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  • 1.
    Agervald, Åsa
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Baebprasert, Wipawee
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Zhang, Xiaohui
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Incharoensakdi, Aran
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Stensjö, Karin
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    The CyAbrB transcription factor CalA regulates the iron superoxide dismutase in Nostoc sp. strain PCC 71202010In: Environmental Microbiology, ISSN 1462-2912, E-ISSN 1462-2920, Vol. 12, no 10, p. 2826-2837Article in journal (Refereed)
    Abstract [en]

    P>In the present investigation the results of induced over-production of the CyAbrB transcription factor CalA (Cyanobacterial AbrB-like, annotated as Alr0946) in the cyanobacterium Nostoc sp. PCC 7120 were analysed. The CalA overexpression strain showed a bleaching phenotype with lower growth rate and truncated filaments 2 days after induction of overexpression. The phenotype was even more pronounced when illumination was increased from 35 to 125 mu mol m-2 s-1. Using gel-based quantitative proteomics, the induced overexpression of CalA was shown to downregulate the abundance of FeSOD, one of two types of superoxide dismutases in Nostoc sp. PCC 7120. The change in protein abundance was also accompanied by lower transcript as well as activity levels. Purified recombinant CalA from Nostoc sp. PCC 7120 was shown to interact with the promoter region of alr2938, encoding FeSOD, indicating a transcriptional regulation of FeSOD by CalA. The bleaching phenotype is in line with a decreased tolerance against oxidative stress and indicates that CalA is involved in regulation of cellular responses in which FeSOD has an important and specific function in the filamentous cyanobacterium Nostoc sp. PCC 7120.

  • 2.
    Agervald, Åsa
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Camsund, Daniel
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Stensjö, Karin
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    CRISPR in the extended hyp-operon of the cyanobacterium Nostoc sp. strain PCC 7120, characteristics and putative function(s)2012In: International journal of hydrogen energy, ISSN 0360-3199, E-ISSN 1879-3487, Vol. 37, no 10, p. 8828-8833Article in journal (Refereed)
    Abstract [en]

    The presence of small RNAs (sRNA) and their functions in transcriptional regulation has lately turned into a hot topic. Since cyanobacteria often face changes in the surrounding environment, they need to have a well working system for stress response. Quick adaption is necessary, and an RNA-based regulatory system is thus useful. One example of these sRNAs is CRISPRs. In this work we report the existence of a CRISPR within the hyp-operon (hyp genes encode proteins responsible for the maturation of hydrogenases) of the filamentous cyanobacterium Nostoc sp. strain PCC 7120. We present data concerning its characteristics and putative function(s) and raise the question concerning the importance of this CRISPR array and other CRISPR systems in general. In addition, we discuss the use of the CRISPR system as a potential bacterial genetic defence mechanism to achieve robust, cyanobacterial cultures in large scale, commercial production units.

  • 3.
    Agervald, Åsa
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science.
    Stensjö, Karin
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science.
    Holmqvist, Marie
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science.
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science.
    Transcription of the extended hyp-operon in Nostoc sp. strain PCC 71202008In: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 8, p. 69-Article in journal (Refereed)
    Abstract [en]

    Background: The maturation of hydrogenases into active enzymes is a complex process and e. g. a correctly assembled active site requires the involvement of at least seven proteins, encoded by hypABCDEF and a hydrogenase specific protease, encoded either by hupW or hoxW. The N2fixing cyanobacterium Nostoc sp. strain PCC 7120 may contain both an uptake and a bidirectional hydrogenase. The present study addresses the presence and expression of hypgenes in Nostoc sp. strain PCC 7120. Results: RTPCRs demonstrated that the six hypgenes together with one ORF may be transcribed as a single operon. Transcriptional start points (TSPs) were identified 280 bp upstream from hypF and 445 bp upstream of hypC, respectively, demonstrating the existence of several transcripts. In addition, five upstream ORFs located in between hupSL, encoding the small and large subunits of the uptake hydrogenase, and the hypoperon, and two downstream ORFs from the hypgenes were shown to be part of the same transcript unit. A third TSP was identified 45 bp upstream of asr0689, the first of five ORFs in this operon. The ORFs are annotated as encoding unknown proteins, with the exception of alr0692 which is identified as a NifUlike protein. Orthologues of the four ORFs asr0689alr0692, with a highly conserved genomic arrangement positioned between hupSL, and the hyp genes are found in several other N2fixing cyanobacteria, but are absent in non N2fixing cyanobacteria with only the bidirectional hydrogenase. Short conserved sequences were found in six intergenic regions of the extended hypoperon, appearing between 11 and 79 times in the genome. Conclusion: This study demonstrated that five ORFs upstream of the hypgene cluster are cotranscribed with the hypgenes, and identified three TSPs in the extended hypgene cluster in Nostoc sp. strain PCC 7120. This may indicate a function related to the assembly of a functional uptake hydrogenase, hypothetically in the assembly of the small subunit of the enzyme.

  • 4.
    Agervald, Åsa
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Zhang, Xiaohui
    Department of Biological Sciences, Purdue University.
    Stensjö, Karin
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Devine, Ellenor
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    CalA, a cyanobacterial AbrB protein, interacts with the upstream region of hypC and acts as a repressor of its transcription in the cyanobacterium Nostoc sp. strain PCC 71202010In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 76, no 3, p. 880-890Article in journal (Refereed)
    Abstract [en]

    The filamentous, heterocystous, nitrogen-fixing cyanobacterium Nostoc sp. strain PCC 7120 may contain, depending on growth condition, up to two hydrogenases directly involved in hydrogen metabolism. HypC is one out of at least seven auxiliary gene products required for synthesis of a functional hydrogenase, specifically involved in the maturation of the large subunit. In this study we present a protein, Alr0946, belonging to the transcription regulator family AbrB, which in protein-DNA assays was found to interact with the upstream region of hypC. Transcriptional investigations showed that alr0946 is co-transcribed with the downstream gene alr0947, which encodes a putative protease from the abortive infection superfamily, Abi. Alr0946 was shown to interact specifically not only with the upstream region of hypC but also with its own upstream region, acting as a repressor on both. The bidirectional hydrogenase activity was significant down-regulated when Alr0946 was over-expressed demonstrating a correlation to the transcription factor, either direct or indirect. In silico studies showed that homologues to both Alr0946 and Alr0947 are highly conserved proteins within cyanobacteria with a very similar physical organisation of the corresponding structural genes. Possible functions of the co-transcribed downstream protein Alr0947 are presented. In addition, we present a 3D model of the CyAbrB domain of Alr0946 and putative DNA-binding mechanisms are discussed.

  • 5. Angermayr, S. Andreas
    et al.
    Hellingwerf, Klaas J.
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science.
    de Mattos, M. Joost Teixeira
    Energy biotechnology with cyanobacteria2009In: Current Opinion in Biotechnology, ISSN 0958-1669, E-ISSN 1879-0429, Vol. 20, no 3, p. 257-263Article, review/survey (Refereed)
    Abstract [en]

    The world's future energy demand calls for a sustainable alternative for the use of fossil fuels, to restrict further global warming. Harvesting solar energy via photosynthesis is one of Nature's remarkable achievements. Existing technologies exploit this process for energy 'production' via processing of, for example, part of plant biomass into ethanol, and of algal biomass into biodiesel. Fortifying photosynthetic organisms with the ability to produce biofuels directly would bypass the need to synthesize all the complex chemicals of 'biomass'. A promising way to achieve this is to redirect cyanobacterial intermediary metabolism by channeling (Calvin cycle) intermediates into fermentative metabolic pathways. This review describes this approach via the biosynthesis of fermentation end products, like alcohols and hydrogen, driven by solar energy, from water (and CO2).

  • 6.
    Antal, Taras
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics. Fysiologisk botanik.
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics. Fysiologisk botanik.
    Production of H2 by sulphur-deprived cells of the unicellular cyanobacteria Gloeocapsa alpicola and Synechocystis sp. PCC 6803 during dark incubation with methane or a various extracellular pH2005In: Journal of Applied Microbiology, Vol. 98, p. 114-120Article in journal (Refereed)
    Abstract [en]

    Aims: To examine sulphur (S) deprivation in combination with the presence of methane (CH4) and changes in extracellular pH as a method to enhance in situ hydrogen (H2) generation during fermentation in the unicellular non-diazotrophic cyanobacteria Gloeocapsa alpicola and Synechocystis PCC 6803.

    Methods and Results: The level of H2 production, measured using a gas chromatography, was determined in S-deprived cells of G. alpicola and Synechocystis PCC 6803 during fermentation. Starvation on S enhanced the rate of H2 production by more than fourfold in both strains. S-deprived cyanobacteria were able to maintain maximum rate of H2 production during at least 8 h of fermentaion representing the entire dark period of a day. Increased H2 production was observed during dark anoxic incubation with a gas phase of 100% CH4 (up to four times) at lower pH of the medium (5.0-5.5)

    Conclusions: S-deprivation in combination with CH4, added or maybe produced by another micro-organisms, and the changes in the pH of the media can be used to further increase the specific capacity of unicellular non-N2-fixing cyanobacteria to produce H2 during fermentaion with the overall aim of applying it for outdoor photobiological H2 production.

    Significance and Impact of the Studies: S-deprivation with respect to H2 production is well studied in the green algae Chlamydomonas reinhardtii while its application for H2 production of cyanobacteria is novel. Similarly, the stimulation of H2 generation in the presence of CH4 opens up new possibilities to increase the H2 production. Natural gas enriched with H2 seems to be a perspective fuel and may be an intermediate step on the pathway to the exploitation of pure biohydrogen.

  • 7. Antal, Taras
    et al.
    Oliveira, Paulo
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science.
    Lindblad, Peter
    The bidirectional hydrogenase in the cyanobacterium Synechocystis sp. strain PCC 68032006In: International journal of hydrogen energy, ISSN 0360-3199, E-ISSN 1879-3487, Vol. 31, no 11, p. 1439-1444Article in journal (Refereed)
  • 8.
    Axelsson, Rikard
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolutionary Biology.
    Lindblad, Peter
    Transcriptional Regulation of Nostoc Hydrogenases: Effects of Hydrogen, Oxygen and Nickel.2002In: Applied and environmental microbiology, ISSN 0099-2240, Vol. 68, p. 444-447Article in journal (Refereed)
  • 9.
    Badary, Amr
    et al.
    Tokyo Univ Agr & Technol, Grad Sch Engn, Dept Biotechnol & Life Sci, 2-24-16 Naka Cho, Koganei, Tokyo 1848588, Japan.;JST, CREST, 2-24-16 Naka Cho, Koganei, Tokyo 1848588, Japan..
    Takamatsu, Shouhei
    Tokyo Univ Agr & Technol, Grad Sch Engn, Dept Biotechnol & Life Sci, 2-24-16 Naka Cho, Koganei, Tokyo 1848588, Japan.;JST, CREST, 2-24-16 Naka Cho, Koganei, Tokyo 1848588, Japan..
    Nakajima, Mitsuharu
    JST, CREST, 2-24-16 Naka Cho, Koganei, Tokyo 1848588, Japan.;Tokyo Univ Agr & Technol, Inst Global Innovat Res, 3-8-1 Harumi Cho, Fuchu, Tokyo 1838538, Japan..
    Ferri, Stefano
    JST, CREST, 2-24-16 Naka Cho, Koganei, Tokyo 1848588, Japan.;Shizuoka Univ, Dept Appl Chem & Biochem Engn, Naka Ku, 3-5-1 Johoku, Hamamatsu, Shizuoka 4328561, Japan..
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Sode, Koji
    Tokyo Univ Agr & Technol, Grad Sch Engn, Dept Biotechnol & Life Sci, 2-24-16 Naka Cho, Koganei, Tokyo 1848588, Japan.;JST, CREST, 2-24-16 Naka Cho, Koganei, Tokyo 1848588, Japan.;Tokyo Univ Agr & Technol, Inst Global Innovat Res, 3-8-1 Harumi Cho, Fuchu, Tokyo 1838538, Japan..
    Glycogen Production in Marine Cyanobacterial Strain Synechococcus sp NKBG 15041c2018In: Marine Biotechnology, ISSN 1436-2228, E-ISSN 1436-2236, Vol. 20, no 2, p. 109-117Article in journal (Refereed)
    Abstract [en]

    An important feature offered by marine cyanobacterial strains over freshwater strains is the capacity to grow in seawater, replacing the need for often-limited freshwater. However, there are only limited numbers of marine cyanobacteria that are available for genetic manipulation and bioprocess applications. The marine unicellular cyanobacteria Synechococcus sp. strain NKBG 15041c (NKBG15041c) has been extensively studied. Recombinant DNA technologies are available for this strain, and its genomic information has been elucidated. However, an investigation of carbohydrate production, such as glycogen production, would provide information for inevitable biofuel-related compound production, but it has not been conducted. In this study, glycogen production by marine cyanobacterium NKBG15041c was investigated under different cultivation conditions. NKBG15041c yielded up to 399 mu g/ml/OD730 when cells were cultivated for 168 h in nitrogen-depleted medium (marine BG11(Delta N)) after medium replacement (336 h after inoculation). Cultivation under nitrogen-limited conditions also yielded an accumulation of glycogen in NKBG15041c cells (1 mM NaNO3, 301 mu g/ml/OD730; 3 mM NaNO3, 393 mu g/ml/OD730; and 5 mM NaNO3, 328 mu g/ml/OD730) under ambient conditions. Transcriptional analyses were carried out for 13 putative genes responsible for glycogen synthesis and catabolism that were predicted based on homology analyses with Synechocystis sp. PCC 6803 (PCC6803) and Synechococcus sp. PCC7002 (PCC7002). The transcriptional analyses revealed that glycogen production in NKBG15041c under nitrogen-depleted conditions can be explained by the contribution of both increased carbon flux towards glycogen synthesis, similar to PCC6803 and PCC7002, and increased transcriptional levels of genes responsible for glycogen synthesis, which is different from the conventionally reported phenomenon, resulting in a relatively high amount of glycogen under ambient conditions compared to PCC6803 and PCC7002.

  • 10.
    Baebprasert, Wipawee
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Molecular Biomimetics.
    Jantaro, Saowarath
    Khetkorn, Wanthanee
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Molecular Biomimetics.
    Incharoensakdi, Aran
    Increased H(2) production in the cyanobacterium Synechocystis sp strain PCC 6803 by redirecting the electron supply via genetic engineering of the nitrate assimilation pathway2011In: Metabolic engineering, ISSN 1096-7176, E-ISSN 1096-7184, Vol. 13, no 5, p. 610-616Article in journal (Refereed)
    Abstract [en]

    The unicellular cyanobacterium Synechocystis sp. strain PCC 6803 contains a single bidirectional NiFe-Hox-hydrogenase, which evolves hydrogen under certain environmental conditions. The nitrate assimilation pathway is a potential competing pathway that may reduce the electron flow to the hydrogenase and thereby limit hydrogen production. To improve H(2) production, the nitrate assimilation pathway was disrupted by genetic engineering to redirect the electron flow towards the Hox-hydrogenase. Mutant strains disrupted in either nitrate reductase (Delta narB) or nitrite reductase (Delta nirA) or both nitrate reductase and nitrite reductase (Delta narB:Delta nirA) were constructed and tested for their ability to produce hydrogen. H(2) production and Hox-hydrogenase activities in all the mutant strains were higher than those in wild-type. Highest H(2) production was observed in the Delta narB:Delta nirA strain. Small changes were observed for Hox-hydrogenase enzyme activities and only minor changes in transcript levels of hoxH and hoxY were not correlated with H(2) production. The results suggest that the high rate of H(2) production observed in the Delta narB:Delta nirA strain of the cyanobacterium Synechocystis sp. strain PCC 6803 is the result of redirecting the electron supply from the nitrate assimilation pathway, through genetic engineering, towards the Hox-hydrogenase.

  • 11.
    Baebprasert, Wipawee
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Molecular Biomimetics.
    Karnchanatat, Aphichart
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Molecular Biomimetics.
    Incharoensakdi, Aran
    Na(+)-stimulated nitrate uptake with increased activity under osmotic upshift in Synechocystis sp strain PCC 68032011In: World Journal of Microbiology & Biotechnology, ISSN 0959-3993, E-ISSN 1573-0972, Vol. 27, no 10, p. 2467-2473Article in journal (Refereed)
    Abstract [en]

    In the non-diazotrophic cyanobacterium Synechocystis sp. strain PCC 6803, an osmolality of 30 and 40 mosmol/kg sorbitol and NaCl resulted in 3.5- and 4.5-fold increase of nitrate uptake, respectively. The NaCl-stimulated uptake was abolished by treatment with chloramphenicol. At 25 mosmol/kg or higher, NaCl induced higher nitrate uptake than sorbitol suggesting an ionic effect of Na(+). The nitrate uptake in Synechocystis showed K (s) and V (max) values of 46 mu M and 1.37 mu mol/min/mg Chl, respectively. Mutants disrupted in nitrate and nitrite reductase exhibited a decreased nitrate uptake. Ammonium, chlorate, and dl-glyceraldehyde caused a reduction of nitrate uptake. Dark treatment caused a drastic reduction of uptake by 70% suggesting an energy-dependent system. Nitrate transport was sensitive to various metabolic inhibitors including those dissipating proton gradients and membrane potential. The results suggest that nitrate uptake in Synechocystis is stimulated by Na(+) ions and requires energy provided by the functioning electron transport chain.

  • 12. Baebprasert, Wipawee
    et al.
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Incharoensakdi, Aran
    Response of H-2 production and Hox-hydrogenase activity to external factors in the unicellular cyanobacterium Synechocystis sp strain PCC 68032010In: International journal of hydrogen energy, ISSN 0360-3199, E-ISSN 1879-3487, Vol. 35, no 13, p. 6611-6616Article in journal (Refereed)
    Abstract [en]

    The effects of external factors on both H-2 production and bidirectional Hox-hydrogenase activity were examined in the non-N-2-fixing cyanobacterium Synechocystis PCC 6803. Exogenous glucose and increased osmolality both enhanced H-2 production with optimal production observed at 0.4% and 20 mosmol kg(-1), respectively. Anaerobic condition for 24 h induced significant higher H(2)ase activity with cells in BC11(0) showing highest activities. Increasing the pH resulted in an increased Hox-hydrogenase activity with an optimum at pH 7.5. The Hox-hydrogenase activity gradually increased with increasing temperature from 30 degrees C to 60 degrees C with the highest activity observed at 70 degrees C. A low concentration at 100 mu M of either DTT or beta-mercaptoethanol resulted in a minor stimulation of H-2 production. beta-Mercaptoethanol added to nitrogen- and sulfur-deprived cells stimulated H-2 production significantly. The highest Hox-hydrogenase activity was observed in cells in BG11(0)-S-deprived condition and 750 mu M beta-mercaptoethanol measured at a temperature of 70 degrees C; 14.32 mu mol H-2 mg chl alpha(-1) min(-1).

  • 13. Berg, Andreas
    et al.
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Svensson, Bo Håkan
    Cyanobacteria as a source of hydrogen for methane formation2014In: World Journal of Microbiology & Biotechnology, ISSN 0959-3993, E-ISSN 1573-0972, Vol. 30, no 2, p. 539-545Article in journal (Refereed)
    Abstract [en]

    In a study during the 1970s co-variation of nitrogenase activity and methane formation associated with Sphagnum riparium was observed. This was suggested as evidence for a possible mechanism of hydrogen transfer from cyanobacteria to methanogens. We show experimentally that such a pathway is feasible. In a series of laboratory experiments, using a hydrogenase deficient strain of the heterocystous cyanobacterium Nostoc punctiforme and the hydrogenotrophic methanogen Methanospirillum hungateii in co-cultures, increasing light intensities resulted in elevated nitrogenase activity and methane production. The increase in methane production can be directly deduced from the nitrogenase activity of the N. punctiforme based on hydrogen balance calculations. These experimental results clearly suggest the possible existence of a novel photosynthetically regulated pathway for methane formation.

  • 14.
    Bhaya, Devaki
    et al.
    Carnegie Inst Sci, Stanford, CA 94305 USA..
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Emerging technologies illuminate facets of photosynthesis in cyanobacteria2015In: Photosynthesis Research, ISSN 0166-8595, E-ISSN 1573-5079, Vol. 126, no 1, p. 1-2Article in journal (Other academic)
  • 15.
    Camsund, Daniel
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Devine, Ellenor
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Holmqvist, Marie
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Peter, Yohanoun
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Stensjö, Karin
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    A HupS-GFP fusion protein demonstrates a heterocyst specific localisation of the uptake hydrogenase in the cyanobacterium Nostoc punctiformeIn: FEMS Microbiology Letters, ISSN 0378-1097, E-ISSN 1574-6968Article in journal (Refereed)
  • 16.
    Camsund, Daniel
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Molecular Biomimetics. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Devine, Ellenor
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science.
    Holmqvist, Marie
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science.
    Yohanoun, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science.
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science.
    Stensjö, Karin
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    A HupS-GFP fusion protein demonstrates a heterocyst-specific localization of the uptake hydrogenase in Nostoc punctiforme2011In: FEMS Microbiology Letters, ISSN 0378-1097, E-ISSN 1574-6968, Vol. 316, no 2, p. 152-159Article in journal (Refereed)
    Abstract [en]

    All diazotrophic filamentous cyanobacteria contain an uptake hydrogenase that is involved in the reoxidation of H-2 produced during N-2-fixation. In Nostoc punctiforme ATCC 29133, N-2-fixation takes place in the microaerobic heterocysts, catalysed by a nitrogenase. Although the function of the uptake hydrogenase may be closely connected to that of nitrogenase, the localization in cyanobacteria has been under debate. Moreover, the subcellular localization is not understood. To investigate the cellular and subcellular localization of the uptake hydrogenase in N. punctiforme, a reporter construct consisting of the green fluorescent protein (GFP) translationally fused to HupS, within the complete hupSL operon, was constructed and transferred into N. punctiforme on a self-replicative vector by electroporation. Expression of the complete HupS-GFP fusion protein was confirmed by Western blotting using GFP antibodies. The N. punctiforme culture expressing HupS-GFP was examined using laser scanning confocal microscopy, and fluorescence was exclusively detected in the heterocysts. Furthermore, the fluorescence in mature heterocysts was localized to several small or fewer large clusters, which indicates a specificity of the subcellular localization of the uptake hydrogenase.

  • 17.
    Camsund, Daniel
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Heidorn, Thorsten
    Bioforsk, Norge.
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Design and analysis of LacI-repressed promoters and DNA-looping in a cyanobacterium2014In: Journal of Biological Engineering, ISSN 1754-1611, E-ISSN 1754-1611, Vol. 8, no 4Article in journal (Refereed)
    Abstract [en]

    Background

    Cyanobacteria are solar-powered prokaryotes useful for sustainable production of valuable molecules, but orthogonal and regulated promoters are lacking. The Lac repressor (LacI) from Escherichia coli is a well-studied transcription factor that is orthogonal to cyanobacteria and represses transcription by binding a primary lac operator (lacO), blocking RNA-polymerase. Repression can be enhanced through DNA-looping, when a LacI-tetramer binds two spatially separated lacO and loops the DNA. Ptrc is a commonly used LacI-repressed promoter that is inefficiently repressed in the cyanobacterium Synechocystis PCC 6803. Ptrc2O, a version of Ptrc with two lacO, is more efficiently repressed, indicating DNA-looping. To investigate the inefficient repression of Ptrc and cyanobacterial DNA-looping, we designed a Ptrc-derived promoter library consisting of single lacO promoters, including a version of Ptrc with a stronger lacO (Ptrc1O-proximal), and dual lacO promoters with varying inter-lacO distances (the Ptrc2O-library).

    Results

    We first characterized artificial constitutive promoters and used one for engineering a LacI-expressing strain of Synechocystis. Using this strain, we observed that Ptrc1O-proximal is similar to Ptrc in being inefficiently repressed. Further, the Ptrc2O-library displays a periodic repression pattern that remains for both non- and induced conditions and decreases with longer inter-lacO distances, in both E. coli and Synechocystis. Repression of Ptrc2O-library promoters with operators out of phase is less efficient in Synechocystis than in E. coli, whereas repression of promoters with lacO in phase is efficient even under induced conditions in Synechocystis. Two well-repressed Ptrc2O promoters were highly active when tested in absence of LacI in Synechocystis.

    Conclusions

    The artificial constitutive promoters herein characterized can be utilized for expression in cyanobacteria, as demonstrated for LacI. The inefficient repression of Ptrc and Ptrc1O-proximal in Synechocystis, as compared to E. coli, may be due to insufficient LacI expression, or differences in RNAP subunits. DNA-looping works as a transcriptional regulation mechanism similarly as in E. coli. DNA-looping contributes strongly to Ptrc2O-library repression in Synechocystis, even though they contain the weakly-repressed primary lacO of Ptrc1O-proximal and relatively low levels of LacI/cell. Hence, Synechocystis RNAP may be more sensitive to DNA-looping than E. coli RNAP, and/or the chromatin torsion resistance could be lower. Two strong and highly repressed Ptrc2O promoters could be used without induction, or together with an unstable LacI.

  • 18. Camsund, Daniel
    et al.
    Lindblad, Peter
    Engineered transcriptional systems for cyanobacterial biotechnology2014In: Frontiers in Bioengineering and Biotechnology, Vol. 2, no 40Article in journal (Refereed)
    Abstract [en]

    Cyanobacteria can function as solar-driven biofactories thanks to their ability to perform photosynthesis and the ease with which they are genetically modified. In this review, we discuss transcriptional parts and promoters available for engineering cyanobacteria. First, we go through special cyanobacterial characteristics that may impact engineering, including the unusual cyanobacterial RNA polymerase, sigma factors and promoter types, mRNA stability, circadian rhythm, and gene dosage effects. Then, we continue with discussing component characteristics that are desirable for synthetic biology approaches, including decoupling, modularity, and orthogonality.We then summarize and discuss the latest promoters for use in cyanobacteria regarding characteristics such as regulation, strength, and dynamic range and suggest potential uses. Finally, we provide an outlook and suggest future developments that would advance the field and accelerate the use of cyanobacteria for renewable biotechnology.

  • 19.
    Camsund, Daniel
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science.
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science.
    Jaramillo, Alfonso
    Genetically engineered light sensors for control of bacterial gene expression2011In: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 6, no 7, p. 826-836Article, review/survey (Refereed)
    Abstract [en]

    Light of different wavelengths can serve as a transient, noninvasive means of regulating gene expression for biotechnological purposes. Implementation of advanced gene regulatory circuits will require orthogonal transcriptional systems that can be simultaneously controlled and that can produce several different control states. Fully genetically encoded light sensors take advantage of the favorable characteristics of light, do not need the supplementation of any chemical inducers or co-factors, and have been demonstrated to control gene expression in Escherichia coli. Herein, we review engineered light-sensor systems with potential for in vivo regulation of gene expression in bacteria, and highlight different means of extending the range of available light input and transcriptional output signals. Furthermore, we discuss advances in multiplexing different light sensors for achieving multichromatic control of gene expression and indicate developments that could facilitate the construction of efficient systems for light-regulated, multistate control of gene expression.

  • 20.
    Cardona, Tanai
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Molecular Biomimetics.
    Battchikova, Natalia
    Agervald, Åsa
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Molecular Biomimetics.
    Zhang, Pengpeng
    Nagel, Erik
    Aro, Eva-Mari
    Styring, Stenbjörn
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Molecular Biomimetics.
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Molecular Biomimetics.
    Magnuson, Ann
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Molecular Biomimetics.
    Isolation and characterization of thylakoid membranes from the filamentous cyanobacterium Nostoc punctiforme2007In: Physiologia Plantarum: An International Journal for Plant Biology, ISSN 0031-9317, E-ISSN 1399-3054, Vol. 131, no 4, p. 622-634Article in journal (Refereed)
    Abstract [en]

    Nostoc punctiforme strain Pasteur Culture Collection (PCC) 73102, a sequenced filamentous cyanobacterium capable of nitrogen fixation, is used as a model organism for characterization of bioenergetic processes during nitrogen fixation in Nostoc. A protocol for isolating thylakoid membranes was developed to examine the biochem. and biophys. aspects of photosynthetic electron transfer. Thylakoids were isolated from filaments of N. punctiforme by pneumatic pressure-drop lysis. The activity of photosynthetic enzymes in the isolated thylakoids was analyzed by measuring oxygen evolution activity, fluorescence spectroscopy and ESR spectroscopy. Electron transfer was found functional in both PSII and PSI. Electron transfer measurements in PSII, using diphenylcarbazide as electron donor and 2,6-dichlorophenolindophenol as electron acceptor, showed that 80% of the PSII centers were active in water oxidn. in the final membrane prepn. Anal. of the membrane protein complexes was made by 2D gel electrophoresis, and identification of representative proteins was made by mass spectrometry. The ATP synthase, several oligomers of PSI, PSII and the NAD(P)H dehydrogenase (NDH)-1L and NDH-1M complexes, were all found in the gels. Some differences were noted compared with previous results from Synechocystis sp. PCC 6803. Two oligomers of PSII were found, monomeric and dimeric forms, but no CP43-less complexes. Both dimeric and monomeric forms of Cyt b6/f could be obsd. In all, 28 different proteins were identified, of which 25 are transmembrane proteins or membrane associated ones.

  • 21.
    Cardona, Tanai
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Molecular Biomimetics.
    Battchikova, Natalia
    Department of Biology, University of Turku.
    Zhang, Pengpeng
    Department of Biology, University of Turku.
    Stensjö, Karin
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Aro, Eva-Mari
    Department of Biology, University of Turku.
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Magnuson, Ann
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Molecular Biomimetics.
    Electron transfer protein complexes in the thylakoid membranes of heterocysts from the cyanobacterium Nostoc punctiforme2009In: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1787, no 4, p. 252-263Article in journal (Refereed)
    Abstract [en]

    Filamentous, heterocystous cyanobacteria are capable of nitrogen fixation and photoautotrophic growth. Nitrogen fixation takes place in heterocysts that differentiate as a result of nitrogen starvation. Heterocysts uphold a microoxic environment to avoid inactivation of nitrogenase, e.g. by downregulation of oxygenic photosynthesis. The ATP and reductant requirement for the nitrogenase reaction is considered to depend on Photosystem I, but little is known about the organization of energy converting membrane proteins in heterocysts. We have investigated the membrane proteome of heterocysts from nitrogen fixing filaments of Nostoc punctiforme sp. PCC 73102, by 2D gel electrophoresis and mass spectrometry. The membrane proteome was found to be dominated by the Photosystem I and ATP-synthase complexes.We could identify asignificant amount of assembled Photosystem II complexes containing the D1, D2, CP43, CP47 and PsbO proteins from these complexes. We could also measure light-driven in vitro electron transfer from Photosystem II in heterocyst thylakoid membranes. We did not find any partially disassembled PhotosystemII complexes lacking the CP43 protein. Several subunits of the NDH-1 complex were also identified. The relative amount of NDH-1M complexes was found to be higher than NDH-1L complexes, which might suggest a role for this complex in cyclic electron transfer in the heterocysts of Nostoc punctiforme.

  • 22. Carrasco, Claudio
    et al.
    Holliday, Scott D.
    Hansel, Alfred
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics. Fysiologisk botanik.
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics. Fysiologisk botanik.
    Golden, James W.
    Heterocyst-Specific Excision of the Anabaena sp. Strain PCC 7120 hupL Element requires xisC2005In: Journal of Bacteriology, Vol. 187, no 17, p. 6031-6038Article in journal (Refereed)
    Abstract [en]

    In nitrogen-limiting conditions, approximately 10% of the vegetative cells in filaments of the cyanobacterium Anabaena (Nostoc) sp. strain PCC7120 differentiate into nitrogen-fixing heterocysts. During the late stages of heterocyst differentiation, three DNA-elements, each embedded within an open reading frame, are programmed to excise from chromosome by site-specific recombination. The DNA elements are named after the genes that they interrupt: nifD, fdxN, and hupL. The nifD and fdxN elements each contain a gene, xisA or xisF, respectively, that encodes the site-specific recombinase required for programmed excision of the element. Here, we show that the xisC gene (alr0677), which is present at one end of the 9,435-bp hupL element, is required for excision of the hupL element. A strain in which the xisC gene was inactivated showed no detactable excision of the hupL element. hupL encodes the large subunit of uptake of hydrogenase. The xisC mutant forms heterocysts and grows diazotrophically, but unlike the wild type, it evolved hydrogen gas under nitrogen-fixing conditions. Overexpression of xisC from a plasmid in the wild-type background caused a low level of hupL rearrangement even in nitrogen-replete conditions. Expression of xisC in Escherichia coli was sufficient to produce rearrangement of an artificial substrate plasmid bearing the hupL elemenat recombination sites. Sequence analysis indicated that XisC is a divergent member of the phage integrase family of recombinases. Site-directed mutagenesis of xisC showed that the XisC recombinase has functional silmilarity to the phage integrase family.

  • 23.
    Costa, José Luis
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Physiological Botany.
    Martínez-Romero, Esperanza
    Lindblad, Peter
    Sequenced based data supports a single Nostoc strain in coralloid roots of cycads2004In: FEMS Microbiology Ecology, ISSN 0168-6496, Vol. 49, p. 481-487Article in journal (Refereed)
  • 24.
    Costa, José Luis
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Physiological Botany.
    Paulsrud, Per
    Lindblad, Peter
    Cyanobiont diversity within coralloid roots of selected cycad species1999In: FEMS Microbiology Ecology, ISSN 0168-6496, Vol. 28, p. 85-91Article in journal (Refereed)
  • 25.
    Costa, José Luis
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Physiological Botany.
    Paulsrud, Per
    Lindblad, Peter
    The cyanobacterial tRNA(Leu) (UAA) intron: Evolutionary patterns in a genetic marker2002In: Molecular Biology and Evolution, ISSN 0737-4038, Vol. 19, p. 850-857Article in journal (Refereed)
  • 26.
    Costa, José Luis
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Physiological Botany.
    Paulsrud, Per
    Rikkinen, Jouko
    Lindblad, Peter
    Genetic diversity of Nostoc symbionts endophytically associated with two bryophyte species2001In: Applied and Environmental Microbiology, ISSN 0099-2240, Vol. 67, p. 4393-4396Article in journal (Refereed)
  • 27.
    Costa, José-Luis
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics. Fysiologisk botanik. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Physiological Botany.
    Martínez Romero, Esperanza
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics. Fysiologisk botanik. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Physiological Botany.
    Sequence based data supports a single Nostoc strain in individual coralloid roots of cycads2004In: FEMS Microbiology: Ecology, Vol. 49, p. 481-487Article in journal (Refereed)
    Abstract [en]

    The genertic diversity of cyanobacteria associated with cycads was examined using the tRNA Leu (UAA) intron as a genetic marker. Coralloid roots of both natural populations of the cycad Macrozamia riedlei (Fischer ex Gaudichaud-Beaupré) C.A. Gardner growing in Perth, Australia and cycads growing in greenhouses, also in Perth, were used and their respective cyanobionts analyzed. Several Nostoc strains were found to be involved in this symbiosis, both in natural populations and greenhouse-orginated cycads. However, only one strain was present in individual coralloid roots and in individual plants, even when analyzing different coralloid roots from the same plant. Moreover, when examining plants growing close to each other (female plants and their respective offspring) the same cyanobacterium was consistently present in the different coralloid roots. Whether this reflects a selective mechanism or merely the availability of Nostoc strains remains to be ascertained. The high cyanobacterial diversity in coralloid roots of cycads revealed by PCR fingerprinting is, therefore, contested. In this study, the potential probems of using different methods (e.g. PCR fingerprinting) to study the genetic diversity of symbiotic cyanobacteria, is also addressed.

  • 28. Dasgupta, Chitralekha Nag
    et al.
    Gilbert, J. Jose
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Heidorn, Thorsten
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Borgvang, Stig A.
    Skjanes, Kari
    Das, Debabrata
    Recent trends on the development of photobiological processes and photobioreactors for the improvement of hydrogen production2010In: International journal of hydrogen energy, ISSN 0360-3199, E-ISSN 1879-3487, Vol. 35, no 19, p. 10218-10238Article in journal (Refereed)
    Abstract [en]

    Hydrogen production through biological routes is promising because they are environmentally friendly. Hydrogen production through biophotolysis or photofermentation is usually a two stage process. In the first stage CO2 is utilized for biomass production which is followed by hydrogen production in the second stage in anaerobic/sulfur-deprived conditions. In addition, one-stage photobiological hydrogen production process can be achieved using selected cyanobacterial strains. The major challenges confronting the large scale production of biomass/hydrogen are limited not only on the performance of the photobioreactors in which light penetration in dense cultures is a major bottleneck but also on the characteristics of the organisms. Other dependable factors include area/volume (AN) ratio, mode of agitation, temperature and gas exchange. Photobioreactors of different geometries are reported for biohydrogen production: Tubular, Flat plate, Fermentor type etc. Every reactor has its own advantages and disadvantages. Airlift, helical tubular and flat plate reactors are found most suitable with respect to biomass production. These bioreactors may be employed for hydrogen production with necessary modifications to overcome the existing bottlenecks like gas hold up, oxygen toxicity and poor agitation. This review article attempts to focus on existing photobioreactors with respect to biomass generation and hydrogen production and the steps taken to improve its performance through engineering innovation that definitely help in the future design and construction of photobioreactors.

  • 29.
    Devine, Ellenor
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science.
    Holmqvist, Marie
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science.
    Stensjö, Karin
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science.
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science.
    Diversity and transcription of proteases involved in the maturation of hydrogenases in Nostoc punctiforme ATCC 29133 and Nostoc sp strain PCC 71202009In: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 9, p. 53-Article in journal (Refereed)
    Abstract [en]

    Background: The last step in the maturation process of the large subunit of [NiFe]-hydrogenases is a proteolytic cleavage of the C-terminal by a hydrogenase specific protease. Contrary to other accessory proteins these hydrogenase proteases are believed to be specific whereby one type of hydrogenases specific protease only cleaves one type of hydrogenase. In cyanobacteria this is achieved by the gene product of either hupW or hoxW, specific for the uptake or the bidirectional hydrogenase respectively. The filamentous cyanobacteria Nostoc punctiforme ATCC 29133 and Nostoc sp strain PCC 7120 may contain a single uptake hydrogenase or both an uptake and a bidirectional hydrogenase respectively. Results: In order to examine these proteases in cyanobacteria, transcriptional analyses were performed of hupW in Nostoc punctiforme ATCC 29133 and hupW and hoxW in Nostoc sp. strain PCC 7120. These studies revealed numerous transcriptional start points together with putative binding sites for NtcA (hupW) and LexA (hoxW). In order to investigate the diversity and specificity among hydrogeanse specific proteases we constructed a phylogenetic tree which revealed several subgroups that showed a striking resemblance to the subgroups previously described for[NiFe]-hydrogenases. Additionally the proteases specificity was also addressed by amino acid sequence analysis and protein-protein docking experiments with 3D-models derived from bioinformatic studies. These studies revealed a so called "HOXBOX"; an amino acid sequence specific for protease of Hox-type which might be involved in docking with the large subunit of the hydrogenase. Conclusion: Our findings suggest that the hydrogenase specific proteases are under similar regulatory control as the hydrogenases they cleave. The result from the phylogenetic study also indicates that the hydrogenase and the protease have co-evolved since ancient time and suggests that at least one major horizontal gene transfer has occurred. This co-evolution could be the result of a close interaction between the protease and the large subunit of the[NiFe]-hydrogenases, a theory supported by protein-protein docking experiments performed with 3D-models. Finally we present data that may explain the specificity seen among hydrogenase specific proteases, the so called "HOXBOX"; an amino acid sequence specific for proteases of Hox-type. This opens the door for more detailed studies of the specificity found among hydrogenase specific proteases and the structural properties behind it.

  • 30.
    Durall de la Fuente, Claudia
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Mechanisms of carbon fixation and engineering for increased carbon fixation in cyanobacteria2015In: ALGAL RESEARCH-BIOMASS BIOFUELS AND BIOPRODUCTS, ISSN 2211-9264, Vol. 11, p. 263-270Article, review/survey (Refereed)
    Abstract [en]

    Cyanobacteria, gram-negative prokaryotic microorganisms, perform oxygenic photosynthesis with a photosynthetic machinery similar to higher plants which includes ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) as the main CO2-fixing enzyme. Currently, there is a growing interest to use cyanobacteria as photosynthetic microbial cell factories for the direct production of solar fuels or other compounds of human interest. However, rates and efficiencies to produce e.g. biofuels are still very low. The amount of available fixed carbon for the synthesis of desired product(s) may be one of the limiting steps. This contribution reviews CO2-fixation in cyanobacteria with focus on CO2-concentrating mechanisms, RuBisCO, phosphoenolpyruvate carboxylase and other carboxylases, engineering approaches for increased carbon fixation, and finally the synthetic malonyl-CoA-oxaloacetate-glyoxylate pathways.

  • 31.
    Durall de la Fuente, Claudia
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Rukminasari, Nita
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics. Hasanuddin Univ, Fac Marine Sci & Fisheries, Jl Perintis Kemerdekaan Km 10, Makassar, South Sulawesi, Indonesia..
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Enhanced growth at low light intensity in the cyanobacterium Synechocystis PCC 6803 by overexpressing phosphoenolpyruvate carboxylase2016In: ALGAL RESEARCH-BIOMASS BIOFUELS AND BIOPRODUCTS, ISSN 2211-9264, Vol. 16, p. 275-281Article in journal (Refereed)
    Abstract [en]

    Synechocystis PCC 6803 strains overexpressing pepc, gene encoding the carbon fixing enzyme phosphoenolpyruvate carboxylase (PEPc), were constructed and characterized for growth, PEPc protein content and in vitro PEPc activities. Synechocystis strains WT + Km(r) - one (native) copy of pepc (control), WT + 2xPEPc - native copy of pepc and two additional native copies of pepc (in total three copies of pepc), and WT + PPM - native copies of ppsa (encoding phosphoenolpyruvate synthase), pepc and mdh (encoding malate dehydrogenase) and one additional copy of each gene (in total two copies each of ppsa, pepc and mdh) were analyzed for growth under normal and low light intensities, and in darkness (no growth). No significant differences in the growth rates were observed when the cells were grown under normal light intensity. However, growth under low light intensity (3 mu mol photons.m(-2).sec(-1)) resulted in increased growth rate, in particular in the strain with 3 copies of pepc. SDS-PAGE/Western immunoblots using antibodies directed against PEPc demonstrated an increased level of PEPc protein with increasing number of copies of pepc. This was followed by increased levels of in vitro PEPc activities. A less efficient ribulose 1,5-bisphosphate carboxylase/oxygenase in combination with reduced levels of NADPH and ATP under low light condition may make the relatively more efficient carbon fixing enzyme PEPc the limiting step for growth under this condition.

  • 32. Elhai, Jeff
    et al.
    Kato, Michiko
    Cousins, Sarah
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Physiological Botany.
    Costa, Jose Luis
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Physiological Botany.
    Very small mobile repeated elements in cyanobacterial genomes2008In: Genome Research, ISSN 1088-9051, E-ISSN 1549-5469, Vol. 18, no 9, p. 1484-1499Article in journal (Refereed)
    Abstract [en]

    Mobile DNA elements play a major role in genome plasticity and other evolutionary processes, an insight gained primarily through the study of transposons and retrotransposons (generally similar to 1000 nt or longer). These elements spawn smaller parasitic versions (generally > ; 100 nt) that propagate through proteins encoded by the full elements. Highly repeated sequences smaller than 100 nt have been described, but they are either nonmobile or their origins are not known. We have surveyed the genome of the multicellular cyanobacterium, Nostoc punctiforme, and its relatives for small dispersed repeat (SDR) sequences and have identified eight families in the range of from 21 to 27 nucleotides. Three of the families (SDR4, SDR5, and SDR6), despite little sequence similarity, share a common predicted secondary structure, a conclusion supported by patterns of compensatory mutations. The SDR elements are found in a diverse set of contexts, often embedded within tandemly repeated heptameric sequences or within minitransposons. One element ( SDR5) is found exclusively within instances of an octamer, HIP1, that is highly over-represented in the genomes of many cyanobacteria. Two elements (SDR1 and SDR4) often are found within copies of themselves, producing complex nested insertions. An analysis of SDR elements within cyanobacterial genomes indicate that they are essentially confined to a coherent subgroup. The evidence indicates that some of the SDR elements, probably working through RNA intermediates, have been mobile in recent evolutionary time, making them perhaps the smallest known mobile elements.

  • 33. Ferreira, Daniela
    et al.
    Leitão, Elsa
    Sjöholm, Johannes
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science.
    Oliveira, Paulo
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science.
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science.
    Moradas-Ferreira, Pedro
    Tamagnini, Paula
    Transcription and regulation of the hydrogenase(s) accessory genes, hypFCDEAB, in the cyanobacterium Lyngbya majuscula CCAP 1446/42007In: Archives of Microbiology, ISSN 0302-8933, E-ISSN 1432-072X, Vol. 188, no 6, p. 609-617Article in journal (Refereed)
    Abstract [en]

    Lyngbya majuscula CCAP 1446/4 is a filamentous cyanobacterium possessing both an uptake and a bi-directional hydrogenase. The presence of a single copy of the hyp operon in the cyanobacterial genomes suggests that these accessory genes might be responsible for the maturation of both hydrogenases. We investigated the concomitant transcription of hypFCDEAB with the hydrogenases structural genes-hup and hox. RT-PCRs performed with L. majuscula cells grown under different physiological conditions showed a substantial decrease in the relative amount of hupL transcript under non-N-2-fixing conditions. In contrast, no significant differences were observed for the transcript levels of hypFCDEAB in all conditions tested, while minor fluctuations could be discerned for hoxH. Previously, it was demonstrated that the transcriptional regulators NtcA and LexA interact with the promoter regions of hup and hox, respectively, and that putative binding sites for both proteins are present in the hyp promoter of L. majuscula. Therefore, a putative involvement of NtcA and LexA in the regulation of the hyp transcription was investigated. Electrophoretic mobility shift assays resulted in NtcA or LexA-bound retarded fragments, suggesting the involvement of these proteins in the transcriptional regulation of hypFCDEAB.

  • 34. Hansel, Alfred
    et al.
    Axelsson, Rikard
    Lindberg, Pia
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolutionary Biology, Physiological Botany.
    Troshina, Olga
    Wünschiers, Röbbe
    Lindblad, Peter
    Cloning and characterization of a hyp gene cluster in the filamentous cyanobacterium Nostoc sp. strain PCC 731022001In: FEMS Microbiology Letters, ISSN 0378-1097, Vol. 201, no 1, p. 59-64Article in journal (Refereed)
  • 35.
    Heidorn, Thorsten
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Camsund, Daniel
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Huang, Hsin-Ho
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Lindberg, Pia
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Oliveira, Paulo
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Stensjö, Karin
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Synthetic Biology in Cyanobacteria: Engineering and Analyzing Novel Functions2011In: Methods in Enzymology, ISSN 0076-6879, E-ISSN 1557-7988, Vol. 497, p. 539-579Article, review/survey (Refereed)
    Abstract [en]

    Cyanobacteria are the only prokaryotes capable of using sunlight as their energy, water as an electron donor, and air as a source of carbon and, for some nitrogen-fixing strains, nitrogen. Compared to algae and plants, cyanobacteria are much easier to genetically engineer, and many of the standard biological parts available for Synthetic Biology applications in Escherichia coli can also be used in cyanobacteria. However, characterization of such parts in cyanobacteria reveals differences in performance when compared to E. coli, emphasizing the importance of detailed characterization in the cellular context of a biological chassis. Furthermore, cyanobacteria possess special characteristics (e.g., multiple copies of their chromosomes, high content of photosynthetically active proteins in the thylakoids, the presence of exopolysaccharides and extracellular glycolipids, and the existence of a circadian rhythm) that have to be taken into account when genetically engineering them. With this chapter, the synthetic biologist is given an overview of existing biological parts, tools and protocols for the genetic engineering, and molecular analysis of cyanobacteria for Synthetic Biology applications.

  • 36.
    Holmqvist, Marie
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Lindberg, Pia
    Agervald, Åsa
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Stensjö, Karin
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Transcript analysis of the extended hyp-operons in the cyanobacteria Nostoc sp. strain PCC 7120 and Nostoc punctiforme ATCC 291332011In: BMC Research Notes, ISSN 1756-0500, E-ISSN 1756-0500, Vol. 4, no 186Article in journal (Other academic)
    Abstract [en]

    The ability of cyanobacteria to capture solar energy, via oxygenic photosynthesis, and convert that energy to molecular hydrogen (H2) has made them an interesting group of organisms with potential as future energy producers. There are three types of enzymes directly involved in the cyanobacterial hydrogen metabolism; nitrogenases that produce H2 as a by-product when fixating atmospheric nitrogen, uptake hydrogenases that catalyze the oxidation of H2,thereby preventing energy losses from the cells, and bidirectional hydrogenases that has the capacity to both oxidize and reduce H2. Hydrogenases are complex metalloenzymes, and the insertion of ligands and correct folding of the proteins require assistance of accessory proteins, the Hyp proteins. Cyanobacterial hydrogenases are NiFe-type hydrogenases and consist of a large and a small subunit. Today, the maturation process of the large subunit has been uncovered to a large extent in cyanobacteria, mostly by analogy assumptions from studies done in other bacteria such as Escherichia coli but also from mutational analyses in cyanobacteria, while the maturation process of the small subunit is still unknown. Recently a set of genes, putatively involved in the maturation process of the small subunit, was discovered in Nostoc sp. PCC 7120 and Nostoc punctiforme ATCC 29133. These five ORFs, encoding unknown proteins, are located in between the uptake hydrogenase structural genes and the hyp-genes were shown to be transcribed together with the hyp-genes in Nostoc PCC 7120. The ORFs upstream the hyp-genes can be found in the same genomic arrangement in other filamentous, nitrogen fixing cyanobacterial strains but are interestingly missing in strains incapable of nitrogen fixation. In this study we have further investigated the function of the ORFs upstream the hyp-genes by studying their transcription pattern after nitrogen depletion in the filamentous, nitrogen fixing strains Nostoc PCC 7120 and N. punctiforme. The transcription pattern were compared to the transcription pattern of hupS and hoxY, encoding the uptake and bidirectional hydrogenase small subunits, nifD, encoding a nitrogenase subunit and hypC and hypF, encoding the maturation process accessory proteins HypC and HypF. All the five ORFs upstream the hyp-genes, in both organisms, were upregulated after nitrogen step down in accordance with the transcription pattern for hupS, nifD, hypC and hypF which support the theory that these genes might be involved in the maturation of the small subunit.

  • 37.
    Huang, Hsin-Ho
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Camsund, Daniel
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Heidorn, Thorsten
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Design and characterization of molecular tools for a Synthetic Biology approach towards developing cyanobacterial biotechnology2010In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 38, no 8, p. 2577-2593Article in journal (Refereed)
    Abstract [en]

    Cyanobacteria are suitable for sustainable, solar-powered biotechnological applications. Synthetic biology connects biology with computational design and an engineering perspective, but requires efficient tools and information about the function of biological parts and systems. To enable the development of cyanobacterial Synthetic Biology, several molecular tools were developed and characterized: (i) a broad-host-range BioBrick shuttle vector, pPMQAK1, was constructed and confirmed to replicate in Escherichia coli and three different cyanobacterial strains. (ii) The fluorescent proteins Cerulean, GFPmut3B and EYFP have been demonstrated to work as reporter proteins in cyanobacteria, in spite of the strong background of photosynthetic pigments. (iii) Several promoters, like P-rnpB and variants of P-rbcL, and a version of the promoter P-trc with two operators for enhanced repression, were developed and characterized in Synechocystis sp. strain PCC6803. (iv) It was shown that a system for targeted protein degradation, which is needed to enable dynamic expression studies, is working in Synechocystis sp. strain PCC6803. The pPMQAK1 shuttle vector allows the use of the growing numbers of BioBrick parts in many prokaryotes, and the other tools herein implemented facilitate the development of new parts and systems in cyanobacteria.

  • 38.
    Huang, Hsin-Ho
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Wide-dynamic-range promoters engineered for cyanobacteria2013In: Journal of Biological Engineering, ISSN 1754-1611, E-ISSN 1754-1611, Vol. 7, no 1, p. 10-Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Cyanobacteria, prokaryotic cells with oxygenic photosynthesis, are excellent bioengineering targets to convert solar energy into solar fuels. Tremendous genetic engineering approaches and tools have been and still are being developed for prokaryotes. However, the progress for cyanobacteria is far behind with a specific lack of non-native inducible promoters.

    RESULTS: We report the development of engineered TetR-regulated promoters with a wide dynamic range of transcriptional regulation. An optimal 239 (±16) fold induction in darkness (white-light-activated heterotrophic growth, 24 h) and an optimal 290 (±93) fold induction in red light (photoautotrophic growth, 48 h) were observed with the L03 promoter in cells of the unicellular cyanobacterium Synechocystis sp. strain ATCC27184 (i.e. glucose-tolerant Synechocystis sp. strain PCC 6803). By altering only few bases of the promoter in the narrow region between the -10 element and transcription start site significant changes in the promoter strengths, and consequently in the range of regulations, were observed.

    CONCLUSIONS: The non-native inducible promoters developed in the present study are ready to be used to further explore the notion of custom designed cyanobacterial cells in the complementary frameworks of metabolic engineering and synthetic biology.

  • 39.
    Khanna, Namita
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Esmieu, Charlène
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Meszaros, Livia S.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Berggren, Gustav
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    In vivo activation of an [FeFe] hydrogenase using synthetic cofactors2017In: Energy & Environmental Science, ISSN 1754-5692, E-ISSN 1754-5706, Vol. 10, no 7, p. 1563-1567Article in journal (Refereed)
    Abstract [en]

    [FeFe] hydrogenases catalyze the reduction of protons, and oxidation of hydrogen gas, with remarkable efficiency. The reaction occurs at the H-cluster, which contains an organometallic [2Fe] subsite. The unique nature of the [2Fe] subsite makes it dependent on a specific set of maturation enzymes for its biosynthesis and incorporation into the apo-enzyme. Herein we report on how this can be circumvented, and the apo-enzyme activated in vivo by synthetic active site analogues taken up by the living cell.

  • 40.
    Khanna, Namita
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Cyanobacterial Hydrogenases and Hydrogen Metabolism Revisited:: Recent Progress and Future Prospects2015In: International Journal of Molecular Sciences, ISSN 1422-0067, E-ISSN 1422-0067, Vol. 16, no 5, p. 10537-10561Article, review/survey (Refereed)
    Abstract [en]

    Cyanobacteria have garnered interest as potential cell factories for hydrogen production. In conjunction with photosynthesis, these organisms can utilize inexpensive inorganic substrates and solar energy for simultaneous biosynthesis and hydrogen evolution. However, the hydrogen yield associated with these organisms remains far too low to compete with the existing chemical processes. Our limited understanding of the cellular hydrogen production pathway is a primary setback in the potential scale-up of this process. In this regard, the present review discusses the recent insight around ferredoxin/flavodoxin as the likely electron donor to the bidirectional Hox hydrogenase instead of the generally accepted NAD(P)H. This may have far reaching implications in powering solar driven hydrogen production. However, it is evident that a successful hydrogen-producing candidate would likely integrate enzymatic traits from different species. Engineering the [NiFe] hydrogenases for optimal catalytic efficiency or expression of a high turnover [FeFe] hydrogenase in these photo-autotrophs may facilitate the development of strains to reach target levels of biohydrogen production in cyanobacteria. The fundamental advancements achieved in these fields are also summarized in this review.

  • 41.
    Khanna, Namita
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Raleiras, Patrícia
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Fundamentals and Recent Advances in Hydrogen Production and Nitrogen Fixation in Cyanobacteria2016In: The Physiology of Microalgae / [ed] Michael A. Borowitzka, John Beardall, John A. Raven, Switzerland: Springer International Publishing , 2016, p. 101-127Chapter in book (Other academic)
  • 42.
    Khetkorn, W.
    et al.
    Chulalongkorn Univ, Fac Sci, Dept Biochem, Lab Cyanobacterial Biotechnol, Bangkok 10330, Thailand.
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Incharoensakdi, A.
    Chulalongkorn Univ, Fac Sci, Dept Biochem, Lab Cyanobacterial Biotechnol, Bangkok 10330, Thailand.
    Inactivation of uptake hydrogenase leads to enhanced and sustained hydrogen production with high nitrogenase activity under high light exposure in the cyanobacterium Anabaena siamensis TISTR 80122012In: Journal of Biological Engineering, ISSN 1754-1611, E-ISSN 1754-1611, Vol. 6, no 1, article id 19Article in journal (Refereed)
    Abstract [en]

    Background: Biohydrogen from cyanobacteria has attracted public interest due to its potential as a renewable energy carrier produced from solar energy and water. Anabaena siamensis TISTR 8012, a novel strain isolated from rice paddy field in Thailand, has been identified as a promising cyanobacterial strain for use as a high-yield hydrogen producer attributed to the activities of two enzymes, nitrogenase and bidirectional hydrogenase. One main obstacle for high hydrogen production by A. siamensis is a light-driven hydrogen consumption catalyzed by the uptake hydrogenase. To overcome this and in order to enhance the potential for nitrogenase based hydrogen production, we engineered a hydrogen uptake deficient strain by interrupting hupS encoding the small subunit of the uptake hydrogenase. Results: An engineered strain lacking a functional uptake hydrogenase ([increment]hupS) produced about 4-folds more hydrogen than the wild type strain. Moreover, the [increment]hupS strain showed long term, sustained hydrogen production under light exposure with 2--3 folds higher nitrogenase activity compared to the wild type. In addition, HupS inactivation had no major effects on cell growth and heterocyst differentiation. Gene expression analysis using RT-PCR indicates that electrons and ATP molecules required for hydrogen production in the [increment]hupS strain may be obtained from the electron transport chain associated with the photosynthetic oxidation of water in the vegetative cells. The [increment]hupS strain was found to compete well with the wild type up to 50 h in a mixed culture, thereafter the wild type started to grow on the relative expense of the [increment]hupS strain. Conclusions: Inactivation of hupS is an effective strategy for improving biohydrogen production, in rates and specifically in total yield, in nitrogen-fixing cultures of the cyanobacterium Anabaena siamensis TISTR 8012.

  • 43. Khetkorn, Wanthanee
    et al.
    Baebprasert, Wipawee
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Incharoensakdi, Aran
    Redirecting the electron flow towards the nitrogenase and bidirectional Hox-hydrogenase by using specific inhibitors results in enhanced H-2 production in the cyanobacterium Anabaena siamensis TISTR 80122012In: Bioresource Technology, ISSN 0960-8524, E-ISSN 1873-2976, Vol. 118, p. 265-271Article in journal (Refereed)
    Abstract [en]

    The inhibition of competitive metabolic pathways by various inhibitors in order to redirect electron flow towards nitrogenase and bidirectional Hox-hydrogenase was investigated in Anabaena siamensis TISTR 8012. Cells grown in BG11(0) supplemented with KCN, rotenone, DCMU, and DL-glyceraldehyde under light condition for 24 h showed enhanced H-2 production. Cells grown in BG11 medium showed only marginal H-2 production and its production was hardly increased by the inhibitors tested. H-2 production with either 20 mM KCN or 50 mu M DCMU in BG11(0) medium was 22 mu mol H-2 mg chl a(-1) h(-1), threefold higher than the control. The increased H-2 production caused by inhibitors was consistent with the increase in the respective Hox-hydrogenase activities and nifD transcript levels, as well as the decrease in hupL transcript levels. The results suggested that interruption of metabolic pathways essential for growth could redirect electrons flow towards nitrogenase and bidirectional Hox-hydrogenase resulting in increased H-2 production. (C) 2012 Elsevier Ltd. All rights reserved.

  • 44.
    Khetkorn, Wanthanee
    et al.
    Chulalongkorn Univ, Dept Biochem, Lab Cyanobacterial Biotechnol, Fac Sci, Bangkok 10330, Thailand.;Rajamangala Univ Technol Thanyaburi, Fac Sci & Technol, Div Biol, Thanyaburi 12110, Pathumthani, Thailand..
    Incharoensakdi, Aran
    Chulalongkorn Univ, Dept Biochem, Lab Cyanobacterial Biotechnol, Fac Sci, Bangkok 10330, Thailand..
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Jantaro, Saowarath
    Chulalongkorn Univ, Dept Biochem, Lab Cyanobacterial Biotechnol, Fac Sci, Bangkok 10330, Thailand..
    Enhancement of poly-3-hydroxybutyrate production in Synechocystis sp PCC 6803 by overexpression of its native biosynthetic genes2016In: Bioresource Technology, ISSN 0960-8524, E-ISSN 1873-2976, Vol. 214, p. 761-768Article in journal (Refereed)
    Abstract [en]

    Synechocystis sp. PCC 6803 strains overexpressing pha genes were constructed and characterized for poly-3- hydroxybutyrate (PHB) production. These pha overexpressing strains showed slightly reduced growth rates. Under N-deprived condition, the strains overexpressing (OE) phaAB, phaEC and phaABEC showed significantly higher PHB contents than the wild type. The maximum PHB content, a 2.6-fold increase producing 26% PHB (dcw), was observed in OE phaAB cells grown for 9 days in N-deprived medium. Under this condition, these OE phaAB cells increased PHB production to 35% PHB (dcw) upon addition of 0.4% (w/v) acetate. Higher PHB granules in OE phaAB cells were clearly visualized by both Nile red staining and TEM imaging. All OE strains under N-deficient condition had increased glgX transcript levels. Overall results demonstrate an enhanced PHB production in Synechocystis cells overexpressing pha genes, particularly phaA and phaB, when grown in N-deprived medium containing 0.4% (w/v) acetate.

  • 45.
    Khetkorn, Wanthanee
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Incharoensakdi, Aran
    Enhanced biohydrogen production by the N-2-fixing cyanobacterium Anabaena siamensis strain TISTR 80122010In: International journal of hydrogen energy, ISSN 0360-3199, E-ISSN 1879-3487, Vol. 35, no 23, p. 12767-12776Article in journal (Refereed)
    Abstract [en]

    The efficiency of hydrogen production depends on several factors We focused on external conditions leading to enhanced hydrogen production when using the N-2 fixing cyanobacterium Anabaena siamensis TISTR 8012 a novel strain isolated from a rice paddy field in Thailand In this study we controlled key factors affecting hydrogen production such as cell age light intensity time of light incubation and source of carbon Our results showed an enhanced hydrogen production when cells at log phase were adapted under N-2 fixing condition using 0 5% fructose as carbon source and a continuous illumination of 200 mu E m(-2) s(-1) for 12 h under anaerobic incubation The maximum hydrogen production rate was 32 mu mol H-2 mg chl a(-1) h(-1) This rate was higher than that observed in the model organisms Anabaena PCC 7120 Nostoc punctiforme ATCC 29133 and Synechocystis PCC 6803 This higher production was likely caused by a higher nitrogenase activity since we observed an upregulation of nifD The production did not increase after 12 h which was probably due to an increased activity of the uptake hydrogenase as evidenced by an increased hupL transcript level Interestingly a proper adjustment of light conditions such as intensity and duration is important to minimize both the photodamage of the cells and the uptake hydrogenase activity Our results indicate that A siamensis TISTR 8012 has a high potential for hydrogen production with the ability to utilize sugars as substrate to produce hydrogen.

  • 46.
    Khetkorn, Wanthanee
    et al.
    Rajamangala Univ Technol Thanyaburi, Fac Sci & Technol, Div Biol, Thanyaburi 12110, Pathumthani, Thailand..
    Rastogi, Rajesh P.
    Minist Environm Forest & Climate Change, Jor Bagh Rd, New Delhi 110003, India..
    Incharoensakdi, Aran
    Chulalongkorn Univ, Fac Sci, Dept Biochem, Lab Cyanobacterial Biotechnol, Phayathai Rd, Bangkok 10330, Thailand..
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Madamwar, Datta
    Sardar Patel Univ, Dept Biosci, UGC Ctr Adv Study, Vadtal Rd,Satellite Campus, Anand 388315, Gujarat, India..
    Pandey, Ashok
    Ctr Innovat & Appl Bioproc, C-127 2nd Floor Phase 8 Ind Area, Mohali 160071, Punjab, India..
    Larroche, Christian
    Labex IMobS3, 4 Ave Blaise Pascal,TSA 60026-CS, F-63178 Aubiere, France.;Inst Pascal, 4 Ave Blaise Pascal,TSA 60026-CS, F-63178 Aubiere, France..
    Microalgal hydrogen production: A review2017In: Bioresource Technology, ISSN 0960-8524, E-ISSN 1873-2976, Vol. 243, p. 1194-1206Article, review/survey (Refereed)
    Abstract [en]

    Bio-hydrogen from microalgae including cyanobacteria has attracted commercial awareness due to its potential as an alternative, reliable and renewable energy source. Photosynthetic hydrogen production from microalgae can be interesting and promising options for clean energy. Advances in hydrogen-fuel-cell technology may attest an eco-friendly way of biofuel production, since, the use of H-2 to generate electricity releases only water as a by-product. Progress in genetic/metabolic engineering may significantly enhance the photobiological hydrogen production from microalgae. Manipulation of competing metabolic pathways by modulating the certain key enzymes such as hydrogenase and nitrogenase may enhance the evolution of H-2 from photoautotrophic cells. Moreover, biological H-2 production at low operating costs is requisite for economic viability. Several photobioreactors have been developed for large-scale biomass and hydrogen production. This review highlights the recent technological progress, enzymes involved and genetic as well as metabolic engineering approaches towards sustainable hydrogen production from microalgae.

  • 47.
    Khetorn, Wanthanee
    et al.
    Chulalongkorn University, Thailand.
    Khanna, Namita
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Incharoensakdi, Aran
    Chulalongkorn University, Thailand.
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Metabolic and genetic engineering of cyanobacteria for enhanced hydrogen production2013In: Biofuels, ISSN 1759-7269, Vol. 4, no 5, p. 535-561Article in journal (Refereed)
    Abstract [en]

    There is an urgent need to develop sustainable solutions to convert solar energy into energy carriers used in the society. In addition to solar cells generating electricity, there are several options to generate solar fuels with molecular hydrogen (H2) being an interesting and promising option. Native and engineered cyanobacteria have been used as model systems to examine, develop and demonstrate photobiological hydrogen production. In the present review we present and discuss recent progress with respect to (i) native biological systems to generate hydrogen, (ii) metabolic modulations, and (iii) genetic engineering of metabolic pathways, as well as the (iv) introduction of custom-designed, non-native enzymes and complexes for enhanced hydrogen production in cyanobacteria. In conclusion, metabolic and genetic engineering of native cyanobacterial hydrogen metabolism can significantly increase the hydrogen production, and introduction of custom-designed non-native capacities open up new possibilities to further enhance cyanobacterial based hydrogen production.

  • 48. Kruse, O.
    et al.
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Editorial - Photosynthetic microorganisms for bio-fuel production2012In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 162, no 1, p. 1-2Article in journal (Refereed)
  • 49. Kumar, Kanhaiya
    et al.
    Dasgupta, Chitralekha Nag
    Nayak, Bikram
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Molecular Biomimetics.
    Das, Debabrata
    Development of suitable photobioreactors for CO2 sequestration addressing global warming using green algae and cyanobacteria2011In: Bioresource Technology, ISSN 0960-8524, E-ISSN 1873-2976, Vol. 102, no 8, p. 4945-4953Article, review/survey (Refereed)
    Abstract [en]

    CO2 sequestration by cyanobacteria and green algae are receiving increased attention in alleviating the impact of increasing CO2 in the atmosphere. They, in addition to CO2 capture, can produce renewable energy carriers such as carbon free energy hydrogen, bioethanol, biodiesel and other valuable biomolecules. Biological fixation of CO2 are greatly affected by the characteristics of the microbial strains, their tolerance to temperature and the CO2 present in the flue gas including SOx, NOR. However, there are additional factors like the availability of light, pH, O-2, removal, suitable design of the photobioreactor, culture density and the proper agitation of the reactor that will affect significantly the CO2 sequestration process. Present paper deals with the photobioreactors of different geometry available for biomass production. It also focuses on the hybrid types of reactors (integrating two reactors) which can be used for overcoming the bottlenecks of a single photobioreactor.

  • 50. Leino, Hannu
    et al.
    Shunmugam, Sumathy
    Isojarvi, Janne
    Oliveira, Paulo
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström.
    Mulo, Paula
    Saari, Lyudmila
    Battchikova, Natalia
    Sivonen, Kaarina
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Aro, Eva-Mari
    Allahverdiyeva, Yagut
    Characterization of ten H-2 producing cyanobacteria isolated from the Baltic Sea and Finnish lakes2014In: International journal of hydrogen energy, ISSN 0360-3199, E-ISSN 1879-3487, Vol. 39, no 17, p. 8983-8991Article in journal (Refereed)
    Abstract [en]

    The genetic background and activities of the enzymes involved in H-2 production were investigated from ten distinct H-2 producing cyanobacteria, revealed by a recent screening. All strains are N-2-fixing, filamentous and heterocystous. Southern hybridization revealed that the tested strains possess the genes encoding the conventional nitrogenase (nifHDK1), and lack the alternative nitrogenases. The high H-2 production rate of these strains was shown not to be dependent on the presence of highly active nitrogenase or bidirectional hydrogenase enzymes. Moreover, most of the strains possessed a highly active uptake hydrogenase enzyme. We also examined the structure of the nif and hup operons encoding nitrogenase and uptake hydrogenase enzymes in the Calothrix 336/3 strain, the best H-2 producer in the screening. We concluded that the ability of the cyanobacteria to produce high levels of H-2 is not directly linked to the maximum capacities of the enzymes involved in H-2 production.

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