over 60 years ago (3). Photosynthetic green algae are unique in that H 2 production by [Fe] hydrogenases is coupled directly to water oxidation through photosystem II and the photosynthetic electron transport chain, providing the means to generate H 2 using sunlight. In the past 5 years, great strides have been made in sustaining H 2 photoproduction activity in C. reinhardtii (4, 5), and intensive research continues to probe novel ways to use this and other photosynthetic organisms to generate renewable H 2 .Hydrogenases containing metallo-catalytic clusters occur as [NiFe] or [Fe]-only enzymes (2, 6, 7). These two forms are phylogenetically distinct (8), which suggests that hydrogenase function is the result of convergent evolution (2). Although [NiFe] and [Fe] hydrogenases are genetically unrelated, similarities between the proteins do exist. First, the active sites of both enzymes contain CO and CN ligands, and, second, each active site contains a binuclear metal center. In general, [NiFe] hydrogenases catalyze H 2 uptake, whereas [Fe] hydrogenases tend to catalyze H 2 evolution (9). Furthermore, the turnover number of [Fe] hydrogenases is 10 -100 times higher than that of [NiFe] hydrogenases (9), making the former one of the most efficient H 2 production catalysts known. Hydrogenases containing NiFeSe (10) and a unique class of hydrogenases containing a single Fe atom bound to a co-factor have also been reported (11).The [NiFe] hydrogenases are found in a variety of anaerobic and facultative archaea, eubacteria, a...

“…Anaerobically maintained Chlamydomonas generates H 2 in the light or dark through photosynthetic and fermentative pathways, respectively. Transient H 2 photoproduction is observed after illumination of dark, anaerobically acclimated cells grown in nutrient-replete medium (4,21,23,24,26,42). However, H 2 production rates rapidly diminish as the O 2 , generated from photosynthesis increases to inhibitory levels.…”

mentioning

confidence: 98%

“…This is achieved in the dark by sparging cultures with an inert gas (or exogenous reductant) to purge them of O 2 (23)(24)(25)(26) or in the light by depriving illuminated, sealed cultures of sulfate (9,(27)(28)(29), which results in attenuated rates of photosynthetic O 2 evolution (30). In the dark, fermentation is coupled to the degradation of starch reserves (23,31,32).…”

mentioning

confidence: 99%

Anaerobic Acclimation in Chlamydomonas reinhardtii

Mus

Dubini

Seibert

et al. 2007

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279

156

Both prokaryotic and eukaryotic photosynthetic microbes experience conditions of anoxia, especially during the night when photosynthetic activity ceases. In Chlamydomonas reinhardtii, dark anoxia is characterized by the activation of an extensive set of fermentation pathways that act in concert to provide cellular energy, while limiting the accumulation of potentially toxic fermentative products. Metabolite analyses, quantitative PCR, and high density Chlamydomonas DNA microarrays were used to monitor changes in metabolite accumulation and gene expression during acclimation of the cells to anoxia. Elevated levels of transcripts encoding proteins associated with the production of H 2 , organic acids, and ethanol were observed in congruence with the accumulation of fermentation products. The levels of over 500 transcripts increased significantly during acclimation of the cells to anoxic conditions. Among these were transcripts encoding transcription/translation regulators, prolyl hydroxylases, hybrid cluster proteins, proteases, transhydrogenase, catalase, and several putative proteins of unknown function. Overall, this study uses metabolite, genomic, and transcriptome data to provide genome-wide insights into the regulation of the complex metabolic networks utilized by Chlamydomonas under the anaerobic conditions associated with H 2 production.