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Keywords:

  • anoxia;
  • Chlamydomonas;
  • fermentative metabolism;
  • hydrogen;
  • hydrogenases

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Fermentation metabolism
  5. Hydrogenase in algae
  6. Hydrogenase structure and oxygen sensitivity
  7. Maturation and interactions with other proteins
  8. Mutants in metabolism that impact fermentation pathways
  9. Conclusions
  10. Acknowledgements
  11. References

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.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Fermentation metabolism
  5. Hydrogenase in algae
  6. Hydrogenase structure and oxygen sensitivity
  7. Maturation and interactions with other proteins
  8. Mutants in metabolism that impact fermentation pathways
  9. Conclusions
  10. Acknowledgements
  11. References

Chlamydomonas reinhardtii is a soil-dwelling photosynthetic organism that shares many metabolic features with vascular plants, although it has certain features (e.g. flagella) that were lost during vascular plant evolution. With the sequencing of the C. reinhardtii nuclear genome (Merchant et al., 2007) and the development of molecular tools applicable to studies of this alga (Harris, 2001; Grossman, 2007; Purton, 2007), and because of its ability to grow on a fixed carbon source in the absence of photosynthesis, C. reinhardtii has become an attractive reference system for dissecting various biological processes including flagellar function and assembly, cell cycle control, photosynthesis, chloroplast biogenesis and acclimation to changing nutrient conditions. Moreover, this green alga can synthesize molecular hydrogen (H2) under anoxic conditions, which suggests its potential use for biofuel production (Melis & Happe, 2001, 2004; Ghirardi et al., 2007).

Fermentation metabolism

  1. Top of page
  2. Summary
  3. Introduction
  4. Fermentation metabolism
  5. Hydrogenase in algae
  6. Hydrogenase structure and oxygen sensitivity
  7. Maturation and interactions with other proteins
  8. Mutants in metabolism that impact fermentation pathways
  9. Conclusions
  10. Acknowledgements
  11. References

In the soil environment, photosynthetic microbes (prokaryotic and eukaryotic) experience hypoxic and anoxic conditions, mainly during the night, when photosynthetic O2 evolution stops and environmental O2 concentrations dramatically decline as a consequence of respiration by various microbes (including C. reinhardtii). However, fermentation can also occur in the light under anoxic conditions (to be further described in this section and in the section designated ‘Hydrogenases in algae’). Several studies have demonstrated that C. reinhardtii rapidly acclimates to anoxia by shifting from aerobic to fermentative metabolism (Gfeller & Gibbs, 1984, 1985; Kreuzberg, 1984; Gibbs et al., 1986; Ohta et al., 1987); genes encoding proteins associated with a diverse set of fermentative pathways have been identified in the C. reinhardtii genome (Grossman et al., 2007; Merchant et al., 2007). Terashima and colleagues recently examined the induction of proteins in C. reinhardtii in response to anaerobiosis (Terashima et al., 2010). While many of the changes observed were expected (e.g. proteins associated with fermentation metabolism), the authors localized a number of these proteins to different cellular compartments and also noted a set of novel proteins of unknown function that respond to anaerobic conditions. When anoxia is triggered in C. reinhardtii in the light under sulfur deprivation conditions (Tsygankov et al., 2002; Zhang et al., 2002), there are major changes in the accumulation of metabolites, including increases in the production of triglycerides and amino acids (Timmins et al., 2009b). In contrast to C. reinhardtii, most vascular plants are not tolerant to anaerobiosis (even though they often can perform fermentation metabolism), although some species such as rice (Oryza sativa) have developed multiple mechanisms to cope with low-oxygen conditions (Magneschi & Perata, 2009).

Based on both experimental and genome information, it is known that C. reinhardtii has a variety of metabolic pathways that enable it to acclimate to hypoxic and anoxic conditions (Tsygankov et al., 2002; Hemschemeier & Happe, 2005; Atteia et al., 2006; Mus et al., 2007; Dubini et al., 2009; Timmins et al., 2009a). Pyruvate, a metabolite generated by glycolysis, is a substrate for a number of fermentation circuits used by C. reinhardtii (Fig. 1), many of which have been detected in other algae and cyanobacteria upon exposure to anoxic conditions. The activities of these circuits are manifested by secretion of organic acids (formate, lactate, malate, acetate and succinate) and alcohols (ethanol and glycerol), and the evolution of H2 and CO2 (Gfeller & Gibbs, 1984; Kreuzberg, 1984; Ohta et al., 1987; Tsygankov et al., 2002; Kosourov et al., 2003; Mus et al., 2007; Dubini et al., 2009). In the environment, many of the fermentation by-products supply heterotrophic microbes with organic compounds and reductant for growth and development.

image

Figure 1. Fermentation pathways present in Chlamydononas reinhardtii cells exposed to dark anoxic conditions. Shown are mutants that we have isolated (green circles; C. Catalanotti, unpublished). Note that the metabolic pathway leading to fermentative succinate production (within the rectangular box delineated with broken lines) only becomes prominent in the hydEF-1 mutant (Dubini et al., 2009). The genes designated are: PEPC, phosphenolpyruvate carboxylase; PYC, pyruvate carboxylase; MME, malic enzyme; MDH, malate dehydrogenase; FUM, fumarase; FMR, fumarate reductase; LDH, lactate dehydrogenase; PDC, pyruvate decarboxylase; ADH2, acetaldehydealcohol dehydrogenase; ADH1, alcohol dehydrogenase; PFL1, pyruvate-formate lyase; PFR1, pyruvate-FDX oxidoreductase; PAT, phosphate acetyltransferase; ACK, acetate kinase; FDX, ferredoxin; HYDA, hydrogenase. In some cases, there are multiple genes encoding distinct enzyme isoforms; the specific gene is indicated by the number at the end of the gene designation (e.g. PDC3 and MME4).

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During dark fermentation, cellular carbohydrate reserves are metabolized to generate ATP, while the reduced pyridine nucleotide that is co-produced with the ATP must be reoxidized to sustain the activity of the fermentation pathways. This reoxidation is linked to both organic acid and H2 formation (Gfeller & Gibbs, 1984; Kreuzberg, 1984; Ohta et al., 1987). A schematic of the various fermentation pathways that operate in C. reinhardtii is presented in Fig. 1. This diagram also shows the range of mutants that we have recently isolated in the various branches of fermentation metabolism (Dubini et al., 2009; C. Catalanotti, unpublished) as well as those for which screens are currently being performed. Often, fixed carbon used for fermentation is derived from starch, which accumulates in cells over the course of the day as a consequence of photosynthetic CO2 fixation. During the evening, much of the starch reserve can be hydrolysed to sugars by amylase activity, and the sugar is converted to pyruvate by glycolysis. Pyruvate is the primary metabolite that fuels fermentation processes, serving as substrate for pathways that generate organic acids, acetyl-CoA, ethanol, CO2 and H2. Two key pyruvate-consuming enzymes encoded in the C. reinhardtii genome are pyruvate formate lyase 1 (PFL1) and pyruvate:ferredoxin:oxidoreductase 1 (PFR1, sometimes designated PFOR). Activity and immunological analyses suggest that PFL1 is located in both mitochondria and chloroplasts (Kreuzberg et al., 1987; Atteia et al., 2006). In the PFL1 reaction, pyruvate is cleaved to acetyl-CoA and formate, while in the PFR1 reaction it is converted to acetyl-CoA, CO2 and reduced ferredoxin (FDX). Hence, the PFR1 reaction oxidizes pyruvate with the concomitant generation of two molecules of reduced FDX, which can be used by the hydrogenases to generate H2 (Müller, 2003), while the PFL1 reaction catalyses the nonoxidative conversion of pyruvate to acetyl-CoA and formate (Wagner et al., 1992). Reduced FDX can be reoxidized by several redox enzymes in addition to hydrogenases; these include nitrite and sulfate/sulfite reductases (Ghirardi et al., 2008). A recent study has demonstrated that PFL1 and PFR1 activities occur concomitantly, with the two enzymes competing for the same substrate (Mus et al., 2007). This finding suggests the potential for rerouting of fermentative electron flow in C. reinhardtii towards PFR1-dependent H2 production. Such a possibility could be tested by disrupting specific pathways in fermentation metabolism (e.g. eliminating the PFL1 reaction) to potentially boost the H2 generation rate. The acetyl-CoA produced by the PFL1 and PFR1 reactions (Fig. 1) is either reduced to ethanol by alcohol/aldehyde dehydrogenase 1 (ADH1) (Hemschemeier & Happe, 2005; Atteia et al., 2006; Dubini et al., 2009), which is thought to be located in mitochondria (Atteia et al., 2006), or metabolized to acetate by the phospho-acetyl transferase (PAT) and acetate kinase (ACK) reactions (Atteia et al., 2006); these reactions appear to occur in both mitochondria (PAT1 and ACK2) and chloroplasts (PAT2 and ACK1). An alternative pathway for ethanol production may be the direct decarboxylation of pyruvate to CO2 and acetaldehyde through the action of pyruvate decarboxylase 3 (PDC3). The acetaldehyde generated in this reaction can be reduced to ethanol by a second ADH enzyme (ADH2), although the identity of this enzyme has not been clearly established (Mus et al., 2007). While the conversion of acetyl-CoA to ethanol by ADH1 oxidizes two NADH molecules, only a single NADH is oxidized in the PDC pathway.

Transcripts encoding many of the enzymes involved in fermentation are elevated in C. reinhardtii during anoxia. The PFR1 transcript and PFR1 protein both increase dramatically at the onset of anoxia (Mus et al., 2007; C. Catalanotti, unpublished data). By contrast, anoxia triggers an increase in PFL1 mRNA with no increase in the PFL1 protein (Atteia et al., 2006). Interestingly, the molecular mass of PFL1 from anoxic cells was slightly less than that from cells maintained in oxic conditions, suggesting that anoxic conditions trigger a post-translational modification that may lead to activation of the protein (Atteia et al., 2006). Transcripts of genes coding for ADH1, PAT1, PAT2, ACK1 and ACK2 are also up-regulated at the onset of anoxia (Mus et al., 2007). Although few studies have been performed to elucidate regulation of genes responsive to anoxic conditions, the region 21–128 nucleotides upstream of the hydrogenase 1 gene (HYDA1) transcription start site was shown to be involved in controlling expression of the hydrogenase genes (Stirnberg & Happe, 2004).

There is still little known about how photosynthetic organisms sense oxic conditions, although the reduction state of the intersystem plastoquinone (PQ) pool (Antal et al., 2003) or the production and detoxification of reactive oxygen species (ROS) may be central for signalling (Bailey-Serres & Chang, 2005; Guzy & Schumacker, 2006; Bell & Chandel, 2007). Hydrogen peroxide (H2O2), synthesized by an NAD(P)H oxidase, is required for controlled induction of ADH expression in Arabidopsis (Baxter-Burrell et al., 2002). In animals, prolyl 4-hydroxylases directly sense O2 and are involved in controlling their responses to anoxia (Guzy & Schumacker, 2006). In Arabidopsis and rice, transcript levels for prolyl 4-hydroxylases are strongly induced by O2 deprivation (Lasanthi-Kudahettige et al., 2007; Vlad et al., 2007), suggesting that their role as sensing elements might be conserved in plants. Interestingly, four prolyl 4-hydroxylases encoded on the C. reinhardtii genome are significantly up-regulated in response to anaerobiosis (Mus et al., 2007); their potential role in sensing O2 is currently being investigated through the generation and phenotypic analysis of insertional mutants (C. Catalanotti, unpublished data). The information presented above suggests that multiple inputs may control the acclimation of organisms to hypoxia/anoxia, and it will be important to determine the precise factors that impact the different responses observed as the cells progress from oxic to hypoxic and finally to anoxic conditions.

Hydrogenase in algae

  1. Top of page
  2. Summary
  3. Introduction
  4. Fermentation metabolism
  5. Hydrogenase in algae
  6. Hydrogenase structure and oxygen sensitivity
  7. Maturation and interactions with other proteins
  8. Mutants in metabolism that impact fermentation pathways
  9. Conclusions
  10. Acknowledgements
  11. References

Over the last 5 yr there have been numerous reviews discussing the structure, function and potential commercial exploitation of hydrogenases (Ghirardi et al., 2007, 2009; Hankamer et al., 2007; Melis et al., 2007; Meyer, 2007; Vignais & Billoud, 2007; Rosenberg et al., 2008; Seibert et al., 2008; Allakhverdiev et al., 2009; Beer et al., 2009; Hemschemeier et al., 2009; Rupprecht, 2009; Stripp & Happe, 2009; Kruse & Hankamer, 2010; Radakovits et al., 2010). Hydrogenase activity is not observed in all genera of green algae (Brand et al., 1989; Boichenko et al., 2004; Melis & Happe, 2004), and the precise physiological function of hydrogenase and H2 synthesis in algal cells is not absolutely resolved. However, H2 production is likely to have significant impacts on redox poising, photoprotection, and fermentative energy production.

Generally, H2 is known to be produced during dark fermentation or by two photoproduction pathways, as shown diagrammatically in Fig. 2. The first of the photoproduction pathways is a consequence of direct biophotolysis and involves the light-dependent oxidation of water by photosystem II (PSII), the transfer of electrons from PSII to photosystem I (PSI), light-dependent excitation of electrons by PSI which causes reduction of FDX (gene designation PETF), and the subsequent transfer of electrons from FDX to the hydrogenases (Benemann et al., 1973; Greenbaum, 1982; Roessler & Lien, 1984; Happe & Naber, 1993; Miura, 1995; Ghirardi et al., 2007; Beer et al., 2009). This pathway can operate under conditions in which the cells have low PSII activity (e.g. sulfur deprivation conditions) (Melis et al., 2000) and relatively high respiration rates, allowing the cultures to become micro-oxic or anoxic (Kosourov et al., 2002; Chochois et al., 2009). When C. reinhardtii cells maintained in acetate-containing medium are deprived of sulfur, PSII activity declines and the cultures become anoxic; batch cultures of C. reinhardtii can sustain H2 production for several days (Ghirardi et al., 2000; Melis et al., 2000). The second photoproduction pathway, indirect biophotolysis, involves nonphotochemical reduction of the PQ pool by NAD(P)H that is generated as a consequence of starch catabolism, followed by light-dependent FDX reduction by PSI, and the subsequent transfer of electrons to hydrogenases (Kosourov et al., 2003; Mus et al., 2005). This pathway in C. reinhardtii depends on NAD(P)H-plastoquinone oxidoreductase (NPQR) activity (Cournac et al., 2000; Mus et al., 2005), and is PSII independent (Kosourov et al., 2003; Chochois et al., 2009). In the third H2-production pathway, starch catabolism provides electrons to the hydrogenases under dark fermentative conditions (Figs 1, 2) (Gfeller & Gibbs, 1984; Kreuzberg, 1984; Ohta et al., 1987; Happe et al., 1994; Ghirardi et al., 1997; Melis & Happe, 2001; Kosourov et al., 2002; Posewitz et al., 2004b; Mus et al., 2007; Dubini et al., 2009).

image

Figure 2. H2 production pathways in Chlamydononas reinhardtii. The two pathways associated with the photoproduction of H2 are photosystem II (PSII)-dependent (red dashed line) or PSII-independent (NAD(P)H-plastoquinone oxidoreductase (NPQR)-dependent; purple dashed line). Both of these pathways require the generation of a reduced plastoquinone (PQ) and plastocyanin (PC) pool, PSI activity (light-blue dashed line), PSI-dependent reduction of ferredoxin (FDX, also PETF; light blue) and the transfer of electrons from FDX to the hydrogenases (pink). A third pathway for H2 production occurs under dark, anoxic conditions (green dashed line); pyruvate oxidation is coupled to FDX reduction through pyruvate-FDX oxidoreductase (PFR1). The electrons from FDX are transferred to the hydrogenases (green line). Electrons used for PSII-independent and dark H2 production are generated by oxidation of organic compounds that are often derived from starch degradation (black).

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H2 photoproduction is significantly influenced by several metabolites. For example, under conditions of limiting CO2, photosynthetic H2 production increases, suggesting that the hydrogenase and CO2 fixation pathways are competing for reductant (Melis et al., 2000). Acetate has been shown to enhance C. reinhardtii H2 photoproduction (Gfeller & Gibbs, 1984; Gibbs et al., 1986; Happe et al., 1994), which may reflect an increased accumulation of cellular carbohydrates that can be oxidized to provide electrons to the photosynthetic electron transport chain at the level of the PQ pool. The reductive utilization of nitrate and nitrite also suppresses H2 photoproduction (Aparicio et al., 1985). The mechanism by which hydrogenase activity is attenuated by nitrate/nitrite is unknown, but probably involves the ability of nitrite reductase, which reduces nitrite to ammonia, to oxidize FDX (Aparicio et al., 1985). FDX-sulfate/sulfite reductases may also compete with the hydrogenases for reductant; however, detailed experiments examining this process have not been reported. The NPQR pathway can be regulated by different electron donors including NAD(P)H (Mus et al., 2005), which may be generated from various reductants that accumulate during anoxia (e.g. reduced ferredoxins and succinate).

The hydrogenases also function in H2 uptake, with two distinct uptake pathways in C. reinhardtii (Gaffron, 1944; Kessler, 1974; Maione & Gibbs, 1986a,b; Chen & Gibbs, 1992). The first pathway, anaerobic CO2 photoreduction, couples H2 oxidation and cyclic PSI activity to RuBisCo-mediated CO2 fixation. Electrons from H2 are used to reduce FDX, which then reduces FDX-NADP oxidoreductase (FNR), leading to NAD(P)H production, which, along with the ATP generated from cyclic electron flow, can be used to fix CO2. H2 can also be taken up in the oxy-hydrogen reaction, which could be coupled to CO2 fixation (Gaffron, 1942, 1944; Kessler, 1974). At low O2 concentrations (below concentrations that inhibit hydrogenase activity), the uptake of both H2 and O2 is observed in the dark (Russell & Gibbs, 1968; Kessler, 1974; Maione & Gibbs, 1986a,b). Maione and Gibbs proposed that FDX is reduced by hydrogenase, which then facilitates reduction of the PQ pool (directly or indirectly) (Maione & Gibbs, 1986a). Electrons in the PQ pool would be used to reduce O2, resulting in the uptake of both H2 and O2. The oxy-hydrogen reaction linked to CO2 reduction has been reported in both the light and the dark (Gaffron & Rubin, 1942; Gaffron, 1944; Maione & Gibbs, 1986a; Chen & Gibbs, 1992). The source of ATP required for CO2 reduction in the dark is probably linked to mitochondrial respiration (Maione & Gibbs, 1986a,b).

Hydrogenase structure and oxygen sensitivity

  1. Top of page
  2. Summary
  3. Introduction
  4. Fermentation metabolism
  5. Hydrogenase in algae
  6. Hydrogenase structure and oxygen sensitivity
  7. Maturation and interactions with other proteins
  8. Mutants in metabolism that impact fermentation pathways
  9. Conclusions
  10. Acknowledgements
  11. References

Two unrelated hydrogenases that catalyse reversible proton reduction and are dominant in microbes are the [NiFe] and [FeFe] hydrogenases (Adams, 1990; Boichenko & Hoffmann, 1994; Frey, 2002). Green algae contain [FeFe] hydrogenases (Ghirardi et al., 2007). Sequencing of genes for algal [FeFe] hydrogenases (Florin et al., 2001; Wunschiers et al., 2001; Happe & Kaminski, 2002; Winkler et al., 2002; Forestier et al., 2003) suggests that most are small, monomeric proteins (45–50 kDa) containing only the H-cluster domain. This domain binds a 4Fe–4S complex co-ordinated with three conserved cysteine residues, and a unique binuclear 2Fe subcluster that is linked to the 4Fe–4S complex by a fourth conserved cysteine (Happe & Naber, 1993; Peters et al., 1998; Nicolet et al., 1999). Terminal CO and CN ligands are bound to each of the Fe atoms of the 2Fe subcluster, while a third CO ligand bridges both of the Fe atoms in this subcluster (Peters et al., 1998; Nicolet et al., 1999) A unique nonprotein dithiolate ligand, postulated to be a dithiomethyl amine, also bridges both of the Fe atoms (Nicolet et al., 2001). Recent spectroscopic evidence is supportive of this hypothesis (Silakov et al., 2009). Two genes encoding [FeFe] hydrogenases, HYDA1 and HYDA2, are present in the C. reinhardtii genome (Happe & Naber, 1993; Happe & Kaminski, 2002; Forestier et al., 2003). The HYDA1 and HYDA2 enzymes (68% identical and 74% similar) both have molecular masses of c. 49 kDa and N-terminal transit peptides that target them to the chloroplast stroma (Happe & Kaminski, 2002; Forestier et al., 2003).

Algal hydrogenases characterized to date are particularly sensitive to O2 and are irreversibly inactivated within minutes after exposure to atmospheric concentrations of O2 (Cohen et al., 2005), with the C. reinhardtii enzymes being the most O2-sensitive among them (Ghirardi et al., 1997; Cohen et al., 2005; King et al., 2006). The green algal hydrogenases may have evolved hypersensitivity to O2 in order to attenuate hydrogenase activity in the presence of O2, when the cells would be able to exploit higher energy-yielding metabolic processes in which O2 would act as the terminal electron acceptor (Posewitz et al., 2008); however, this idea has been discussed previously by many groups on an informal basis. Recent studies suggest that O2 inhibition of C. reinhardtii HYDA1 activity involves binding of the O2 to the 2Fe centre of the H cluster with the subsequent destruction of the 4Fe4S cluster. As the gases have access to the active site of the enzyme through gas channels (Cohen et al., 2005), steric factors associated with the channels, and especially immediately around the active site, are likely to affect the ability of O2 to reach the H cluster (Stripp et al., 2009). A restriction in the gas channel leading to the active site has been proposed to be the explanation for O2 tolerance of some of the H2-sensing [NiFe] hydrogenases (Buhrke et al., 2005; Duche et al., 2005). Replacing small amino acids within a gas channel with methionine can decrease the O2 sensitivity of some [NiFe] hydrogenases (Dementin et al., 2009).

Maturation and interactions with other proteins

  1. Top of page
  2. Summary
  3. Introduction
  4. Fermentation metabolism
  5. Hydrogenase in algae
  6. Hydrogenase structure and oxygen sensitivity
  7. Maturation and interactions with other proteins
  8. Mutants in metabolism that impact fermentation pathways
  9. Conclusions
  10. Acknowledgements
  11. References

The maturation of [FeFe] hydrogenases (Bock et al., 2006) involves the activities of three auxiliary proteins; two of these proteins, HydE and HydG, belong to the radical S-adenosylmethionine (radical SAM) superfamily, while the third (HydF) has a GTPase domain (Posewitz et al., 2004a). These proteins are involved in the formation of the H cluster, which requires the generation and assembly of specific ligands (CN, CO and the dithiolate linkage) (Posewitz et al., 2004a). HydG catalyses the synthesis of the CO and CN ligands using tyrosine as a substrate (Driesener et al., 2010; Shepard et al., 2010a) while HydF, which may serve as the scaffold for H-cluster synthesis, associates with an iron cofactor co-ordinated to CO and CN that is transferred to the hydrogenase active site (Czech et al., 2010; Shepard et al., 2010b). In C. reinhardtii, the HYDE and HYDF proteins (protein designations are capitalized in C. reinhardtii) are fused (Posewitz et al., 2004a), and both HYDEF and HYDG are necessary for the synthesis of an active C. reinhardtii hydrogenase in Escherichia coli (Posewitz et al., 2004a).

There are six [2Fe-2S] FDXs encoded in the C. reinhardtii genome (Jacobs et al., 2009; Terauchi et al., 2009; Winkler et al., 2009), with PETF serving as the likely physiological donor to the hydrogenases (Winkler et al., 2009). The FDX5 transcript increases significantly as cell cultures become anoxic (Mus et al., 2007; Jacobs et al., 2009; Terauchi et al., 2009). However, the specificity of the FDXs in the dark- or light-mediated H2-production and H2-uptake pathways and to a large extent their in vivo interactions with specific electron donors and acceptors is just beginning to be elucidated (Terauchi et al., 2009).

Mutants in metabolism that impact fermentation pathways

  1. Top of page
  2. Summary
  3. Introduction
  4. Fermentation metabolism
  5. Hydrogenase in algae
  6. Hydrogenase structure and oxygen sensitivity
  7. Maturation and interactions with other proteins
  8. Mutants in metabolism that impact fermentation pathways
  9. Conclusions
  10. Acknowledgements
  11. References

Metabolic mutants have been isolated that exhibit attenuated HYDA transcription under anaerobic conditions. Two C. reinhardtii mutants that do not accumulate starch, the starch 6 (sta6) (Zabawinski et al., 2001) and sta7 (Poswitz et al., 2004b), have reduced hydrogenase activity under dark, anaerobic conditions and the levels of the HYDA1 and HYDA2 transcripts decline (Posewitz et al., 2004b). This mutant phenotype may be a consequence of altered metabolite pools and/or changes in cellular redox and energy status. Starch degradation under anaerobic conditions can influence intracellular NAD(P)H concentrations and/or the oxidation state of the PQ pool, both of which have been shown previously to regulate transcriptional processes (Escoubas et al., 1995; Rutter et al., 2001; Pfannschmidt & Liere, 2005). Recently, Chochois et al. demonstrated that the C. reinhardtii sta6 and sta7 mutants had near wild-type levels of hydrogenase activity when anaerobic cells were sulfur-starved in the light (Chochois et al., 2009). Under these conditions the redox and energetic states of the cell are influenced by both linear and cyclic photosynthetic electron transport. Although 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) severely attenuated H2 photoproduction in these cells, the in vitro hydrogenase activity was similar to that measured in uninhibited cells (Chochois et al., 2009). These results suggest that, although DCMU blocked the flow of reductant to the hydrogenases, the expression of the HYDA genes was not significantly affected. Additional analysis of the sta mutants indicated that starch is not absolutely necessary for high levels of H2-photoproduction activity; increased acetate utilization by the sta mutants allowed high respiratory activity, which allowed anaerobiosis and H2 production (Chochois et al., 2009), similar to what has been demonstrated in immobilized wild-type cells (Kosourov & Seibert, 2009).

Another example that demonstrates the impact of a metabolic change on hydrogenase activity comes from studies of the state transition mutant 6 (stm6) of C. reinhardtii (Schonfeld et al., 2004; Kruse et al., 2005a). This mutant, disrupted for a homologue of the human mitochondrial transcription termination factor, has a variety of phenotypes, including inhibition of cyclic electron transport under anaerobic conditions, hyper-accumulation of starch, a decreased number of active PSII reaction centres, an increased rate of respiration, and an elevated rate of and more sustained photo-H2 production during sulfur deprivation relative to its low H2-producing parental, cell wall-less strain (Kruse et al., 2005a,b; Rupprecht et al., 2006). Increased H2 production by the stm6 strain probably reflects the aggregate mutant phenotype, which includes attenuated cyclic electron transport (eliminating competition between the hydrogenase and PSI-dependent cyclic electron transport), over-accumulation of starch (providing additional reductant to the PQ pool) and reduced O2 evolution by PSII (which delays accumulation of O2 in the culture) (Kruse et al., 2005a).

A more direct effect on hydrogenase activity and fermentation metabolism has recently been noted for the hydEF-1 mutant (Posewitz et al., 2004a; Dubini et al., 2009), which has no hydrogenase activity. Anoxic cultures of the mutant also exhibit lower CO2 evolution, less accumulation of extracellular formate, acetate and ethanol, and high concentrations of extracellular succinate; the accumulation of succinate was not detected in the parental strain (Dubini et al., 2009). Transcript and metabolite analysis strongly suggests that in the hydEF-1 mutant carboxylation of pyruvate leads to the generation of either malate or oxaloacetate, which is subsequently converted to succinate by reverse tricarboxylic acid cycle (TCA) reactions (Fig. 1 and Dubini et al., 2009). The results of this study suggest that fermentative metabolism in C. reinhardtii has significant flexibility and that alternative metabolic pathways may be activated as conditions in the environment change. The exploitation of reverse TCA cycle reactions to dispose of excess reductant has been previously observed in anaerobic bacteria (Gray & Gest, 1965; Schauder et al., 1987; Buchanan & Arnon, 1990; Beh et al., 1993). It has also has been suggested to occur in the green alga Selenastrum minutum (Vanlerberghe et al., 1990) and in vascular plants (Sweetlove et al., 2010), but was not previously known to occur in C. reinhardtii. These results also suggest that, when the hydrogenase is unable to accept electrons from FDX and there are no other electron acceptors available (e.g. nitrite or sulfate), cellular redox increases, which might lead to activation of the thioredoxin system through coupling with reduced FDX. These regulatory molecules might in turn control the activities of genes associated with the various metabolic pathways capable of oxidizing NAD(P)H; one such pathway would result in the generation of succinate. These observations raise the (speculative) possibility that both the MME4 (NADP malic enzyme) gene (encoding the malic enzyme, which catalyses the formation of malate from pyruvate with the concomitant uptake of CO2; Fig. 1) and the FMR (fumarate reductase) gene (encoding fumarate reductase, which reduces fumarate to succinate; Fig. 1), which are both dramatically up-regulated specifically in the hydEF-1 mutant during anoxia (Dubini et al., 2009), may be under the control of the FDX-thioredoxin regulatory system.

Conclusions

  1. Top of page
  2. Summary
  3. Introduction
  4. Fermentation metabolism
  5. Hydrogenase in algae
  6. Hydrogenase structure and oxygen sensitivity
  7. Maturation and interactions with other proteins
  8. Mutants in metabolism that impact fermentation pathways
  9. Conclusions
  10. Acknowledgements
  11. References

Chlamydononas reinhardtii is a metabolically versatile organism that can perform photosynthetic CO2 fixation, aerobic respiration, and anaerobic fermentation metabolism. Many of the pathways and specific enzymes associated with fermentation metabolisms in this organism are just being defined, and there is little known about the mechanisms by which these pathways are regulated. The generation of mutations that block each of these pathways, as depicted in Fig. 1, will provide a wealth of information about the ways in which fermentation metabolism is regulated, the compensatory responses that reveal novel mechanisms for balancing ATP production with the elimination of reducing equivalents, and possible strategies for rerouting electrons through the various branches of fermentation metabolism for H2 and biofuel production.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Fermentation metabolism
  5. Hydrogenase in algae
  6. Hydrogenase structure and oxygen sensitivity
  7. Maturation and interactions with other proteins
  8. Mutants in metabolism that impact fermentation pathways
  9. Conclusions
  10. Acknowledgements
  11. References

This work was supported by the Office of Biological and Environmental Research, GTL Program, Office of Science, US Department of Energy (grants to A.R.G., M.C. P. and M.S.), by National Science Foundation Grant MCB 0235878 and US Department of Energy Grant DE-FG02-07ER64427 (to A.R.G.), and by Air Force Office of Scientific Research Grant FA9550-05-1-0365 (to M.C.P.). L.M. was supported by Scuola Superiore Sant’Anna and Regione Toscana POR OB.2 F.S.E. The work at the National Renewable Energy Laboratory was performed under US Department of Energy contract number DE-AC36-08GO28308. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked ‘advertisement’ in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Fermentation metabolism
  5. Hydrogenase in algae
  6. Hydrogenase structure and oxygen sensitivity
  7. Maturation and interactions with other proteins
  8. Mutants in metabolism that impact fermentation pathways
  9. Conclusions
  10. Acknowledgements
  11. References
  • Adams MW. 1990. The structure and mechanism of iron-hydrogenases. Biochimica et Biophysica Acta 1020: 115145.
  • Allakhverdiev SI, Kreslavski VD, Thavasi V, Zharmukhamedov SK, Klimov VV, Nagata T, Nishihara H, Ramakrishna S. 2009. Hydrogen photoproduction by use of photosynthetic organisms and biomimetic systems. Photochemical & Photobiological Sciences 8: 148156.
  • Antal TK, Krendeleva TE, Laurinavichene TV, Makarova VV, Ghirardi ML, Rubin AB, Tsygankov AA, Seibert M. 2003. The dependence of algal H2 production on photosystem II and O2 consumption activities in sulfur-deprived Chlamydomonas reinhardtii cells. Biochimica et Biophysica Acta 1607: 153160.
  • Aparicio PJ, Azuara MP, Ballesteros A, Fernandez VM. 1985. Effects of light intensity and oxidized nitrogen sources on hydrogen production by Chlamydomonas reinhardii. Plant Physiology 78: 803806.
  • Atteia A, van Lis R, Gelius-Dietrich G, Adrait A, Garin J, Joyard J, Rolland N, Martin W. 2006. Pyruvate formate-lyase and a novel route of eukaryotic ATP synthesis in Chlamydomonas mitochondria. Journal of Biological Chemistry 281: 99099918.
  • Bailey-Serres J, Chang R. 2005. Sensing and signalling in response to oxygen deprivation in plants and other organisms. Annals of Botany 96: 507518.
  • Baxter-Burrell A, Yang Z, Springer PS, Bailey-Serres J. 2002. RopGAP4-dependent Rop GTPase rheostat control of Arabidopsis oxygen deprivation tolerance. Science 296: 20262028.
  • Beer LL, Boyd ES, Peters JW, Posewitz MC. 2009. Engineering algae for biohydrogen and biofuel production. Current Opinion in Biotechnology 20: 264271.
  • Beh M, Strauss G, Huber R, Stetter KO, Fuchs G. 1993. Enzymes of the reductive citric acid cycle in the autotrophic eubacterium Aquifex pyrophilus and in the archaebacterium Thermoproteus neutrophilus. Archives of Microbiology 160: 306311.
  • Bell EL, Chandel NS. 2007. Mitochondrial oxygen sensing: regulation of hypoxia-inducible factor by mitochondrial generated reactive oxygen species. Essays in Biochemistry 43: 1727.
  • Benemann JR, Berenson JA, Kaplan NO, Kamen MD. 1973. Hydrogen evolution by a chloroplast-ferredoxin-hydrogenase system. Proceedings of the National Academy of Sciences, USA 70: 23172320.
  • Bock A, King PW, Blokesch M, Posewitz MC. 2006. Maturation of hydrogenases. Advances in Microbial Physiology 51: 171.
  • Boichenko VA, Greenbaum E, Seibert M. 2004. Hydrogen production by photosynthetic microorganisms. In: ArcherMD, BarberJ, eds. Photoconversion of solar energy: molecular to global photosynthesis. London, UK: Imperial College Press, 397452.
  • Boichenko VA, Hoffmann P. 1994. Photosynthetic hydrogen production in prokaryotes and eukaryotes: occurrence, mechanism, and functions. Photosynthetica 30: 527552.
  • Brand JJ, Wright J, Lien S. 1989. Hydrogen production by eukaryotic algae. Biotechnology and Bioengineering 33: 14821488.
  • Buchanan BB, Arnon DI. 1990. A reverse KREBS cycle in photosynthesis: consensus at last. Photosynthesis Research 24: 4753.
  • Buhrke T, Lenz O, Krauss N, Friedrich B. 2005. Oxygen tolerance of the H2-sensing [NiFe] hydrogenase from Ralstonia eutropha H16 is based on limited access of oxygen to the active site. Journal of Biological Chemistry 280: 2379123796.
  • Chen C, Gibbs M. 1992. Coupling of carbon dioxide fixation to the oxyhydrogen reaction in the isolated chloroplast of Chlamydomonas reinhardtii. Plant Physiology 100: 13611365.
  • Chochois V, Dauvillee D, Beyly A, Tolleter D, Cuine S, Timpano H, Ball S, Cournac L, Peltier G. 2009. Hydrogen production in Chlamydomonas: photosystem II-dependent and -independent pathways differ in their requirement for starch metabolism. Plant Physiology 151: 631640.
  • Cohen J, Kim K, Posewitz M, Ghirardi ML, Schulten K, Seibert M, King P. 2005. Molecular dynamics and experimental investigation of H2 and O2 diffusion in [Fe]-hydrogenase. Biochemical Society Transactions 33: 8082.
  • Cournac L, Redding K, Ravenel J, Rumeau D, Josse EM, Kuntz M, Peltier G. 2000. Electron flow between photosystem II and oxygen in chloroplasts of photosystem I-deficient algae is mediated by a quinol oxidase involved in chlororespiration. Journal of Biological Chemistry 275: 1725617262.
  • Czech I, Silakov A, Lubitz W, Happe T. 2010. The [FeFe]-hydrogenase maturase HydF from Clostridium acetobutylicum contains a CO and CN- ligated iron cofactor. FEBS Letters 584: 638642.
  • Dementin S, Leroux F, Cournac L, de Lacey AL, Volbeda A, Leger C, Burlat B, Martinez N, Champ S, Martin L. 2009. Introduction of methionines in the gas channel makes [NiFe] hydrogenase aero-tolerant. Journal of the American Chemical Society 131: 1015610164.
  • Driesener RC, Challand MR, McGlynn SE, Shepard EM, Boyd ES, Broderick JB, Peters JW, Roach PL. 2010. [FeFe]-hydrogenase cyanide ligands derived from S-adenosylmethionine-dependent cleavage of tyrosine. Angewandte Chemie (International Ed. in English) 49: 16871690.
  • Dubini A, Mus F, Seibert M, Grossman AR, Posewitz MC. 2009. Flexibility in anaerobic metabolism as revealed in a mutant of Chlamydomonas reinhardtii lacking hydrogenase activity. Journal of Biological Chemistry 284: 72017213.
  • Duche O, Elsen S, Cournac L, Colbeau A. 2005. Enlarging the gas access channel to the active site renders the regulatory hydrogenase HupUV of Rhodobacter capsulatus O2 sensitive without affecting its transductory activity. The FEBS Journal 272: 38993908.
  • Escoubas JM, Lomas M, LaRoche J, Falkowski PG. 1995. Light intensity regulation of cab gene transcription is signaled by the redox state of the plastoquinone pool. Proceedings of the National Academy of Sciences, USA 92: 1023710241.
  • Florin L, Tsokoglou A, Happe T. 2001. A novel type of iron hydrogenase in the green alga Scenedesmus obliquus is linked to the photosynthetic electron transport chain. Journal of Biological Chemistry 276: 61256132.
  • Forestier M, King P, Zhang L, Posewitz M, Schwarzer S, Happe T, Ghirardi ML, Seibert M. 2003. Expression of two [Fe]-hydrogenases in Chlamydomonas reinhardtii under anaerobic conditions. European Journal of Biochemistry 270: 27502758.
  • Frey M. 2002. Hydrogenases: hydrogen-activating enzymes. Chembiochem 3: 153160.
  • Gaffron H. 1942. The effect of specific poisons upon the photo-reduction with hydrogen in green algae. Journal of General Physiology 26: 195217.
  • Gaffron H. 1944. Photosynthesis, photoreduction and dark reduction of carbon dioxide in certain algae. Biological Reviews of the Cambridge Philosophical Society 19: 120.
  • Gaffron H, Rubin J. 1942. Fermentative and photochemical production of hydrogen in algae. Journal of General Physiology 26: 219240.
  • Gfeller RP, Gibbs M. 1984. Fermentative metabolism of Chlamydomonas reinhardtii: I. Analysis of fermentative products from starch in dark and light. Plant Physiology 75: 212218.
  • Gfeller RP, Gibbs M. 1985. Fermentative metabolism of Chlamydomonas reinhardtii: II. Role of plastoquinone. Plant Physiology 77: 509511.
  • Ghirardi ML, Dubini A, Yu J, Maness PC. 2009. Photobiological hydrogen-producing systems. Chemical Society Reviews 38: 5261.
  • Ghirardi ML, Maness P-C, Seibert M. 2008. Photobiological methods of renewable hydrogen production. In: RajeshwarK, McConnellR, LichtS, eds. Solar generation of hydrogen. New York, NY, USA: Springer, 229271.
  • Ghirardi ML, Posewitz MC, Maness PC, Dubini A, Yu J, Seibert M. 2007. Hydrogenases and hydrogen photoproduction in oxygenic photosynthetic organisms. Annual Review of Plant Biology 58: 7191.
  • Ghirardi ML, Togasaki RK, Seibert M. 1997. Oxygen sensitivity of algal H2-production. Applied Biochemistry and Biotechnology 63: 141151.
  • Ghirardi ML, Zhang L, Lee JW, Flynn T, Seibert M, Greenbaum E, Melis A. 2000. Microalgae: a green source of renewable H2. Trends in Biotechnology 18: 506511.
  • Gibbs M, Gfeller RP, Chen C. 1986. Fermentative metabolism of Chlamydomonas reinhardii: III. Photoassimilation of acetate. Plant Physiology 82: 160166.
  • Gray CT, Gest H. 1965. Biological formation of molecular hydrogen. Science 148: 186192.
  • Greenbaum E. 1982. Photosynthetic hydrogen and oxygen production: kinetic studies. Science 215: 291293.
  • Grossman AR. 2007. In the grip of algal genomics. Advances in Experimental Medicine and Biology 616: 5476.
  • Grossman AR, Croft M, Gladyshev VN, Merchant SS, Posewitz MC, Prochnik S, Spalding MH. 2007. Novel metabolism in Chlamydomonas through the lens of genomics. Current Opinion in Plant Biology 10: 190198.
  • Guzy RD, Schumacker PT. 2006. Oxygen sensing by mitochondria at complex III: the paradox of increased reactive oxygen species during hypoxia. Experimental Physiology 91: 807819.
  • Hankamer B, Lehr F, Rupprecht J, Mussgnug JH, Posten C, Kruse O. 2007. Photosynthetic biomass and H2 production by green algae: from bioengineering to bioreactor scale-up. Physiologia Plantarum 131: 1021.
  • Happe T, Kaminski A. 2002. Differential regulation of the Fe-hydrogenase during anaerobic adaptation in the green alga Chlamydomonas reinhardtii. European Journal of Biochemistry 269: 10221032.
  • Happe T, Mosler B, Naber JD. 1994. Induction, localization and metal content of hydrogenase in the green alga Chlamydomonas reinhardtii. European Journal of Biochemistry 222: 769774.
  • Happe T, Naber JD. 1993. Isolation, characterization and N-terminal amino acid sequence of hydrogenase from the green alga Chlamydomonas reinhardtii. European Journal of Biochemistry 214: 475481.
  • Harris EH. 2001. Chlamydomonas as a model organism. Annual Review of Plant Physiology and Plant Molecular Biology 52: 363406.
  • Hemschemeier A, Happe T. 2005. The exceptional photofermentative hydrogen metabolism of the green alga Chlamydomonas reinhardtii. Biochemical Society Transactions 33: 3941.
  • Hemschemeier A, Melis A, Happe T. 2009. Analytical approaches to photobiological hydrogen production in unicellular green algae. Photosynthesis Research 102: 523540.
  • Jacobs J, Pudollek S, Hemschemeier A, Happe T. 2009. A novel, anaerobically induced ferredoxin in Chlamydomonas reinhardtii. FEBS Letters 583: 325329.
  • Kessler E. 1974. Hydrogenase, photoreduction and anaerobic growth. In: StewardWDP, ed. Algal physiology and biochemistry. Berkeley and Los Angeles, CA, USA: University of California Press, Blackwell Scientific Publications Ltd, 456473.
  • King PW, Posewitz MC, Ghirardi ML, Seibert M. 2006. Functional studies of [FeFe] hydrogenase maturation in an Escherichia coli biosynthetic system. Journal of Bacteriology 188: 21632172.
  • Kosourov S, Seibert M, Ghirardi ML. 2003. Effects of extracellular pH on the metabolic pathways in sulfur-deprived, H2-producing Chlamydomonas reinhardtii cultures. Plant and Cell Physiology 44: 146155.
  • Kosourov SN, Seibert M. 2009. Hydrogen photoproduction by nutrient-deprived Chlamydomonas reinhardtii cells immobilized within thin alginate films under aerobic and anaerobic conditions. Biotechnology and Bioengineering 102: 5058.
  • Kosourov SN, Tsygankov A, Seibert M, Ghirardi ML. 2002. Sustained hydrogen photoproduction by Chlamydomonas reinhardtii – effects of culture parameters. Biotechnology and Bioengineering 78: 731740.
  • Kreuzberg K. 1984. Starch fermentation via formate producing pathway in Chlamydomonas reinhardtii, Chlorogonium elongatum and Chlorella fusca. Physiologia Plantarum 61: 8794.
  • Kreuzberg K, Klöck G, Grobheiser D. 1987. Subcellular distribution of pyruvate-degrading enzymes in Chlamydomonas reinhardtii studied by an improved protoplast fractionation procedure. Physiologia Plantarum 69: 481488.
  • Kruse O, Hankamer B. 2010. Microalgal hydrogen production. Current Opinion in Biotechnology 21: 238243.
  • Kruse O, Rupprecht J, Bader KP, Thomas-Hall S, Schenk PM, Finazzi G, Hankamer B. 2005a. Improved photobiological H2 production in engineered green algal cells. Journal of Biological Chemistry 280: 3417034177.
  • Kruse O, Rupprecht J, Mussgnug J, Dismukes G, Hankamer B. 2005b. Photosynthesis: a blueprint for solar energy capture and biohydrogen production technologies. Photochemical & Photobiological Sciences 4: 957970.
  • Lasanthi-Kudahettige R, Magneschi L, Loreti E, Gonzali S, Licausi F, Novi G, Beretta O, Vitulli F, Alpi A, Perata P. 2007. Transcript profiling of the anoxic rice coleoptile. Plant Physiology 144: 218231.
  • Magneschi L, Perata P. 2009. Rice germination and seedling growth in the absence of oxygen. Annals of Botany 103: 181196.
  • Maione TE, Gibbs M. 1986a. Association of the chloroplastic respiratory and photosynthetic electron transport chains of Chlamydomonas reinhardii with photoreduction and the oxyhydrogen reaction. Plant Physiology 80: 364368.
  • Maione TE, Gibbs M. 1986b. Hydrogenase-mediated activities in isolated chloroplasts of Chlamydomonas reinhardii. Plant Physiology 80: 360363.
  • Melis A, Happe T. 2001. Hydrogen production. Green algae as a source of energy. Plant Physiology 127: 740748.
  • Melis A, Happe T. 2004. Trails of green alga hydrogen research – from Hans Gaffron to new frontiers. Photosynthesis Research 80: 401409.
  • Melis A, Seibert M, Ghirardi ML. 2007. Hydrogen fuel production by transgenic microalgae. Advances in Experimental Medicine and Biology 616: 110121.
  • Melis A, Zhang L, Forestier M, Ghirardi ML, Seibert M. 2000. Sustained photobiological hydrogen gas production upon reversible inactivation of oxygen evolution in the green alga Chlamydomonas reinhardtii. Plant Physiology 122: 127136.
  • Merchant SS, Prochnik SE, Vallon O, Harris EH, Karpowicz SJ, Witman GB, Terry A, Salamov A, Fritz-Laylin LK, Marechal-Drouard L et al. 2007. The Chlamydomonas genome reveals the evolution of key animal and plant functions. Science 318: 245250.
  • Meyer J. 2007. [FeFe] hydrogenases and their evolution: a genomic perspective. Cellular and Molecular Life Sciences 64: 10631084.
  • Miura Y. 1995. Hydrogen production by biophotolysis based on microalgal photosynthesis. Process Biochemistry 30: 17.
  • Müller M. 2003. Energy metabolism. Part 1: anaerobic protozoa. In: MarrJJ, NilsenTW, KomunieckR, eds. Molecular medical parasitolgy. Amsterdam, the Netherlands: Academic Press, 125139.
  • Mus F, Cournac L, Cardettini V, Caruana A, Peltier G. 2005. Inhibitor studies on non-photochemical plastoquinone reduction and H2 photoproduction in Chlamydomonas reinhardtii. Biochimica et Biophysica Acta 1708: 322332.
  • Mus F, Dubini A, Seibert M, Posewitz MC, Grossman AR. 2007. Anaerobic acclimation in Chlamydomonas reinhardtii: anoxic gene expression, hydrogenase induction, and metabolic pathways. Journal of Biological Chemistry 282: 2547525486.
  • Nicolet Y, de Lacey AL, Vernede X, Fernandez VM, Hatchikian EC, Fontecilla-Camps JC. 2001. Crystallographic and FTIR spectroscopic evidence of changes in Fe coordination upon reduction of the active site of the Fe-only hydrogenase from Desulfovibrio desulfuricans. Journal of the American Chemical Society 123: 15961601.
  • Nicolet Y, Piras C, Legrand P, Hatchikian CE, Fontecilla-Camps JC. 1999. Desulfovibrio desulfuricans iron hydrogenase: the structure shows unusual coordination to an active site Fe binuclear center. Structure 7: 1323.
  • Ohta S, Miyamoto K, Miura Y. 1987. Hydrogen evolution as a consumption mode of reducing equivalents in green algal fermentation. Plant Physiology 83: 10221026.
  • Peters JW, Lanzilotta WN, Lemon BJ, Seefeldt LC. 1998. X-ray crystal structure of the Fe-only hydrogenase (CpI) from Clostridium pasteurianum to 1.8 angstrom resolution. Science 282: 18531858.
  • Pfannschmidt T, Liere K. 2005. Redox regulation and modification of proteins controlling chloroplast gene expression. Antioxidants Redox Signaling 7: 607618.
  • Posewitz MC, Dubini A, Meuser JE, Seibert M, Ghirardi ML. 2008. Hydrogenases, hydrogen production and anoxia. In: HarrisE, SternD, eds. The Chlamydomonas sourcebook. Amsterdam, the Netherlands: Elsevier, 217255.
  • Posewitz MC, King PW, Smolinski SL, Zhang L, Seibert M, Ghirardi ML. 2004a. Discovery of two novel radical S-adenosylmethionine proteins required for the assembly of an active [Fe] hydrogenase. Journal of Biological Chemistry 279: 2571125720.
  • Posewitz MC, Smolinski SL, Kanakagiri S, Melis A, Seibert M, Ghirardi ML. 2004b. Hydrogen photoproduction is attenuated by disruption of an isoamylase gene in Chlamydomonas reinhardtii. Plant Cell 16: 21512163.
  • Purton S. 2007. Tools and techniques for chloroplast transformation of Chlamydomonas. Advances in Experimental Medicine and Biology 616: 3445.
  • Radakovits R, Jinkerson RE, Darzins A, Posewitz MC. 2010. Genetic engineering of algae for enhanced biofuel production. Eukaryotic Cell 9: 486501.
  • Roessler P, Lien S. 1984. Effects of electron mediator charge properties on the reaction kinetics of hydrogenase from Chlamydomonas. Archives of Biochemistry and Biophysics 230: 103109.
  • Rosenberg JN, Oyler GA, Wilkinson L, Betenbaugh MJ. 2008. A green light for engineered algae: redirecting metabolism to fuel a biotechnology revolution. Current Opinion in Biotechnology 19: 430436.
  • Rupprecht J. 2009. From systems biology to fuel –Chlamydomonas reinhardtii as a model for a systems biology approach to improve biohydrogen production. Journal of Biotechnology 142: 1020.
  • Rupprecht J, Hankamer B, Mussgnug JH, Ananyev G, Dismukes C, Kruse O. 2006. Perspectives and advances of biological H2 production in microorganisms. Applied Microbiology and Biotechnology 72: 442449.
  • Russell GK, Gibbs M. 1968. Evidence for the participation of the reductive pentose phosphate cycle in photoreduction and the oxyhydrogen reaction. Plant Physiology 43: 649652.
  • Rutter J, Reick M, Wu LC, McKnight SL. 2001. Regulation of clock and NPAS2 DNA binding by the redox state of NAD cofactors. Science 293: 510514.
  • Schauder R, Widdel F, Fuchs G. 1987. Carbon assimilation pathways in sulfate-reducing bacteria. II. Enzymes of a reductive citric acid cycle in the autotrophic Desulfobacter hydrogenophilus. Archives of Microbiology 148: 218225.
  • Schonfeld C, Wobbe L, Borgstadt R, Kienast A, Nixon PJ, Kruse O. 2004. The nucleus-encoded protein MOC1 is essential for mitochondrial light acclimation in Chlamydomonas reinhardtii. Journal of Biological Chemistry 279: 5036650374.
  • Seibert M, King P, Posewitz MC, Melis A, Ghirardi ML. 2008. Photosynthetic water-splitting for hydrogen production. In: WallJ, HarwoodC, DemainA, eds. Bioenergy. Washington, DC, USA: ASM Press, 273291.
  • Shepard EM, Duffus BR, George SJ, McGlynn SE, Challand MR, Swanson KD, Roach PL, Cramer SP, Peters JW, Broderick JB. 2010a. [FeFe]-hydrogenase maturation: HydG-catalyzed synthesis of carbon monoxide. Journal of the American Chemical Society 132: 92479249.
  • Shepard EM, McGlynn SE, Bueling AL, Grady-Smith CS, George SJ, Winslow MA, Cramer SP, Peters JW, Broderick JB. 2010b. Synthesis of the 2Fe subcluster of the [FeFe]-hydrogenase H cluster on the HydF scaffold. Proceedings of the National Academy of Sciences, USA 107: 1044810453.
  • Silakov A, Wenk B, Reijerse E, Lubitz W. 2009. 14N HYSCORE investigation of the H-cluster of [FeFe] hydrogenase: evidence for a nitrogen in the dithiol bridge. Physical Chemistry Chemical Physics 11: 65926599.
  • Stirnberg M, Happe T. 2004. Identification of a cis-acting element controlling anaerobic expression of the HydA gene from Chlamydomonas reinhardtii. In: MiyakeJ, IgarashiY, RoegnerM, eds. Biohydrogen III. Oxford, UK: Elsevier Science, 117127.
  • Stripp ST, Goldet G, Brandmayr C, Sanganas O, Vincent KA, Haumann M, Armstrong FA, Happe T. 2009. How oxygen attacks [FeFe] hydrogenases from photosynthetic organisms. Proceedings of the National Academy of Sciences, USA 106: 1733117336.
  • Stripp ST, Happe T. 2009. How algae produce hydrogen-news from the photosynthetic hydrogenase. Dalton Transactions 45: 99609969.
  • Sweetlove LJ, Beard KF, Nunes-Nesi A, Fernie AR, Ratcliffe RG. 2010. Not just a circle: flux modes in the plant TCA cycle. Trends in Plant Science 15: 462470.
  • Terashima M, Specht M, Naumann B, Hippler M. 2010. Characterizing the anaerobic response of Chlamydomonas reinhardtii by quantitative proteomics. Molecular & Cellular Proteomics 9: 15141532.
  • Terauchi AM, Lu SF, Zaffagnini M, Tappa S, Hirasawa M, Tripathy JN, Knaff DB, Farmer PJ, Lemaire SD, Hase T et al. 2009. Pattern of expression and substrate specificity of chloroplast ferredoxins from Chlamydomonas reinhardtii. Journal of Biological Chemistry 284: 2586725878.
  • Timmins M, Thomas-Hall SR, Darling A, Zhang E, Hankamer B, Marx UC, Schenk PM. 2009a. Phylogenetic and molecular analysis of hydrogen-producing green algae. Journal of Experimental Botany 60: 16911702.
  • Timmins M, Zhou W, Rupprecht J, Lim L, Thomas-Hall SR, Doebbe A, Olaf Kruse O, Hankamer B, Marx UC, Smith SM et al. 2009b. The metabolome of Chlamydomonas reinhardtii following induction of anaerobic hydrogen production by sulfur deprivation. Journal of Biological Chemistry 284: 2341523425.
  • Tsygankov AA, Kosourov S, Seibert M, Ghirardi ML. 2002. Hydrogen photoproduction under continuous illumination by sulfur-deprived, synchronous Chlamydomonas reinhardtii cultures. International Journal of Hydrogen Energy 27: 12391244.
  • Vanlerberghe GC, Feil R, Turpin DH. 1990. Anaerobic metabolism in the N-limited green alga Selenastrum minutum: I. Regulation of carbon metabolism and succinate as a fermentation product. Plant Physiology 94: 11161123.
  • Vignais PM, Billoud B. 2007. Occurrence, classification, and biological function of hydrogenases: an overview. Chemical Reviews 107: 42064272.
  • Vlad F, Spano T, Vlad D, Daher FB, Ouelhadj A, Kalaitzis P. 2007. Arabidopsis prolyl 4-hydroxylases are differentially expressed in response to hypoxia, anoxia and mechanical wounding. Physiologia Plantarum 130: 471483.
  • Wagner AF, Frey M, Neugebauer FA, Schafer W, Knappe J. 1992. The free radical in pyruvate formate-lyase is located on glycine-734. Proceedings of the National Academy of Sciences, USA 89: 9961000.
  • Winkler M, Heil B, Heil B, Happe T. 2002. Isolation and molecular characterization of the [Fe]-hydrogenase from the unicellular green alga Chlorella fusca. Biochimica et Biophysica Acta 1576: 330334.
  • Winkler M, Kuhlgert S, Hippler M, Happe T. 2009. Characterization of the key step for light-driven hydrogen evolution in green algae. Journal of Biological Chemistry 284: 3662036627.
  • Wunschiers R, Stangier K, Senger H, Schulz R. 2001. Molecular evidence for a Fe-hydrogenase in the green alga Scenedesmus obliquus. Current Microbiology 42: 353360.
  • Zabawinski C, Van Den Koornhuyse N, D’Hulst C, Schlichting R, Giersch C, Delrue B, Lacroix JM, Preiss J, Ball S. 2001. Starchless mutants of Chlamydomonas reinhardtii lack the small subunit of a heterotetrameric ADP-glucose pyrophosphorylase. Journal of Bacteriology 183: 10691077.
  • Zhang L, Happe T, Melis A. 2002. Biochemical and morphological characterization of sulfur deprived and H2- producing Chlamydomonas reinhardtii (green algae). Planta 214: 552561.