RESUMEN
A naturally occurring multispecies bacterial community composed of Bacillus cereus and two novel bacteria (Microbacterium forte sp. nov. and Stenotrophomonas goyi sp. nov.) has been identified from a contaminated culture of the microalga Chlamydomonas reinhardtii. When incubated in mannitol- and yeast extract-containing medium, this bacterial community can promote and sustain algal hydrogen production up to 313 mL H2·L-1 for 17 days and 163.5 mL H2·L-1 for 25 days in high-cell (76.7 µg·mL-1 of initial chlorophyll) and low-cell density (10 µg·mL-1 of initial chlorophyll) algal cultures, respectively. In low-cell density algal cultures, hydrogen production was compatible with algal growth (reaching up to 60 µg·mL-1 of chlorophyll). Among the bacterial community, M. forte sp. nov. was the sole responsible for the improvement in hydrogen production. However, algal growth was not observed in the Chlamydomonas-M. forte sp. nov. consortium during hydrogen-producing conditions (hypoxia), suggesting that the presence of B. cereus and S. goyi sp. nov. could be crucial to support the algal growth during hypoxia. Still, under nonhydrogen producing conditions (aerobiosis) the Chlamydomonas-M. forte sp. nov. consortium allowed algal growth (up to 40 µg·mL-1 of chlorophyll) and long-term algal viability (>45 days). The genome sequence and growth tests of M. forte sp. nov. have revealed that this bacterium is auxotroph for biotin and thiamine and unable to use sulfate as sulfur source; it requires S-reduced forms such as cysteine and methionine. Cocultures of Chlamydomonas reinhardtii and M. forte sp. nov. established a mutualistic association: the alga complemented the nutrient deficiencies of the bacterium, while the bacterium released ammonium (0.19 mM·day-1) and acetic acid (0.15 mM·day-1) for the alga. This work offers a promising avenue for photohydrogen production concomitant with algal biomass generation using nutrients not suitable for mixotrophic algal growth.
Asunto(s)
Chlamydomonas reinhardtii , Chlamydomonas , Microbacterium , Clorofila , Ácido Acético , Bacterias , Hipoxia , HidrógenoRESUMEN
Background: A culture of the green algae Chlamydomonas reinhardtii was accidentally contaminated with three different bacteria in our laboratory facilities. This contaminated alga culture showed increased algal biohydrogen production. These three bacteria were independently isolated. Methods: The chromosomic DNA of one of the isolated bacteria was extracted and sequenced using PacBio technology. Tentative genome annotation (RAST server) and phylogenetic trees analysis (TYGS server) were conducted. Diverse growth tests were assayed for the bacterium and for the alga-bacterium consortium. Results: Phylogenetic analysis indicates that the bacterium is a novel member of the Stenotrophomonas genus that has been termed in this work as S. goyi sp. nov. A fully sequenced genome (4,487,389 base pairs) and its tentative annotation (4,147 genes) are provided. The genome information suggests that S. goyi sp. nov. is unable to use sulfate and nitrate as sulfur and nitrogen sources, respectively. Growth tests have confirmed the dependence on the sulfur-containing amino acids methionine and cysteine. S. goyi sp. nov. and Chlamydomonas reinhardtii can establish a mutualistic relationship when cocultured together. Conclusions: S. goyi sp. nov. could be of interest for the design of biotechnological approaches based on the use of artificial microalgae-bacteria multispecies consortia that take advantage of the complementary metabolic capacities of their different microorganisms.
Asunto(s)
Chlamydomonas reinhardtii , Chlamydomonas reinhardtii/genética , Stenotrophomonas , Filogenia , Bacterias/genética , Azufre/metabolismoRESUMEN
In the context of algal wastewater bioremediation, this study has identified a novel consortium formed by the bacterium Methylobacterium oryzae and the microalga Chlamydomonas reinhardtii that greatly increase biomass generation (1.22 g L-1·d-1), inorganic nitrogen removal (>99%), and hydrogen production (33 mL·L-1) when incubated in media containing ethanol and methanol. The key metabolic aspect of this relationship relied on the bacterial oxidation of ethanol to acetate, which supported heterotrophic algal growth. However, in the bacterial monocultures the acetate accumulation inhibited bacterial growth. Moreover, in the absence of methanol, ethanol was an unsuitable carbon source and its incomplete oxidation to acetaldehyde had a toxic effect on both the alga and the bacterium. In cocultures, both alcohols were used as carbon sources by the bacteria, the inhibitory effects were overcome and both microorganisms mutually benefited. Potential biotechnological applications in wastewater treatment, biomass generation and hydrogen production are discussed.
Asunto(s)
Chlamydomonas , Acetatos , Biomasa , Carbono , Chlamydomonas/metabolismo , Desnitrificación , Etanol , Hidrógeno , Metanol , Methylobacterium , Nitrógeno/metabolismoRESUMEN
Biological hydrogen production by microalgae is a potential sustainable, renewable and clean source of energy. However, many barriers limiting photohydrogen production in these microorganisms remain unsolved. In order to explore this potential and make biohydrogen industrially affordable, the unicellular microalga Chlamydomonas reinhardtii is used as a model system to solve barriers and identify new approaches that can improve hydrogen production. Recently, Chlamydomonas-bacteria consortia have opened a new window to improve biohydrogen production. In this study, we review the different consortia that have been successfully employed and analyze the factors that could be behind the improved H2 production.
Asunto(s)
Bacterias/metabolismo , Chlamydomonas reinhardtii/metabolismo , Hidrógeno/metabolismo , Absorción de Radiación , Oxígeno/metabolismo , Luz SolarRESUMEN
Ferredoxin5 (FDX5), a minor ferredoxin protein in the alga Chlamydomonas (Chlamydomonas reinhardtii), helps maintain thylakoid membrane integrity in the dark. Sulfur (S) deprivation has been used to achieve prolonged hydrogen production in green algae. Here, we propose that FDX5 is involved in algal responses to S-deprivation as well as to the dark. Specifically, we tested the role of FDX5 in both the initial aerobic and subsequent anaerobic phases of S-deprivation. Under S-deprived conditions, absence of FDX5 causes a distinct delay in achieving anoxia by affecting photosynthetic O2 evolution, accompanied by reduced acetate uptake, lower starch accumulation, and delayed/lower fermentative metabolite production, including photohydrogen. We attribute these differences to transcriptional and/or posttranslational regulation of acetyl-CoA synthetase and ADP-Glc pyrophosphorylase, and increased stability of the PSII D1 protein. Interestingly, increased levels of FDX2 and FDX1 were observed in the mutant under oxic, S-replete conditions, strengthening our previously proposed hypothesis that other ferredoxins compensate in response to a lack of FDX5. Taken together, the results of our omics and pull-down experiments confirmed biochemical and physiological results, suggesting that FDX5 may have other effects on Chlamydomonas metabolism through its interaction with multiple redox partners.
Asunto(s)
Chlamydomonas reinhardtii/metabolismo , Ferredoxinas/metabolismo , Azufre/metabolismo , Chlamydomonas reinhardtii/genética , Clorofila/metabolismo , Fermentación , Ferredoxinas/genética , Expresión Génica , Metaboloma , Oxígeno/metabolismo , Almidón/metabolismoRESUMEN
This study is a proof of concept for the synergetic biohydrogen production in alga-bacteria co-cultures. Algal hydrogen photoproduction was obtained in sugar-containing media only when the green alga Chlamydomonas reinhardtii was co-cultured with Pseudomonas putida (40.8â¯ml H2·L-1), Escherichia coli (35.1â¯ml H2·L-1) and Rhizobium etli (16.1â¯ml H2·L-1). Hydrogen photo-production in these co-cultures was not only linked to the induction of hypoxia, but to the ability of the bacteria to produce acetic acid from sugars. Synergetic hydrogen production was achieved by integrating the photobiological and fermentative production in Chlamydomonas and Escherichia coli co-cultures supplemented with glucose, which resulted in 60% more H2 production than the sum of the respective monocultures. This cooperation relied on the ability of the alga to consume the excreted bacterial acetic acid, which benefited both bacterial and algal hydrogen production. This knowledge may open new possibilities for the biohydrogen production from industrial wastes.
Asunto(s)
Chlamydomonas reinhardtii , Chlamydomonas , Ácido Acético , Bacterias , Técnicas de Cocultivo , HidrógenoRESUMEN
BACKGROUND: A recent Commentary article entitled "On the pathways feeding the H2 production process in nutrient-replete, hypoxic conditions" by Dr. Scoma and Dr. Tóth, Biotechnology for Biofuels (2017), opened a very interesting debate about the H2 production photosynthetic-linked pathways occurring in Chlamydomonas cultures grown in acetate-containing media and incubated under hypoxia/anoxia conditions. This Commentary article mainly focused on the results of our previous article "Low oxygen levels contribute to improve photohydrogen production in mixotrophic non-stressed Chlamydomonas cultures," by Jurado-Oller et al., Biotechnology for Biofuels (7, 2015; 8:149). MAIN BODY: Here, we review some previous knowledge about the H2 production pathways linked to photosynthesis in Chlamydomonas, especially focusing on the role of the PSII-dependent and -independent pathways in acetate-containing nutrient-replete cultures. The potential contributions of these pathways to H2 production under anoxia/hypoxia are discussed. CONCLUSION: Despite the fact that the PSII inhibitor DCMU is broadly used to discern between the two different photosynthetic pathways operating under H2 production conditions, its use may lead to distinctive conclusions depending on the growth conditions. The different potential sources of reductive power needed for the PSII-independent H2 production in mixotrophic nutrient-replete cultures are a matter of debate and conclusive evidences are still missing.
RESUMEN
The green alga Chlamydomonas reinhardtii contains six plastidic [2Fe2S]-cluster ferredoxins (FDXs), with FDX1 as the predominant isoform under photoautotrophic growth. FDX2 is highly similar to FDX1 and has been shown to interact with specific enzymes (such as nitrite reductase), as well as to share interactors with FDX1, such as the hydrogenases (HYDA), ferredoxin:NAD(P) reductase I (FNR1), and pyruvate:ferredoxin oxidoreductase (PFR1), albeit performing at low catalytic rates. Here we report the FDX2 crystal structure solved at 1.18 Å resolution. Based on differences between the Chlorella fusca FDX1 and C. reinhardtii FDX2 structures, we generated and purified point-mutated versions of the FDX2 protein and assayed them in vitro for their ability to catalyze hydrogen and NADPH photo-production. The data show that structural differences at two amino acid positions contribute to functional differences between FDX1 and FDX2, suggesting that FDX2 might have evolved from FDX1 toward a different physiological role in the cell. Moreover, we demonstrate that the mutations affect both the midpoint potentials of the FDX and kinetics of the FNR reaction, possibly due to altered binding between FDX and FNR. An effect on H2 photo-production rates was also observed, although the kinetics of the reaction were not further characterized.
Asunto(s)
Chlamydomonas reinhardtii/química , Ferredoxinas/química , Ferredoxinas/metabolismo , Sitios de Unión , Dominio Catalítico , Cristalografía por Rayos X , Escherichia coli/genética , Ferredoxina-NADP Reductasa/metabolismo , Ferredoxinas/genética , Hidrógeno/metabolismo , Modelos Moleculares , Mutagénesis Sitio-Dirigida , NADP/metabolismo , Oxidación-Reducción , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Homología de Secuencia de Aminoácido , Homología Estructural de ProteínaRESUMEN
Photosynthetic microorganisms typically have multiple isoforms of the electron transfer protein ferredoxin, although we know little about their exact functions. Surprisingly, a Chlamydomonas reinhardtii mutant null for the ferredoxin-5 gene (FDX5) completely ceased growth in the dark, with both photosynthetic and respiratory functions severely compromised; growth in the light was unaffected. Thylakoid membranes in dark-maintained fdx5 mutant cells became severely disorganized concomitant with a marked decrease in the ratio of monogalactosyldiacylglycerol to digalactosyldiacylglycerol, major lipids in photosynthetic membranes, and the accumulation of triacylglycerol. Furthermore, FDX5 was shown to physically interact with the fatty acid desaturases CrΔ4FAD and CrFAD6, likely donating electrons for the desaturation of fatty acids that stabilize monogalactosyldiacylglycerol. Our results suggest that in photosynthetic organisms, specific redox reactions sustain dark metabolism, with little impact on daytime growth, likely reflecting the tailoring of electron carriers to unique intracellular metabolic circuits under these two very distinct redox conditions.
Asunto(s)
Chlamydomonas reinhardtii/enzimología , Ácido Graso Desaturasas/metabolismo , Ferredoxinas/metabolismo , Galactolípidos/metabolismo , Proteínas de Plantas/metabolismo , Tilacoides/metabolismo , Chlamydomonas reinhardtii/genética , Ácido Graso Desaturasas/genética , Ferredoxinas/genética , Galactolípidos/genética , Oxidación-Reducción , Proteínas de Plantas/genética , Tilacoides/genéticaRESUMEN
BACKGROUND: Currently, hydrogen fuel is derived mainly from fossil fuels, but there is an increasing interest in clean and sustainable technologies for hydrogen production. In this context, the ability of some photosynthetic microorganisms, particularly cyanobacteria and microalgae, to produce hydrogen is a promising alternative for renewable, clean-energy production. Among a diverse array of photosynthetic microorganisms able to produce hydrogen, the green algae Chlamydomonas reinhardtii is the model organism widely used to study hydrogen production. Despite the well-known fact that acetate-containing medium enhances hydrogen production in this algae, little is known about the precise role of acetate during this process. RESULTS: We have examined several physiological aspects related to acetate assimilation in the context of hydrogen production metabolism. Measurements of oxygen and CO2 levels, acetate uptake, and cell growth were performed under different light conditions, and oxygenic regimes. We show that oxygen and light intensity levels control acetate assimilation and modulate hydrogen production. We also demonstrate that the determination of the contribution of the PSII-dependent hydrogen production pathway in mixotrophic cultures, using the photosynthetic inhibitor DCMU, can lead to dissimilar results when used under various oxygenic regimes. The level of inhibition of DCMU in hydrogen production under low light seems to be linked to the acetate uptake rates. Moreover, we highlight the importance of releasing the hydrogen partial pressure to avoid an inherent inhibitory factor on the hydrogen production. CONCLUSION: Low levels of oxygen allow for low acetate uptake rates, and paradoxically, lead to efficient and sustained production of hydrogen. Our data suggest that acetate plays an important role in the hydrogen production process, during non-stressed conditions, other than establishing anaerobiosis, and independent of starch accumulation. Potential metabolic pathways involved in hydrogen production in mixotrophic cultures are discussed. Mixotrophic nutrient-replete cultures under low light are shown to be an alternative for the simultaneous production of hydrogen and biomass.
RESUMEN
Oxygenic photosynthetic organisms such as green algae are capable of absorbing sunlight and converting the chemical energy into hydrogen gas. This process takes advantage of the photosynthetic apparatus of these organisms which links water oxidation to H2 production. Biological H2 has therefore the potential to be an alternative fuel of the future and shows great promise for generating large scale sustainable energy. Microalgae are able to produce H2 under light anoxic or dark anoxic condition by activating 3 different pathways that utilize the hydrogenases as catalysts. In this review, we highlight the principal barriers that prevent hydrogen production in green algae and how those limitations are being addressed, through metabolic and genetic engineering. We also discuss the major challenges and bottlenecks facing the development of future commercial algal photobiological systems for H2 production. Finally we provide suggestions for future strategies and potential new techniques to be developed towards an integrated system with optimized hydrogen production.
Asunto(s)
Biocombustibles , Chlorophyta/metabolismo , Ingeniería Genética/métodos , Hidrógeno/metabolismo , Microalgas/metabolismo , Fotosíntesis/fisiología , Chlorophyta/genética , Hidrogenasas/metabolismo , Microalgas/genéticaRESUMEN
Chlamydomonas reinhardtii is well adapted to survive under different environmental conditions due to the unique flexibility of its metabolism. Here we report metabolic pathways that are active during acclimation to anoxia, but were previously not thoroughly studied under dark, anoxic H2-producing conditions in this model green alga. Proteomic analyses, using 2D-differential in-gel electrophoresis in combination with shotgun mass fingerprinting, revealed increased levels of proteins involved in the glycolytic pathway downstream of 3-phosphoglycerate, the glyoxylate pathway, and steps of the tricarboxylic acid (TCA) reactions. Upregulation of the enzyme, isocitrate lyase (ICL), was observed, which was accompanied by increased intracellular succinate levels, suggesting the functioning of glyoxylate pathway reactions. The ICL-inhibitor study revealed presence of reverse TCA reactions under these conditions. Contributions of the serine-isocitrate lyase pathway, glycine cleavage system, and c1-THF/serine hydroxymethyltransferase pathway in the acclimation to dark anoxia were found. We also observed increased levels of amino acids (AAs) suggesting nitrogen reorganization in the form of de novo AA biosynthesis during anoxia. Overall, novel routes for reductant utilization, in combination with redistribution of carbon and nitrogen, are used by this alga during acclimation to O2 deprivation in the dark.
Asunto(s)
Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Perfilación de la Expresión Génica/métodos , Hidrógeno/metabolismo , Metabolómica/métodos , Proteómica/métodos , Proteínas Algáceas/genética , Proteínas Algáceas/metabolismo , Aminoácidos/metabolismo , Anaerobiosis , Western Blotting , Carbono/metabolismo , Oscuridad , Electroforesis en Gel Bidimensional , Cromatografía de Gases y Espectrometría de Masas , Espectrometría de Masas , Redes y Vías Metabólicas/genética , Metaboloma , Nitrógeno/metabolismo , Proteoma/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Ferredoxins (FDXs) can distribute electrons originating from photosynthetic water oxidation, fermentation, and other reductant-generating pathways to specific redox enzymes in different organisms. The six FDXs identified in Chlamydomonas reinhardtii are not fully characterized in terms of their biological function. In this report, we present data from the following: (a) yeast two-hybrid screens, identifying interaction partners for each Chlamydomonas FDX; (b) pairwise yeast two-hybrid assays measuring FDX interactions with proteins from selected biochemical pathways; (c) affinity pulldown assays that, in some cases, confirm and even expand the interaction network for FDX1 and FDX2; and (d) in vitro NADP(+) reduction and H2 photo-production assays mediated by each FDX that verify their role in these two pathways. Our results demonstrate new potential roles for FDX1 in redox metabolism and carbohydrate and fatty acid biosynthesis, for FDX2 in anaerobic metabolism, and possibly in state transition. Our data also suggest that FDX3 is involved in nitrogen assimilation, FDX4 in glycolysis and response to reactive oxygen species, and FDX5 in hydrogenase maturation. Finally, we provide experimental evidence that FDX1 serves as the primary electron donor to two important biological pathways, NADPH and H2 photo-production, whereas FDX2 is capable of driving these reactions at less than half the rate observed for FDX1.
Asunto(s)
Proteínas Algáceas/metabolismo , Chlamydomonas reinhardtii/metabolismo , Ferredoxinas/metabolismo , Proteínas Algáceas/genética , Chlamydomonas reinhardtii/genética , Ferredoxinas/genética , Hidrogenasas/metabolismo , Modelos Biológicos , NADP/metabolismo , Oxidación-Reducción , Mapas de Interacción de Proteínas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Técnicas del Sistema de Dos HíbridosRESUMEN
Hydrogenases are metalloenzymes that catalyze 2H(+) + 2e(-) â H(2). A multisubunit, bidirectional [NiFe]-hydrogenase has been identified and characterized in a number of bacteria, including cyanobacteria, where it is hypothesized to function as an electron valve, balancing reductant in the cell. In cyanobacteria, this Hox hydrogenase consists of five proteins in two functional moieties: a hydrogenase moiety (HoxYH) with homology to heterodimeric [NiFe]-hydrogenases and a diaphorase moiety (HoxEFU) with homology to NuoEFG of respiratory Complex I, linking NAD(P)H â NAD(P)(+) as a source/sink for electrons. Here, we present an extensive study of Hox hydrogenase in the cyanobacterium Synechocystis sp. PCC 6803. We identify the presence of HoxEFUYH, HoxFUYH, HoxEFU, HoxFU, and HoxYH subcomplexes as well as association of the immature, unprocessed large subunit (HoxH) with other Hox subunits and unidentified factors, providing a basis for understanding Hox maturation and assembly. The analysis of mutants containing individual and combined hox gene deletions in a common parental strain reveals apparent alterations in subunit abundance and highlights an essential role for HoxF and HoxU in complex/subcomplex association. In addition, analysis of individual and combined hox mutant phenotypes in a single strain background provides a clear view of the function of each subunit in hydrogenase activity and presents evidence that its physiological function is more complicated than previously reported, with no outward defects apparent in growth or photosynthesis under various growth conditions.
Asunto(s)
Proteínas Bacterianas/metabolismo , Hidrogenasas/metabolismo , Complejos Multienzimáticos/metabolismo , Synechocystis/enzimología , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Eliminación de Gen , Hidrogenasas/química , Hidrogenasas/genética , Complejos Multienzimáticos/química , Complejos Multienzimáticos/genética , Synechocystis/genéticaRESUMEN
Chlamydomonas reinhardtii, a unicellular green alga, often experiences hypoxic/anoxic soil conditions that activate fermentation metabolism. We isolated three Chlamydomonas mutants disrupted for the pyruvate formate lyase (PFL1) gene; the encoded PFL1 protein catalyzes a major fermentative pathway in wild-type Chlamydomonas cells. When the pfl1 mutants were subjected to dark fermentative conditions, they displayed an increased flux of pyruvate to lactate, elevated pyruvate decarboxylation, ethanol accumulation, diminished pyruvate oxidation by pyruvate ferredoxin oxidoreductase, and lowered H(2) production. The pfl1-1 mutant also accumulated high intracellular levels of lactate, succinate, alanine, malate, and fumarate. To further probe the system, we generated a double mutant (pfl1-1 adh1) that is unable to synthesize both formate and ethanol. This strain, like the pfl1 mutants, secreted lactate, but it also exhibited a significant increase in the levels of extracellular glycerol, acetate, and intracellular reduced sugars and a decrease in dark, fermentative H(2) production. Whereas wild-type Chlamydomonas fermentation primarily produces formate and ethanol, the double mutant reroutes glycolytic carbon to lactate and glycerol. Although the metabolic adjustments observed in the mutants facilitate NADH reoxidation and sustained glycolysis under dark, anoxic conditions, the observed changes could not have been predicted given our current knowledge of the regulation of fermentation metabolism.
Asunto(s)
Acetiltransferasas/metabolismo , Alcohol Deshidrogenasa/metabolismo , Chlamydomonas reinhardtii/metabolismo , Fermentación , Acetiltransferasas/genética , Alcohol Deshidrogenasa/genética , Chlamydomonas reinhardtii/enzimología , Chlamydomonas reinhardtii/genética , Etanol/metabolismo , Formiatos/metabolismo , Cromatografía de Gases y Espectrometría de Masas , Hidrógeno/metabolismo , Ácido Láctico/metabolismo , Datos de Secuencia Molecular , Mutagénesis Insercional , Oxidación-Reducción , Ácido Pirúvico/metabolismoRESUMEN
The green alga Chlamydomonas reinhardtii has numerous genes encoding enzymes that function in fermentative pathways. Among these, the bifunctional alcohol/acetaldehyde dehydrogenase (ADH1), highly homologous to the Escherichia coli AdhE enzyme, is proposed to be a key component of fermentative metabolism. To investigate the physiological role of ADH1 in dark anoxic metabolism, a Chlamydomonas adh1 mutant was generated. We detected no ethanol synthesis in this mutant when it was placed under anoxia; the two other ADH homologs encoded on the Chlamydomonas genome do not appear to participate in ethanol production under our experimental conditions. Pyruvate formate lyase, acetate kinase, and hydrogenase protein levels were similar in wild-type cells and the adh1 mutant, while the mutant had significantly more pyruvate:ferredoxin oxidoreductase. Furthermore, a marked change in metabolite levels (in addition to ethanol) synthesized by the mutant under anoxic conditions was observed; formate levels were reduced, acetate levels were elevated, and the production of CO(2) was significantly reduced, but fermentative H(2) production was unchanged relative to wild-type cells. Of particular interest is the finding that the mutant accumulates high levels of extracellular glycerol, which requires NADH as a substrate for its synthesis. Lactate production is also increased slightly in the mutant relative to the control strain. These findings demonstrate a restructuring of fermentative metabolism in the adh1 mutant in a way that sustains the recycling (oxidation) of NADH and the survival of the mutant (similar to wild-type cell survival) during dark anoxic growth.
Asunto(s)
Alcohol Deshidrogenasa/metabolismo , Chlamydomonas reinhardtii/metabolismo , Glicerol/metabolismo , Proteínas de Plantas/metabolismo , Acetato Quinasa/genética , Acetato Quinasa/metabolismo , Acetatos/metabolismo , Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Alcohol Deshidrogenasa/genética , Alcohol Deshidrogenasa/fisiología , Anaerobiosis , Western Blotting , Dióxido de Carbono/metabolismo , Chlamydomonas reinhardtii/enzimología , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/fisiología , Etanol/metabolismo , Fermentación , Formiatos/metabolismo , Genes de Plantas , Hidrógeno/metabolismo , Ácido Láctico/metabolismo , Metaboloma , NAD/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/fisiología , Piruvato-Sintasa/metabolismo , Transcripción GenéticaRESUMEN
Many microbes in the soil environment experience micro-oxic or anoxic conditions for much of the late afternoon and night, which inhibit or prevent respiratory metabolism. To sustain the production of energy and maintain vital cellular processes during the night, organisms have developed numerous pathways for fermentative metabolism. This review discusses fermentation pathways identified for the soil-dwelling model alga Chlamydomonas reinhardtii, its ability to produce molecular hydrogen under anoxic conditions through the activity of hydrogenases, and the molecular flexibility associated with fermentative metabolism that has only recently been revealed through the analysis of specific mutant strains.
Asunto(s)
Chlamydomonas reinhardtii/citología , Chlamydomonas reinhardtii/metabolismo , Hidrógeno/metabolismo , Anaerobiosis , Chlamydomonas reinhardtii/enzimología , Chlamydomonas reinhardtii/genética , Fermentación , Hidrogenasas/química , Hidrogenasas/metabolismo , Mutación/genéticaRESUMEN
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 CO2 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 O2. Fermentative succinate production potentially proceeds via the formation of malate, and increases in the abundance of mRNAs encoding two malate-forming 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.
Asunto(s)
Proteínas Algáceas/metabolismo , Chlamydomonas reinhardtii/enzimología , Endopeptidasas/metabolismo , Hidrogenasas/metabolismo , Proteínas Protozoarias/metabolismo , Ácido Pirúvico/metabolismo , Proteínas Algáceas/genética , Anaerobiosis/fisiología , Animales , Dióxido de Carbono/metabolismo , Chlamydomonas reinhardtii/genética , Endopeptidasas/genética , Hidrogenasas/genética , Malato Deshidrogenasa/genética , Malato Deshidrogenasa/metabolismo , Mutación , NADP/genética , NADP/metabolismo , Oxidación-Reducción , Proteínas Protozoarias/genética , Succinato Deshidrogenasa/genética , Succinato Deshidrogenasa/metabolismo , Ácido Succínico/metabolismoRESUMEN
Hydrogen photoproduction by micro-organisms combines the photosynthetic properties of oxygenic and non-oxygenic microbes with the activity of H2-producing enzymes in nature: hydrogenases and nitrogenases. The overall efficiency of the process depends on the separate efficiencies of photosynthesis and enzymatic catalysis. This tutorial review discusses the biochemical pathways for H2 production in different organisms, barriers to be overcome, and possible suggestions for integrating photobiological H2 production with fermentative, anaerobic systems for a potentially more efficient process.