international journal of hydrogen energy 36 (2011) 10648e10654

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Introducing pyruvate oxidase into the chloroplast of Chlamydomonas reinhardtii increases consumption and promotes hydrogen production

Fu-Qiao Xu a,b,c, Wei-Min Ma d, Xin-Guang Zhu a,b,c,* a Key Laboratory of Computational Biology, Chinese Academy of Sciences, Yueyang Road 320, Shanghai 200031, China b Chinese Academy of Sciences and Max Planck Society (CAS-MPG) Partner Institute for Computational Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yueyang Road, Shanghai 200031, China c Shanghai Institute of Plant Physiology and Ecology, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, China d Colleague of Life and Environmental Sciences, Shanghai Normal University, Guilian Road 100, Shanghai 200234, China article info abstract

Article history: In an anaerobic environment, the unicellular green algae Chlamydomonas reinhardtii can

Received 4 February 2011 produce hydrogen (H2) using hydrogenase. The activity of hydrogenase is inhibited at the Received in revised form presence of molecular oxygen, forming a major barrier for large scale production of 17 May 2011 hydrogen in autotrophic organisms. In this study, we engineered a novel pathway to Accepted 22 May 2011 consume oxygen and correspondingly promote hydrogen production in Chlamydomonas Available online 30 June 2011 reinhardtii. The pyruvate oxidase from Escherichia coli and catalase from Synechococcus elongatus PCC 7942 were cloned and integrated into the chloroplast of Chlamydomonas Keywords: reinhardtii. These two foreign genes are driven by a HSP70A/RBCS2 promoter, a heat shock Chlamydomonas reinhardtii inducing promoter. After continuous heat shock treatments, the foreign genes showed Chloroplast transformation high expression levels, while the growth rate of transgenic algal cells was slightly inhibited Photosynthetic hydrogen compared to the wild type. Under low light, transgenic algal cells consumed more oxygen production than wild type. This resulted in lower oxygen content in sealed culture conditions, espe- Pyruvate oxidase cially under low light condition, and dramatically increased hydrogen production. These results demonstrate that pyruvate oxidase expressed in Chlamydomonas reinhardtii increases oxygen consumption and has potential for improving photosynthetic hydrogen production in Chlamydomonas reinhardtii. Copyright ª 2011, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.

1. Introduction photosynthetic microorganisms [2e5]. Different biological pathways of hydrogen production have been reported in Hydrogen is an ideal, clean, and potentially sustainable energy different biological organisms [4]. Generally, biological source. It is currently produced primarily from fossil fuel (oil, hydrogen generation can be classified into four categories: coal, natural gas) [1], but can also be produced by (1) biophotolysis of water using algae and cyanobacteria [6e8],

* Corresponding author. CAS-MPG Partner Institute for Computational Biology, Physiology building, Room 359, Yueyang Road 320, Shanghai 200031, China. Tel.: þ86 21 5492 0486. E-mail address: [email protected] (X.-G. Zhu). 0360-3199/$ e see front matter Copyright ª 2011, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijhydene.2011.05.130 international journal of hydrogen energy 36 (2011) 10648e10654 10649

(2) photodecomposition of organic compounds by photosyn- In this study, we introduced a new way to decrease cellular thetic bacteria [9e11], (3) fermentative hydrogen production oxygen levels and increase the hydrogen productivity. This way from organic compounds [12e14] and (4) hybrid systems using utilizes pyruvate oxidase (POX), an that catalyzes the photosynthetic organisms and fermentative bacteria [15]. decarboxylation of pyruvate to acetyl-phosphate and CO2 [29]. Though much research has been done on the process of þ þ 5 hydrogen production, so far, large scale biological production Pyruvate Orthophosphate O2 Acetyl phosphate þ þ has not yet been accomplished mainly due to the low H2O2 CO2 production efficiency [3]. This enzyme has been characterized in E. coli [30e33]. One of the primary organisms for studying hydrogen Though previously this enzyme has been considered nones- production is Chlamydomonas reinhardtii, a green alga capable sential in the early stationary phase of E. coli [34], more recent of producing hydrogen under stress conditions. A sustained studies suggest that pyruvate oxidase might play an impor- hydrogen production for about 50e100 h has been reported in tant role in the aerobic growth of E. coli, possibly related to Chlamydomonas reinhardtii under anaerobic and sulfur preserving the pool of free CoA [35]. Here we hypothesize that deprived conditions [16]. C. reinhardtii uses solar energy to split þ the expression of E. coli POX in the chloroplast of C. reinhardtii water (H2O) into protons (H ), electrons (e ), and oxygen (O2) can increases the oxygen consumption. Given that H2O2, [6]. The [FeeFe] hydrogenase [17] then catalyzes the synthesis þ a of POX, can potentially damage the cell [36],we of hydrogen from H and e . This reaction is inhibited in the introduced enzyme catalase (CAT) to reduce H2O2. presence of oxygen, which diffuses into the enzyme’s cata- lytic center and irreversibly binds to it, halting the catalytic capacity [18]. Furthermore, oxygen was found to inhibit the 2. Material and methods mRNA synthesis of hydrogenase as well [19]. One of the major research focuses on hydrogen production is identifying ways 2.1. POX and CAT cloning and vector construction to overcome the inhibition of oxygen on hydrogen production, through either engineering the oxygen sensitivity of the The full-length fragments of pyruvate oxidase (POX) from enzyme or decreasing the oxygen levels in the cell [20]. Escherichia coli (Genbank accession No. M28208) and catalase Depletion of sulfur in the medium of C. reinhardtii is shown (CAT) from Synechococcus elongatus PCC 7942 (Genbank to affect photosystem II (PSII) and consequently dramatically accession No. D61378) were cloned by PCR. Primers are decreases O production [18], resulting in a significant 2 shown in Table 1. The forward primers were inserted with increase in the hydrogenase activity and an extended H 2 restriction enzyme sites and ribosomal binding sequence production for over 4e5 days. This sulfur-deprivation process (AGGGAGGG) in front of ATG. The coding sequence frag- consists of two phases: in the initial phase O evolution 2 ments were confirmed by sequencing. The POX fragment gradually declines and there is over-accumulation of carbo- was inserted into a modified pBluescript SK vector contain- hydrates in the form of starch, followed by a H -production 2 ing HSP70A/RBSC2 promoters (heat shock inducible phase driven by residual photosynthetic water oxidation and promoter) [37] using restriction endonucleases XhoI and ClaI. the anaerobic degradation of starch [18]. The observed Subsequently the CAT fragment was inserted using restric- decrease in the water oxidation activity of PSII is correlated tion endonucleases SpeI and NotI. The fragment, containing with a decrease in the turnover of the D1 protein, an essential the HSP70A/RBSC2 promoter, POX and CAT, was inserted component of PSII that is inactivated in the light and re- into the chloroplast transformation vector pcg40 [38] using synthesized at very high rates in the dark subsequently [21,22]. At the absence of sulfur, the D1 turnover is impaired due to the lack of sulfurylated residues and PSII activity is gradually lost [23]. Though this method can be used to produce hydrogen, the productivity was much lower than the pre- Table 1 e The primers used in the full length cds cloning dicted capacity due to the depletion of electron sources from of POX and CAT and the expression level analysis of foreign genes of transgenic algae using RT-PCR. PSII as a result of the decreased activity [16,24]. 0 0 Removing sulfur from the medium is a potential useful Gene Length of Primer sequences (5 -3 ) way to decrease oxygen concentration in the cells, which is name fragments required for hydrogen production [18]. There are additional Full length fragments cloning methods to decrease the oxygen concentration inside C. rein- POX 1776 bp ACTCGAGGGAGGGAACCATGAAACA hardtii, either in the mitochondria or the chloroplast. In the AACGGT mitochondria, oxygen is reduced by the complex IV, i.e. TATCGATGTCCTTATTATGACGGGA CAT 2225 bp AACTAGTAGGGAGGGAACCATGACA cytochrome oxidase, which is at the end of oxidative electron GCAACCCAG transport chain, or by alternative oxidase (AOX), that bypasses TGCGGCCGCTAACAATGTGGGGATGGA the proton-pumping complexes III and IV [25]. Plastid terminal Gene expression oxidase (PTOX) is the third way to consume oxygen [26]. Under POX 515 bp CGCCTGTTAGGTTCGTTT normal conditions these reactions can not consume all the TTTGTTTTCGCCAGTTCG oxygen generated by photosynthesis, therefore identifying CAT 446 bp TGAGCAAACGGATACGGA new metabolic engineering options to further decrease the CGACAAAGTCTCGCACAAA atpB 596 bp CTGGTGGTTCGTTCATTTG cellular oxygen level and enhance biohydrogen production is CGCTCTAACTATTCGTGCTAA a major research focus [27,28]. 10650 international journal of hydrogen energy 36 (2011) 10648e10654

2.5. Measurement of photosynthetic oxygen evolution and respiration rates

Photosynthetic oxygen evolution and respiration rates were measured at 25 C with a Clark-type oxygen electrode (Han- satech, UK). Specifically, 2 ml algal cells were taken after growing in the dark for 24 h and heat-shocked once every 6 h. Fig. 1 e A Diagram of the pcg40-HPC construct used in the The cells were placed in an O2-electrode chamber and equili- chloroplast transformation of Chlamydomonas reinhardtii. brated with NaHCO3 which has a final concentration of 10 mM. cpDNA: fragment of chloroplast DNA; aadA: 0 Aminoglycoside 3 -Adenyltransferase, a spectinomycin- 2.6. Measurement of oxygen evolution and hydrogen resistance gene; HSP70A/RBCS2: heat-shock promoter; production ecoPOX: pyruvate oxidase from E. coli; 7942CAT: catalase from Synechococcus elongatus PCC 7942. 30 ml algal cells in the middle of the exponential growth phase were transferred to cylindrical glass bottles. The glass bottles were sealed with a rubber gas-tight septum and incubated in the dark for 24 h to create an anaerobic liquid medium. The volume of restriction endonucleases SacII and NotI to create pcg40-HPC. the top air space of the bottle is 30 ml. From the second day, the The complete construct can be seen in Fig. 1. sealed bottles were placed under continuous illumination of 30 mmol photons m 2 s 1 (LL, low light) or 2.2. Chloroplast transformation and transformant 100 mmol photons m 2 s 1 (HL, high light). Evolved gas was screening collected from the top air space of the bottle using a gas-tight lockable syringe and injected into a gas chromatograph Particle bombardment transformation was conducted on C. (Agilent 7890) with a thermal conductivity detector to measure reinhardtii strain cc503 using a helium-driven particle gun (Bio- gas composition. Rad Model PDS1000/He Biolistic particle delivery system) as described by Purton [39]. Before transformation, 10 mgDNAof 2.7. Statistical analysis plasmid pcg40-HPC was linearized by the restriction enzyme EcoRI. After transformation, the algal cells were grown on Two-way ANOVA was used to analyze the effects of time and normal TAP (Tris-Acetate-Phosphate) plates in the dark for 18 h strain type on the growth rates, oxygen evolution and at 25 C, and washed with fresh TAP liquid medium once. hydrogen production rates. Afterwards, these cells were cultured on TAP plates containing 100 mg/ml spectinomycin under continuous illumination at 25 C for 1e2 weeks to screen transformants. After selection, trans- 3. Results formants were subcultured on TAP plates containing 100 mg/ml spectinomycin for at least six times to reach homoplasmicity. 3.1. Identification of transformant and foreign gene The transgenic strain was named ccHPC. expression

2.3. PCR analysis Fig. 2A shows the timescale of the heat shock treatments and moments of algal sample taking. We took samples at three DNA extraction from C. reinhardtii was performed according to time points corresponding to the end of a dark growth period, Ramesh [40]. Total RNA was extracted from 1.5 ml of C. rein- after one-hour heat shock treatment and at the end of 6 h light hardtii culture using EZ Spin Column total RNA Purification Kit treatment. No fragments of either POX or CAT were detected (Shanghai Sangon Ltd., China) and was subsequently treated in the wild-type cc503 (Fig. 2B, lane 1). However specific POX with RNase-free DNase I (Fermentas Canada Inc.) for the and CAT fragments were found in the chloroplastic DNA of removal of DNA from the total RNA. The first strand cDNAs the transgenic algae ccHPC (Fig. 2B, lane 2). RT-PCR products were synthesized by RevertAid First Strand cDNA Synthesis were amplified from Dnase-treated RNA using specific Kit (Fermentas Canada Inc.). PCR was carried out using the primers for POX and CAT to ensure no DNA contamination. primers listed in Table 1. The PCR program was: 30 s at 95 C, The mRNA expressions of foreign were kept at high 30 s at 56 C, and 30 s at 72 C for 30 cycles. levels after different heat shock treatments (Fig. 2B). The selected house-keeping gene atpB gene showed consistent 2.4. Algal culture and growth measurement high expression levels (Fig. 2B). These results indicated that the POX and CAT genes were integrated into chloroplastic C. reinhardtii strain cc503 and transgenic strain ccHPC were DNA of the transgenic algae and transcribed with high effi- grown photoheterotrophically on TAP agar plates or in TAP ciency after continuous heat shock treatments. liquid medium under continuous illumination (about 75 mmol photons m 2 s 1)at25C. To induce expression of POX 3.2. Growth rate of the transgenic algae and CAT, heat shock treatments were performed at 40 C for 1 h every 6 h. Cell density was checked by measuring absorbance We examined the growth rate to analyze whether the expres- at 750 nm. sion of two foreign genes affected the growth of algal cells. The international journal of hydrogen energy 36 (2011) 10648e10654 10651

e Fig. 2 e Identification of the transgenic algae by PCR and Fig. 4 Photosynthesis light response curves of the RT-PCR. (A) Treatment of C. reinhardtii to identify succesful transgenic algae ccHPC (circles) and wild type algae cc503 transformation. Sample after heat shock. Red: 40 C heat (triangles) using a Clark-type oxygen electrode. All data are shock for 1 h; White: under the light; Black: in the dark. (B) the mean of three independent experiments with PCR analysis. Lane 1: cc503 DNA; Lane 2: ccHPC DNA; triplicates performed for each experiment. The bars Lane3: cc503 cDNA (HS-A); Lane4: ccHPC cDNA (HS-A); represent the standard errors at each light level. The Lane5: cc503 cDNA (HS-B); Lane6: ccHPC cDNA (HS-B); oxygen evolution rates are statistically different between Lane7: cc503 cDNA (HS-C); Lane8: ccHPC cDNA (HS-C). For the wild-type and transgenic strains. interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article. transgenic algae cells, oxygen evolution rates of algal cells were measured by a Clark-type oxygen electrode. The results showed that oxygen evolution rates of transgenic algae were absorbance at 750 nm was used to estimate cell density, the significantly decreased at different light levels (Fig. 4). At change of which was correlated with growth rate. The growth m 2 1 30 mol photons m s illumination, the O2 consumption rate of transgenic algae ccHPC was slightly slower compared to m 1 1 rate of ccHPC was about 25 mol O2 mg Chl h , while the wild type cc503 (Fig. 3). No statistically significant difference in m 1 1 wild type had a rate of about 11 mol O2 mg Chl h .At chlorophyll content was identified between transgenic strain m 2 1 100 mol photons m s illumination, the difference in O2 ccHPC and wild type strain cc503 (data not shown). evolution rates is much smaller than that at 30 mmol photons m 2 s 1 illumination. 3.3. Reduced oxygen evolution of the transgenic algae

To examine whether the POX expression in the Chlamydomo- 3.4. Hydrogen production in the transgenic algae nas chloroplast affects oxygen concentration inside After algae cells were incubated in the dark for 24 h, the content of dissolved oxygen in the algal cultures gradually decreased. The oxygen content of the top air space of transgenic algal cultures was lower than that of wild type algal cultures, both under low light (30 mmol photons m 2 s 1, Fig. 5A) and high light (100 mmol photons m 2 s 1, Fig. 5A). The hydrogen production of transgenic algae was higher than of the wild type under low light (30 mmol photons m 2 s 1, Fig. 5C). After 48 h, the hydrogen production in the sealed culture of the transgenic algae was about three times higher than the wild type algae under low light. Hydrogen production had a low efficiency under high light (100 mmol photons m 2 s 1, Fig. 5D), either in transgenic algae or in the wild type. No statistical difference was detected between the hydrogen production rate under high light between the wild type and the transgenic algae cells.

Fig. 3 e Growth curve of the wild-type cc503 and transgenic strain ccHPC under continuous illumination. Every 6 h the 4. Discussion temperature was changed to 40 C for 1 h to induce heat shock. The bars represent the standard errors at each time. The hydrogenase of C. reinhardtii is irreversibly inactivated The differences between growth rates in the wild-type and in the presence of oxygen [17]. In an anaerobic environment transgenic strains are not statistically different. the photosynthetic hydrogen production in C. reinhardtii is 10652 international journal of hydrogen energy 36 (2011) 10648e10654

e Fig. 5 Comparison of O2 content (A and B) and H2 production (C and D) between transgenic algae ccHPC (triangles) and wild L L type algae cc503 (circles) under dark respiration-induced anaerobic conditions under low light (30 mmol photons m 2 s 1, L L a and c) and high light (100 mmol photons m 2 s 1, b and d). The bars represent the standard errors for each light level. The p values for the strain effect obtained using the two-way ANOVA are: Oxygen content: under low light, p [ 0.0003; under high L light, p [ 0.47; Accumulated hydrogen content: under low light, p [ 8 3 10 7; under high light, p [ 0.73.

e induced [41 43]. Sulfur deprivation inhibits the photosyn- [44]. Furthermore it is also oxidized to acetyl-CoA and CO2 by thetic oxygen evolution and also slightly decreases respira- the complex (PDC), a cluster of tion, and therefore can assist in the formation of an anaerobic enzymes located in the mitochondria of eukaryotic cells and state [18]. However, inhibiting photosynthesis also decreased in the cytosol of prokaryotes. While PDC and PFL are regarded production of the protons and electrons required for hydrogen as major enzymes catalyzing pyruvate oxidation to acetyl- production. Furthermore, sulfur deprivation also decreases CoA under aerobic and anaerobic conditions in E. coli [45,46], the lifetime of algae [16,24]. To obtain a sustainable hydrogen pyruvate oxidase is usually regarded as a non-essential photoproduction in C. reinhardtii, many researchers have been enzyme [30]. In our study, the transgenic algae which focusing on creating an oxygen-tolerant hydrogenase [20].In expressed foreign POX have low oxygen evolution (Fig. 4), but this study we designed a novel method to decrease the oxygen did not show a significant inhibition in growth rate (Fig. 3). concentration inside the cell. Specifically, we introduced This shows that POX in C. reinhardtii improves hydrogen a fused pyruvate oxidase and catalase gene cassette into the production without decreasing growth. On this aspect, it is chloroplast of C. reinhardtii. Under the low light conditions, also worth noting that previous research demonstrated that transgenic algal cells evolved less oxygen than wild type C. PoxB (POX in E. coli) is used preferentially at low growth rates reinhardtii cc503 (Fig. 4) and correspondingly the hydrogen and that E. coli benefited from being able to convert pyruvate production rate dramatically increased (Fig. 5). to acetyl-CoA [2]. In this study, the foreign genes in transgenic algae were In this study, though the oxygen evolution rate was increased driven by a heat shock inducible promoter. Under normal by expressing POX in C. reinhardtii at all light levels, we did not temperature (25 C), the foreign genes will not be expressed. obtain a statistically significant enhanced rate of hydrogen After continuous heat shock treatments, POX and CAT were production under high light (100 mmol photons m 2 s 1, Fig. 5D). expressed at high levels (Fig. 2). We also analyzed the pyruvate This might be because under low light levels, oxygen evolution concentration, and did not find a significant difference rate is low; therefore enhancing oxygen consumption rate can between transgenic and wild type. The pyruvate concentra- greatly influence the oxygen concentration in the algae cell tion of cc503 and ccHPC is 0.167 0.026 fmol/cell and culture. Under high light levels, the rate of oxygen generation 0.167 0.010 fmol/cell, respectively. This might because there is high; therefore much greater enhancement of oxygen are many reactions that pyruvate participates in and its consumption rate is required to generate the same degree of concentration might be well regulated. For example, in C. decrease in the oxygen content in the algae cell culture as under reinhardtii, pyruvate can be oxidized by pyruvate formatelyase low light. (PFL) and pyruvate-ferredoxin (PFR), and also Though this new approach holds potential to enhance the be generated by pyruvate kinase (PYK) and malic enzyme (ME) hydrogen production, the rate of hydrogen production international journal of hydrogen energy 36 (2011) 10648e10654 10653

Table 2 e Comparison of the maximal hydrogen production rates using different methods with different algae strains. Maximal hydrogen production (ml hydrogen/L culture)

Expression of the pyruvate cc503 0.411 0.488 oxidation pathway (this work) ccHPC 1.056 0.553 light level (mmol photons m 2 s 1) 30 100 cell density (mg chl/L) 10 10 cell density (cells/L) 5 10 6 5 10 6 Sulfur concentration (mM) 405 405 -S culture method [18] cc137 139 light level (mmol photons m 2 s 1) 200 cell density (cells/L) 6 10 6 Sulfur concentration (mM) 0 Expression of lehemoglaobin cc849 1.2 3 Gene [48] light level (mmol photons m 2 s 1) 100 100 cell density (mg chl/L) 7.5 7.5 cell density (cells/L) 4 10 6 4 10 6 Sulfur concentration (mM) 100 0 achieved is still much lower compared to that obtained in -S correspondingly increase the hydrogen production. Much treatment (Table 2). This might be partially due to different more work is still needed to further optimize this approach to chlamydomonas strains and different culture medium used. enhance the hydrogen production efficiency. However, compared to the wild-type, the transgenic strain increased the hydrogen production rate by about 2 times, suggesting that this new methodology still holds potential as another additional approach to increase hydrogen produc- Acknowledgment tion. There are a few strategies to further improve the hydrogen production efficiency of the transgenic POX strain. We thank Professor Michel Goldschmidt-Clermont for kindly The first approach is to use a constitutive promoter to drive providing plasmid pcg40. We thank Ms. Boom Carolina for POX expression. While the heat shock inducing promoter proof-reading. This work was supported by the Postdoctoral can induce foreign enzymes to express at a high level 2 h Research Program of Shanghai Institutes for Biological after heat shock and keep the activity high for 6 h [37], Sciences, Chinese Academy of Sciences. continuous heat shock causes damage to the cell. A consti- tutive promoter would drive foreign genes to express at high level without adding external stress. The second approach is references to express a codon-optimized fragment of POX. A proof of concept of using such an optimized codon to improve enzyme expression has been shown in a study, where [1] Edwards PP, Kuznetsov VL, David WIF. Hydrogen energy. dramatically increased GFP expression level, i.e. with 80-fold Philos Trans R Soc Lond B Biol Sci 2007;365(1853):1043e56. increase in C. reinhardtii, has been achieved by optimizing its [2] Abdel-Hamid AM, Attwood MM, Guest JR. Pyruvate oxidase codon bias [47]. Both approaches can potentially increase the contributes to the aerobic growth efficiency of Escherichia hydrogen production. Thirdly, new approaches to combine coli. Microbiology 2001;147(6):1483e98. the advantage of both the current transgenic approach [3] Akkerman I, Janssen M, Rocha J, Wijffels RH. Photobiological with previous engineering or culturing methods might be hydrogen production: photochemical efficiency and bioreactor design. Int J Hydrogen Energy 2001;27(11e12):1195e208. an area to concentrate future research on, given that [4] Das D, Veziroglu TN. Hydrogen production by biological these different approaches have different advantage and processes: a survey of literature. Int J Hydrogen Energy 2001; disadvantages. 26(1):13e28. [5] Levin DB, Pitt L, Love M. Biohydrogen production: prospects and limitations to practical application. Int J Hydrogen e 5. Conclusion Energy 2004;29(2):173 85. [6] Miura Y, Yagi K, Shoga M, Miyamoto K. Hydrogen production by a green alga, Chlamydomonas reinhardtii, in an alternating In this work, we designed a novel approach to decrease the light/dark cycle. Biotechnol Bioeng 1982;24(7):1555e63. oxygen levels in C. reinhardtii and increase hydrogen produc- [7] Kumar D, Kumar HD. Light-dependent acetylene reduction tion without decreasing any essential ingredients of the and hydrogen production by the cyanobacterium, Anabaena culture medium. This approach holds potential to become sp. (strain CA). Int J Hydrogen Energy 1988;13(9):573e5. a new approach towards sustainable large scale production of [8] Sarkar S, Pandey KD, Kashyap AK. Hydrogen hydrogen using C. reinhardtii. The advantage of this engi- photoproduction by filamentous non-heterocystous cyanobacterium Plectonema boryanum and simultaneous neering approach is that the modified cells have a higher release of ammonia. Int J Hydrogen Energy 1992;17(9):689e94. oxygen consumption rate without disrupting the photosyn- [9] Hillmer P, Gest H. H2 metabolism in the photosynthetic thetic rate. Results from this work suggest that this new bacterium Rhodopseudomonas capsulata: H2 production by approach can decrease the cellular oxygen concentration and growing cultures. J Bacteriol 1977;129(2):724e31. 10654 international journal of hydrogen energy 36 (2011) 10648e10654

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