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  • 1.
    Dewez, David
    et al.
    Department of Plant and Microbial Biology, University of California, Berkeley.
    Park, Sungsoon
    Department of Plant and Microbial Biology, University of California, Berkeley.
    García-Cerdán, Jose Gines
    Department of Plant and Microbial Biology, University of California, Berkeley.
    Lindberg, Pia
    Department of Plant and Microbial Biology, University of California, Berkeley.
    Melis, Anastasios
    Department of Plant and Microbial Biology, University of California, Berkeley.
    Mechanism of REP27 protein action in the D1 protein turnover and photosystem II repair from photodamage.2009In: Plant Physiology, ISSN 0032-0889, E-ISSN 1532-2548, Vol. 151, no 1, p. 88-99Article in journal (Refereed)
    Abstract [en]

    The function of the REP27 protein (GenBank accession no. EF127650) in the photosystem II (PSII) repair process was elucidated. REP27 is a nucleus-encoded and chloroplast-targeted protein containing two tetratricopeptide repeat (TPR) motifs, two putative transmembrane domains, and an extended carboxyl (C)-terminal region. Cell fractionation and western-blot analysis localized the REP27 protein in the Chlamydomonas reinhardtii chloroplast thylakoids. A folding model for REP27 suggested chloroplast stroma localization for amino- and C-terminal regions as well as the two TPRs. A REP27 gene knockout strain of Chlamydomonas, termed the rep27 mutant, was employed for complementation studies. The rep27 mutant was aberrant in the PSII-repair process and had substantially lower than wild-type levels of D1 protein. Truncated REP27 cDNA constructs were made for complementation of rep27, whereby TPR1, TPR2, TPR1+TPR2, or the C-terminal domains were deleted. rep27-complemented strains minus the TPR motifs showed elevated levels of D1 in thylakoids, comparable to those in the wild type, but the PSII photochemical efficiency of these strains was not restored, suggesting that the functionality of the PSII reaction center could not be recovered in the absence of the TPR motifs. It is suggested that TPR motifs play a role in the functional activation of the newly integrated D1 protein in the PSII reaction center. rep27-complemented strains missing the C-terminal domain showed low levels of D1 protein in thylakoids as well as low PSII photochemical efficiency, comparable to those in the rep27 mutant. Therefore, the C-terminal domain is needed for a de novo biosynthesis and/or assembly of D1 in the photodamaged PSII template. We conclude that REP27 plays a dual role in the regulation of D1 protein turnover by facilitating cotranslational biosynthesis insertion (C-terminal domain) and activation (TPR motifs) of the nascent D1 during the PSII repair process.

  • 2.
    Englund, Elias
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Andersen-Ranberg, Johan
    Miao, Rui
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Hamberger, Björn
    Lindberg, Pia
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Metabolic Engineering of Synechocystis sp. PCC 6803 for Production of the Plant Diterpenoid Manoyl Oxide2015In: ACS Synthetic Biology, E-ISSN 2161-5063, Vol. 4, no 12, p. 1270-1278Article in journal (Refereed)
    Abstract [en]

    Forskolin is a high value diterpenoid with a broad range of pharmaceutical applications, naturally found in root bark of the plant Coleus forskohlii. Because of its complex molecular structure, chemical synthesis of forskolin is not commercially attractive. Hence, the labor and resource intensive extraction and purification from C. forskohlii plants remains the current source of the compound. We have engineered the unicellular cyanobacterium Synechocystis sp. PCC 6803 to produce the forskolin precursor 13R-manoyl oxide (13R-MO), paving the way for light driven biotechnological production of this high value compound. In the course of this work, a new series of integrative vectors for use in Synechocystis was developed and used to create stable lines expressing chromosomally integrated CfTPS2 and CfTPS3, the enzymes responsible for the formation of 13R-MO in C. forskohlii. The engineered strains yielded production titers of up to 0.24 mg g(-1) DCW 13R-MO. To increase the yield, 13R-MO producing strains were further engineered by introduction of selected enzymes from C. forskohlii, improving the titer to 0.45 mg g(-1) DCW. This work forms a basis for further development of production of complex plant diterpenoids in cyanobacteria.

  • 3.
    Englund, Elias
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Liang, Feiyan
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Lindberg, Pia
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Evaluation of promoters and ribosome binding sites for biotechnological applications in the unicellular cyanobacterium Synechocystis sp. PCC 68032016In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 6, article id 36640Article in journal (Refereed)
    Abstract [en]

    For effective metabolic engineering, a toolbox of genetic components that enables predictable control of gene expression is needed. Here we present a systematic study of promoters and ribosome binding sites in the unicellular cyanobacterium Synechocystis sp. PCC 6803. A set of metal ion inducible promoters from Synechocystis were compared to commonly used constitutive promoters, by measuring fluorescence of a reporter protein in a standardized setting to allow for accurate comparisons of promoter activity. The most versatile and useful promoter was found to be PnrsB, which from a relatively silent expression could be induced almost 40-fold, nearly up to the activity of the strong psbA2 promoter. By varying the concentrations of the two metal ion inducers Ni(2+) and Co(2+), expression from the promoter was highly tunable, results that were reproduced with PnrsB driving ethanol production. The activities of several ribosomal binding sites were also measured, and tested in parallel in Synechocystis and Escherichia coli. The results of the study add useful information to the Synechocystis genetic toolbox for biotechnological applications.

  • 4.
    Englund, Elias
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Pattanaik, Bagmi
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Ubhayasekera, Sarojini J.K.A.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Stensjö, Karin
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Lindberg, Pia
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Production of Squalene in Synechocystis sp. PCC 68032014In: PLoS ONE, Vol. 9, no 3, p. e90270-Article in journal (Refereed)
    Abstract [en]

    In recent years, there has been an increased interest in the research and development of sustainable alternatives to fossil fuels. Using photosynthetic microorganisms to produce such alternatives is advantageous, since they can achieve direct conversion of carbon dioxide from the atmosphere into the desired product, using sunlight as the energy source. Squalene is a naturally occurring 30-carbon isoprenoid, which has commercial use in cosmetics and in vaccines. If it could be produced sustainably on a large scale, it could also be used instead of petroleum as a raw material for fuels and as feedstock for the chemical industry. The unicellular cyanobacterium Synechocystis PCC 6803 possesses a gene, slr2089, predicted to encode squalene hopene cyclase (Shc), an enzyme converting squalene into hopene, the substrate for forming hopanoids. Through inactivation of slr2089 (shc), we explored the possibility to produce squalene using cyanobacteria. The inactivation led to accumulation of squalene, to a level over 70 times higher than in wild type cells, reaching 0.67 mg OD750−1 L−1. We did not observe any significant growth deficiency in the Δshc strain compared to the wild type Synechocystis, even at high light conditions, suggesting that the observed squalene accumulation was not detrimental to growth, and that formation of hopene by Shc is not crucial for growth under normal conditions, nor for high-light stress tolerance. Effects of different light intensities and growth stages on squalene accumulation in the Δshc strain were investigated. We also identified a gene, sll0513, as a putative squalene synthase in Synechocystis, and verified its function by inactivation. In this work, we show that it is possible to use the cyanobacterium Synechocystis to generate squalene, a hydrocarbon of commercial interest and a potential biofuel. We also report the first identification of a squalene hopene cyclase, and the second identification of squalene synthase, in cyanobacteria.

  • 5.
    Englund, Elias
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Shabestary, Kiyan
    Hudson, Elton P
    Lindberg, Pia
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Systematic overexpression study to find target enzymes enhancing production of terpenes in Synechocystis PCC 6803, using isoprene as a model compound.2018In: Metabolic engineering, ISSN 1096-7176, E-ISSN 1096-7184, Vol. 49, p. 164-177Article in journal (Refereed)
    Abstract [en]

    Of the two natural metabolic pathways for making terpenoids, biotechnological utilization of the mevalonate (MVA) pathway has enabled commercial production of valuable compounds, while the more recently discovered but stoichiometrically more efficient methylerythritol phosphate (MEP) pathway is underdeveloped. We conducted a study on the overexpression of each enzyme in the MEP pathway in the unicellular cyanobacterium Synechocystis sp. PCC 6803, to identify potential targets for increasing flux towards terpenoid production, using isoprene as a reporter molecule. Results showed that the enzymes Ipi, Dxs and IspD had the biggest impact on isoprene production. By combining and creating operons out of those genes, isoprene production was increased 2-fold compared to the base strain. A genome-scale model was used to identify targets upstream of the MEP pathway that could redirect flux towards terpenoids. A total of ten reactions from the Calvin-Benson-Bassham cycle, lower glycolysis and co-factor synthesis pathways were probed for their effect on isoprene synthesis by co-expressing them with the MEP enzymes, resulting in a 60% increase in production from the best strain. Lastly, we studied two isoprene synthases with the highest reported catalytic rates. Only by expressing them together with Dxs and Ipi could we get stable strains that produced 2.8 mg/g isoprene per dry cell weight, a 40-fold improvement compared to the initial strain.

  • 6. 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)
  • 7.
    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.

  • 8.
    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.

  • 9.
    Holmqvist, Marie
    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.
    Oliviera, Paulo
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science.
    Lindberg, Pia
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science.
    Characterization of the hupSL promoter activity in Nostoc punctiforme ATCC 291332009In: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 11, no 9, p. 54-Article in journal (Refereed)
    Abstract [en]

    BACKGROUND:

    In cyanobacteria three enzymes are directly involved in the hydrogen metabolism; a nitrogenase that produces molecular hydrogen, H2, as a by-product of nitrogen fixation, an uptake hydrogenase that recaptures H2 and oxidize it, and a bidirectional hydrogenase that can both oxidize and produce H2.Nostoc punctiforme ATCC 29133 is a filamentous dinitrogen fixing cyanobacterium containing a nitrogenase and an uptake hydrogenase but no bidirectional hydrogenase. Generally, little is known about the transcriptional regulation of the cyanobacterial uptake hydrogenases. In this study gel shift assays showed that NtcA has a specific affinity to a region of the hupSL promoter containing a predicted NtcA binding site. The predicted NtcA binding site is centred at 258.5 bp upstream the transcription start point (tsp). To further investigate the hupSL promoter, truncated versions of the hupSL promoter were fused to either gfp or luxAB, encoding the reporter proteins Green Fluorescent Protein and Luciferase, respectively.

    RESULTS:

    Interestingly, all hupsSL promoter deletion constructs showed heterocyst specific expression. Unexpectedly the shortest promoter fragment, a fragment covering 57 bp upstream and 258 bp downstream the tsp, exhibited the highest promoter activity. Deletion of the NtcA binding site neither affected the expression to any larger extent nor the heterocyst specificity.

    CONCLUSION:

    Obtained data suggest that the hupSL promoter in N. punctiforme is not strictly dependent on the upstream NtcA cis element and that the shortest promoter fragment (-57 to tsp) is enough for a high and heterocyst specific expression of hupSL. This is highly interesting because it indicates that the information that determines heterocyst specific gene expression might be confined to this short sequence or in the downstream untranslated leader sequence.

  • 10.
    Liang, Feiyan
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Englund, Elias
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Lindberg, Pia
    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.
    Engineered cyanobacteria with enhanced growth show increased ethanol production and higher biofuel to biomass ratio2018In: Metabolic engineering, ISSN 1096-7176, E-ISSN 1096-7184, Vol. 46, p. 51-59Article in journal (Refereed)
    Abstract [en]

    The Calvin-Benson-Bassham (CBB) cycle is the main pathway to fix atmospheric CO2 and store energy in carbon bonds, forming the precursors of most primary and secondary metabolites necessary for life. Speeding up the CBB cycle theoretically has positive effects on the subsequent growth and/or the end metabolite(s) production. Four CBB cycle enzymes, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), fructose-1,6/sedoheptulose-1,7-bisphosphatase (FBP/SBPase), transketolase (TK) and aldolase (FBA) were selected to be co-overexpressed with the ethanol synthesis enzymes pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) in the cyanobacterium Synechocystis PCC 6803. An inducible promoter, PnrsB, was used to drive PDC and ADH expression. When PnrsB was induced and cells were cultivated at 65 µmol photons m−2 s−1, the RuBisCO-, FBP/SBPase-, TK-, and FBA-expressing strains produced 55%, 67%, 37% and 69% more ethanol and 7.7%, 15.1%, 8.8% and 10.1% more total biomass (the sum of dry cell weight and ethanol), respectively, compared to the strain only expressing the ethanol biosynthesis pathway. The ethanol to total biomass ratio was also increased in CBB cycle enzymes overexpressing strains. This study experimentally demonstrates that using the cells with enhanced carbon fixation, when the product synthesis pathway is not the main bottleneck, can significantly increase the generation of a product (exemplified with ethanol), which acts as a carbon sink.

  • 11.
    Liang, Feiyan
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Lindberg, Pia
    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.
    Engineering photoautotrophic carbon fixation for enhanced growth and productivity2018In: Sustainable Energy & Fuels, ISSN 2398-4902, Vol. 2, no 12, p. 2583-2600Article, review/survey (Refereed)
    Abstract [en]

    Oxygenic photosynthesis is the origin of most organic carbon compounds on Earth and an essential part of the natural carbon cycle. Cyanobacteria, the only oxygenic photoautotrophic prokaryotes, are important in several natural processes: as primary sustainable producers, in providing oxygen to the atmosphere, and in nitrogen fixation. From a biotechnological perspective, cyanobacteria are ideal cell factories since (i) the required energy and carbon source, sunlight and CO2, are abundant and freely available, (ii) cyanobacteria are capable of producing a variety of natural products, which can be used as fuels, medicines, cosmetics etc., and (iii) metabolic engineering and synthetic biology tools of cyanobacteria are being developed rapidly, making them feasible as host organisms for heterologous production of interesting compounds. However, compared to commercially employed heterotrophic microorganisms, the growth and productivity of cyanobacteria are currently not competitive. Therefore, improving cyanobacterial growth and productivity is an important task to enable commercialization of cyanobacterial bioproducts. Such studies also offer important clues for increasing the photosynthesis and yield of crop plants, which is important in view of providing food for a rapidly increasing world population. There are many strategies targeting this task, such as optimizing cultivation conditions, engineering native pathways, and introducing synthetic pathways based on an understanding of overall metabolic networks. One major limitation of cyanobacterial productivity, however, is the low efficiency of carbon fixation through the Calvin-Benson-Bassham (CBB) cycle. In this review, we introduce and discuss the possibilities to enhance growth and productivity by engineering the CBB cycle. We also give a brief discussion of options to further extend the capabilities of cells to fix inorganic carbon by the introduction of other native and artificial carbon fixation cycles.

  • 12.
    Lindberg, Pia
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Devine, Ellenor
    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.
    HupW Protease Specifically Required for Processing of the Catalytic Subunit of the Uptake Hydrogenase in the Cyanobacterium Nostoc sp Strain PCC 71202012In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 78, no 1, p. 273-276Article in journal (Refereed)
    Abstract [en]

    The maturation process of [NiFe] hydrogenases includes a proteolytic cleavage of the large subunit. We constructed a mutant of Nostoc strain PCC 7120 in which hupW, encoding a putative hydrogenase-specific protease, is inactivated. Our results indicate that the protein product of hupW selectively cleaves the uptake hydrogenase in this cyanobacterium.

  • 13.
    Lindberg, Pia
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolutionary Biology, Physiological Botany.
    Hansel, Alfred
    Lindblad, Peter
    hupS and hupL constitute a transcription unit in the cyanobacterium Nostoc sp. PCC 731022000In: Archives of Microbiology, ISSN 0302-8933, Vol. 174, no 1-2, p. 129-133Article in journal (Refereed)
  • 14.
    Lindberg, Pia
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Physiological Botany. Fysiologisk botanik.
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Physiological Botany. Fysiologisk botanik.
    Cournac, Laurent
    Gas Exchange in the Filamentous Cyanobacterium Nostoc punctiforme Strain ATCC 29133 and Its Hydrogenase-Deficient Mutant Strain NHM52004In: Applied and Environmental Microbiology, Vol. 70, no 4, p. 2137-2145Article in journal (Refereed)
    Abstract [en]

    Nostoc punctiforme ATCC 29133 is a nitrogen-fixing, heterocystous cyanobacterium of symbiotic origin. During nitrogen fixation, it produces molecular hydrogen (H2), which is recaptured by an uptake hydrogenase. Gas exchange in cultures of N. punctiforme ATCC 29133 and its hydrogenase-free mutant strain NHM5 was studied. Exchange of O2, CO2, N2 and H2 was followed simultaneously with mass spectrometer in cultures grown under nitrogen-fixing conditions. Isotopic tracing was used to separate evolution and uptake of CO2 and O2. The amount of H2 produced per molecule of N2 fixed was found to vary with light conditions, high light giving a greater increase in H2 production than N2 fixation. The ratio under low light and high light was approximately 1.4 and 6.1 molecules of H2 produced per molecule of N2 fixed, respectively. Incubation under high light for a longer time, until the culture was depleted of CO2, caused a decrease in the nitrogen fixation rate. At the same time, hydrogen production in the hydrgenase-deficient strain was increased from an initial rate of approximately 6 umol (mg of chlorophyll a)-1h-1 to 9 umol (mg of chlorophyll a)-1h-1 after about 50 min. A light-stimulated hydrogen-deuterium exchange activity stemming from the nitrogenase was observed in the two strains. The present findings are important for understanding this nitrogenase-based system, aiming at photobiological hydrogen production, as we have identified the conditions under which the energy flow through the nitrogenase can be directed towards hydrogen production rather than nitrogen fixation.

  • 15.
    Lindberg, Pia
    et al.
    Department of Plant and Microbial Biology, University of California, Berkeley.
    Melis, Anastasios
    Department of Plant and Microbial Biology, University of California, Berkeley.
    The chloroplast sulfate transport system in the green alga Chlamydomonas reinhardtii.2008In: Planta, ISSN 0032-0935, E-ISSN 1432-2048, Vol. 228, no 6, p. 951-61Article in journal (Refereed)
    Abstract [en]

    The genome of the model unicellular green alga Chlamydomonas reinhardtii contains four distinct genes, SulP, SulP2, Sbp and Sabc, which together are postulated to encode a chloroplast envelope-localized sulfate transporter holocomplex. In this work, evidence is presented that regulation of expression of SulP2, Sbp and Sabc is specifically modulated by sulfur availability to the cells. Induction of transcription and higher steady-state levels of the respective mRNAs are reported under S-deprivation conditions. No such induction could be observed under N or P deprivation conditions. Expression, localization, and complex-association of the Sabc protein was specifically investigated using cellular and chloroplast fractionations, BN-PAGE, SDS-PAGE and Western blot analyses. It is shown that Sabc protein levels in the cells increased under S-deprivation conditions, consistent with the observed induction of Sabc gene transcription. It is further shown that the Sabc protein co-localizes with SulP to the chloroplast envelope. Blue-native PAGE followed by Western blot analysis revealed the presence of an apparent 380 kDa complex in C. reinhardtii, specifically recognized by polyclonal antibodies against SulP and Sabc. These results suggest the presence and function in C. reinhardtii of a Sbp-SulP-SulP2-Sabc chloroplast envelope sulfate transporter holocomplex.

  • 16.
    Lindberg, Pia
    et al.
    Department of Plant and Microbial Biology, University of California, Berkeley.
    Park, Sungsoon
    Department of Plant and Microbial Biology, University of California, Berkeley.
    Melis, Anastasios
    Department of Plant and Microbial Biology, University of California, Berkeley.
    Engineering a platform for photosynthetic isoprene production in cyanobacteria, using Synechocystis as the model organism.2010In: Metabolic engineering, ISSN 1096-7176, E-ISSN 1096-7184, Vol. 12, no 1, p. 70-9Article in journal (Refereed)
    Abstract [en]

    The concept of "photosynthetic biofuels" envisions application of a single organism, acting both as photo-catalyst and producer of ready-made fuel. This concept was applied upon genetic engineering of the cyanobacterium Synechocystis, conferring the ability to generate volatile isoprene hydrocarbons from CO(2) and H(2)O. Heterologous expression of the Pueraria montana (kudzu) isoprene synthase (IspS) gene in Synechocystis enabled photosynthetic isoprene generation in these cyanobacteria. Codon-use optimization of the kudzu IspS gene improved expression of the isoprene synthase in Synechocystis. Use of the photosynthesis psbA2 promoter, to drive the expression of the IspS gene, resulted in a light-intensity-dependent isoprene synthase expression. Results showed that oxygenic photosynthesis can be re-directed to generate useful small volatile hydrocarbons, while consuming CO(2), without a prior requirement for the harvesting, dewatering and processing of the respective biomass.

  • 17.
    Lindberg, Pia
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolutionary Biology, Physiological Botany.
    Schütz, Kathrin
    Happe, Thomas
    Lindblad, Peter
    A hydrogen-producing, hydrogenase-free mutant strain of Nostoc punctiforme ATCC 291332002In: International Journal of Hydrogen Energy, ISSN 0360-3199, Vol. 27, no 11-12, p. 1291-1296Article in journal (Refereed)
  • 18. Lindblad, Peter
    et al.
    Christensson, Kjell
    Lindberg, Pia
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolutionary Biology, Physiological Botany.
    Fedorov, Alexander
    Lopes Pinto, Fernando
    Tsygankov, Anatoly
    Photoproduction of H2 by wildtype Anabaena PCC 7120 and a hydrogen uptake deficient mutant: From laboratory experiments to outdoor culture2002In: International Journal of Hydrogen Energy, ISSN 0360-3199, Vol. 27, no 11-12, p. 1271-1281Article in journal (Refereed)
  • 19.
    Lindblad, Peter
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Lindberg, Pia
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Oliveira, Paulo
    Stensjö, Karin
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Heidorn, Thorsten
    Design, Engineering, and Construction of Photosynthetic Microbial Cell Factories for Renewable Solar Fuel Production2012In: Ambio, ISSN 0044-7447, E-ISSN 1654-7209, Vol. 41, no Suppl 2, p. 163-168Article 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. This paper outlines and discusses the design and engineering of photosynthetic microbial systems for the generation of renewable solar fuels, with a focus on cyanobacteria. Cyanobacteria are prokaryotic microorganisms with the same type of photosynthesis as higher plants. Native and engineered cyanobacteria have been used by us and others as model systems to examine, demonstrate, and develop photobiological H-2 production. More recently, the production of carbon-containing solar fuels like ethanol, butanol, and isoprene have been demonstrated. We are using a synthetic biology approach to develop efficient photosynthetic microbial cell factories for direct generation of biofuels from solar energy. Present progress and advances in the design, engineering, and construction of such cyanobacterial cells for the generation of a portfolio of solar fuels, e. g., hydrogen, alcohols, and isoprene, are presented and discussed. Possibilities and challenges when introducing and using synthetic biology are highlighted.

  • 20.
    Liu, Xufeng
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Miao, Rui
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Lindberg, Pia
    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.
    Metabolic engineering of Synechocystis PCC 6803 for photosynthetic 1-butanol productionManuscript (preprint) (Other academic)
  • 21.
    Liu, Xufeng
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Miao, Rui
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Lindberg, Pia
    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.
    Modular engineering for efficient photosynthetic biosynthesis of 1-butanol from CO2 in cyanobacteria2019In: Energy & Environmental Science, ISSN 1754-5692, E-ISSN 1754-5706, Vol. 12, no 9, p. 2765-2777Article in journal (Refereed)
    Abstract [en]

    Cyanobacteria are photoautotrophic microorganisms which can be engineered to directly convert CO2 and water into biofuels and chemicals via photosynthesis using sunlight as energy. However, the product titers and rates are the main challenges that need to be overcome for industrial applications. Here we present systematic modular engineering of the cyanobacterium Synechocystis PCC 6803, enabling efficient biosynthesis of 1-butanol, an attractive commodity chemical and gasoline substitute. Through introducing and re-casting the 1-butanol biosynthetic pathway at the gene and enzyme levels, optimizing the 5 '-regions of expression units for tuning transcription and translation, rewiring the carbon flux and rewriting the photosynthetic central carbon metabolism to enhance the precursor supply, and performing process development, we were able to reach a cumulative 1-butanol titer of 4.8 g L-1 with a maximal rate of 302 mg L-1 day(-1) from the engineered Synechocystis. This represents the highest 1-butanol production from CO2 reported so far. Our multi-level modular strategy for high-level production of chemicals and advanced biofuels represents a blue-print for future systematic engineering in photosynthetic microorganisms.

  • 22.
    Miao, Rui
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Liu, Xufeng
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Englund, Elias
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Lindberg, Pia
    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.
    Isobutanol production in Synechocystis PCC 6803 using heterologous and endogenous alcohol dehydrogenases2017In: Metabolic Engineering Communications, ISSN 2214-0301, Vol. 5, p. 45-53Article in journal (Refereed)
    Abstract [en]

    Isobutanol is a flammable compound that can be used as a biofuel due to its high energy density and suitable physical and chemical properties. In this study, we examined the capacity of engineered strains of Synechocystis PCC 6803 containing the α-ketoisovalerate decarboxylase from Lactococcus lactis and different heterologous and endogenous alcohol dehydrogenases (ADH) for isobutanol production. A strain expressing an introduced kivdwithout any additional copy of ADH produced 3 mg L−1 OD750−1 isobutanol in 6 days. After the cultures were supplemented with external addition of isobutyraldehyde, the substrate for ADH, 60.8 mg L−1 isobutanol was produced after 24 h when OD750 was 0.8. The in vivo activities of four different ADHs, two heterologous and two putative endogenous in Synechocystis, were examined and the Synechocystis endogenous ADH encoded by slr1192 showed the highest efficiency for isobutanol production. Furthermore, the strain overexpressing the isobutanol pathway on a self-replicating vector with the strong Ptrc promoter showed significantly higher gene expression and isobutanol production compared to the corresponding strains expressing the same operon introduced on the genome. Hence, this study demonstrates that Synechocystis endogenous AHDs have a high capacity for isobutanol production, and identifies kivd encoded α-ketoisovalerate decarboxylase as one of the likely bottlenecks for further isobutanol production.

  • 23.
    Miranda, Hélder
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Immerzeel, Peter
    Department of Forest Genetics and Plant Physiology, Umeå Plant Science Centre, Swedish University of Agricultural Sciences, Umeå, Sweden.
    Gerber, Lorenz
    Department of Forest Genetics and Plant Physiology, Umeå Plant Science Centre, Swedish University of Agricultural Sciences, Umeå, Sweden.
    Hörnaeus, Katarina
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Bergström, Sara K.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Pattanaik, Bagmi
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Lindberg, Pia
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Mamedov, Fikret
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Sll1783, a monooxygenase associated with polysaccharide processing in the unicellular cyanobacterium Synechocystis PCC 68032017In: Physiologia Plantarum: An International Journal for Plant Biology, ISSN 0031-9317, E-ISSN 1399-3054, Vol. 161, no 2, p. 182-195Article in journal (Refereed)
    Abstract [en]

    Cyanobacteria play a pivotal role as the primary producer in many aquatic ecosystems. The knowledge on the interacting processes of cyanobacteria with its environment - abiotic and biotic factors - is still very limited. Many potential exocytoplasmic proteins in the model unicellular cyanobacterium Synechocystis PCC 6803 have unknown functions and their study is essential to improve our understanding of this photosynthetic organism and its potential for biotechnology use. Here we characterize a deletion mutant of Synechocystis PCC 6803, Δsll1783, a strain that showed a remarkably high light resistance which is related with its lower thylakoid membrane formation. Our results suggests Sll1783 to be involved in a mechanism of polysaccharide degradation and uptake and we hypothesize it might function as a sensor for cell density in cyanobacterial cultures.

  • 24.
    Pattanaik, Bagmi
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Lindberg, Pia
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Terpenoids and their biosynthesis in cyanobacteria.2015In: Life (Basel, Switzerland), ISSN 2075-1729, Vol. 5, no 1, p. 269-93Article in journal (Refereed)
    Abstract [en]

    Terpenoids, or isoprenoids, are a family of compounds with great structural diversity which are essential for all living organisms. In cyanobacteria, they are synthesized from the methylerythritol-phosphate (MEP) pathway, using glyceraldehyde 3-phosphate and pyruvate produced by photosynthesis as substrates. The products of the MEP pathway are the isomeric five-carbon compounds isopentenyl diphosphate and dimethylallyl diphosphate, which in turn form the basic building blocks for formation of all terpenoids. Many terpenoid compounds have useful properties and are of interest in the fields of pharmaceuticals and nutrition, and even potentially as future biofuels. The MEP pathway, its function and regulation, and the subsequent formation of terpenoids have not been fully elucidated in cyanobacteria, despite its relevance for biotechnological applications. In this review, we summarize the present knowledge about cyanobacterial terpenoid biosynthesis, both regarding the native metabolism and regarding metabolic engineering of cyanobacteria for heterologous production of non-native terpenoids.

  • 25.
    Sybirna, Kateryna
    et al.
    CEA Saclay, DSV.
    Antoine, Tatiana
    CEA Saclay, DSV.
    Lindberg, Pia
    CEA Saclay, DSV.
    Fourmond, Vincent
    CEA Saclay, DSV.
    Rousset, Marc
    CNRS.
    Méjean, Vincent
    CNRS.
    Bottin, Hervé
    CEA Saclay, DSV.
    Shewanella oneidensis: a new and efficient system for expression and maturation of heterologous [Fe-Fe] hydrogenase from Chlamydomonas reinhardtii.2008In: BMC biotechnology, ISSN 1472-6750, Vol. 8, p. 73-Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: The eukaryotic green alga, Chlamydomonas reinhardtii, produces H2 under anaerobic conditions, in a reaction catalysed by a [Fe-Fe] hydrogenase HydA1. For further biochemical and biophysical studies a suitable expression system of this enzyme should be found to overcome its weak expression in the host organism. Two heterologous expression systems used up to now have several advantages. However they are not free from some drawbacks. In this work we use bacterium Shewanella oneidensis as a new and efficient system for expression and maturation of HydA1 from Chlamydomonas reinhardtii.

    RESULTS: Based on codon usage bias and hydrogenase maturation ability, the bacterium S. oneidensis, which possesses putative [Fe-Fe] and [Ni-Fe] hydrogenase operons, was selected as the best potential host for C. reinhardtii [Fe-Fe] hydrogenase expression. Hydrogen formation by S. oneidensis strain AS52 (Delta hydA Delta hyaB) transformed with a plasmid bearing CrHydA1 and grown in the presence of six different substrates for anaerobic respiration was determined. A significant increase in hydrogen evolution was observed for cells grown in the presence of trimethylamine oxide, dimethylsulfoxide and disodium thiosulfate, showing that the system of S. oneidensis is efficient for heterologous expression of algal [Fe-Fe] hydrogenase.

    CONCLUSION: In the present work a new efficient system for heterologous expression and maturation of C. reinhardtii hydrogenase has been developed. HydA1 of C. reinhardtii was purified and shown to contain 6 Fe atoms/molecule of protein, as expected. Using DMSO, TMAO or thiosulfate as substrates for anaerobic respiration during the cell growth, 0.4 - 0.5 mg l(-1)(OD600 = 1) of catalytically active HydA1 was obtained with hydrogen evolution rate of approximately 700 micromol H2 mg(-1) min(-1).

  • 26. Tamagnini, Paula
    et al.
    Axelsson, Rikard
    Lindberg, Pia
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolutionary Biology, Physiological Botany.
    Oxelfelt, Fredrik
    Wünschiers, Röbbe
    Lindblad, Peter
    Hydrogenases and Hydrogen Metabolism of Cyanobacteria2002In: Microbiology and Molecular Biology Reviews, ISSN 1092-2172, Vol. 66, no 1, p. 1-20Article in journal (Refereed)
  • 27.
    Vorndran, Elke
    et al.
    Univ Clin, Dept Funct Mat Med & Dent, Wurzburg, Germany..
    Lindberg, Pia
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    In situ-immobilization of two model cyanobacterial strains in ceramic structures: A new biohybrid material for photobioreactor applications2016In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 223, p. 1-5Article in journal (Refereed)
    Abstract [en]

    Two cyanobacterial strains, Synechocystis sp. PCC 6803 and Nostoc punctiforme ATCC 29133 were immobilized within magnesium phosphate based cements, showing a viability and activity for at least 4 weeks. These biohybrids are considered as an alternative photobioreactor material for bioremediation or an improved yield of biotechnologically relevant molecules. 

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