Evidence for Electron Transfer from the Bidirectional Hydrogenase to the Photosynthetic Complex I (NDH-1) in the Cyanobacterium Synechocystis sp. PCC 6803

The cyanobacterial bidirectional [NiFe]-hydrogenase is a pentameric enzyme. Apart from the small and large hydrogenase subunits (HoxYH) it contains a diaphorase module (HoxEFU) that interacts with NAD(P)+ and ferredoxin. HoxEFU shows strong similarity to the outermost subunits (NuoEFG) of canonical respiratory complexes I. Photosynthetic complex I (NDH-1) lacks these three subunits. This led to the idea that HoxEFU might interact with NDH-1 instead. HoxEFUYH utilizes excited electrons from PSI for photohydrogen production and it catalyzes the reverse reaction and feeds electrons into the photosynthetic electron transport. We analyzed hydrogenase activity, photohydrogen evolution and hydrogen uptake, the respiration and photosynthetic electron transport of ΔhoxEFUYH, and a knock-out strain with dysfunctional NDH-1 (ΔndhD1/ΔndhD2) of the cyanobacterium Synechocystis sp. PCC 6803. Photohydrogen production was prolonged in ΔndhD1/ΔndhD2 due to diminished hydrogen uptake. Electrons from hydrogen oxidation must follow a different route into the photosynthetic electron transport in this mutant compared to wild type cells. Furthermore, respiration was reduced in ΔhoxEFUYH and the ΔndhD1/ΔndhD2 localization of the hydrogenase to the membrane was impaired. These data indicate that electron transfer from the hydrogenase to the NDH-1 complex is either direct, by the binding of the hydrogenase to the complex, or indirect, via an additional mediator

Physiologische Studien und Ansätze zur Maximierung der Wasserstoffproduktion im Cyanobakterium Synechocystis sp. PCC 6803

This work shows approaches to produce hydrogen sustainably and environmentally friendly by exploiting cyanobacteria. The cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis) possesses an oxygen-sensitive bidirectional [NiFe]-hydrogenase, which is either able to produce hydrogen at the onset of photosynthesis (photohydrogen) or during carbohydrate oxidation in darkness (fermentative hydrogen). Previous experiments already showed that the hydrogenase is essential for the growth of Synechocystis cells on arginine and glucose in the presence of oxygen. However, the hydrogenase is oxygen-sensitive and is not able to either produce or take up hydrogen under these conditions (Burgstaller, 2017). It must, therefore, fulfill a different function at the given condition, which led to the hypothesis that the hydrogenase might function as an oxygenase in the presence of oxygen by working as an electron valve. Experimental results, however, indicated that the hydrogenase could have the opposite function and might feed electron into the electron transport chain at the thylakoid membrane, probably through complex 1 (NDH1). The hydrogenase obviously fine-tunes the respiratory and photosynthetic electron transfer under these conditions. In order to increase the electron flux to the hydrogenase and enhance the hydrogen production, fusion constructs were designed and constructed, which directly link photosystem I (PSI) to the hydrogenase subunits (HoxYH) of Synechocystis to maximize the photohydrogen production. Measurements of the production in those mutants revealed an increase in hydrogen production in comparison to the Synechocystis wild-type (WT) in the absence of oxygen. These results show a successful generation of a fusion protein in Synechocystis cells in vivo and could be the first step towards a sustainable and CO2 neutral way to produce hydrogen as an alternative and renewable energy source

Comparison of angiogenic potential in vitrified vs. slow frozen human ovarian tissue

Abstract Vitrification of ovarian tissue is a promising alternative approach to the traditional slow freezing method. Few empirical investigations have been conducted to determine the angiogenic profiles of these two freezing methods. In this study we aimed to answer the question whether one of the cryopreservation methods should be preferred based on the secretion of angiogenic factors. Tissue culture with reduced oxygen (5%) was conducted for 48 h with samples of fresh, slow frozen/thawed and vitrified/rapid warmed ovarian cortex tissue from 20 patients. From each patient, tissue was used in all three treatment groups. Tissue culture supernatants were determined regarding cytokine expression profiles of angiogenin, angiopoietin-2, epidermal growth factor, basic fibroblast growth factor, heparin binding epidermal growth factor, hepatocyte growth factor, Leptin, Platelet-derived growth factor B, placental growth factor and vascular endothelial growth factor A via fluoroimmunoassay. Apoptotic changes were assessed by TUNEL staining of cryosections and supplemented by hematoxylin and eosin and proliferating cell nuclear antigen staining. Comparing the angiogenic expression profiles of vitrified/rapid warmed tissue with slow frozen/thawed tissue samples, no significant differences were observed. Detection of apoptotic DNA fragmentation via TUNEL indicated minor apoptotic profiles that were not significantly different comparing both cryopreservation methods. Vitrification of ovarian cortical tissue does not appear to impact negatively on the expression profile of angiogenic factors and may be regarded as an effective alternative approach to the traditional slow freezing method

Evidence for Electron Transfer from the Bidirectional Hydrogenase to the Photosynthetic Complex I (NDH-1) in the Cyanobacterium Synechocystis sp. PCC 6803

The cyanobacterial bidirectional [NiFe]-hydrogenase is a pentameric enzyme. Apart from the small and large hydrogenase subunits (HoxYH) it contains a diaphorase module (HoxEFU) that interacts with NAD(P)+ and ferredoxin. HoxEFU shows strong similarity to the outermost subunits (NuoEFG) of canonical respiratory complexes I. Photosynthetic complex I (NDH-1) lacks these three subunits. This led to the idea that HoxEFU might interact with NDH-1 instead. HoxEFUYH utilizes excited electrons from PSI for photohydrogen production and it catalyzes the reverse reaction and feeds electrons into the photosynthetic electron transport. We analyzed hydrogenase activity, photohydrogen evolution and hydrogen uptake, the respiration and photosynthetic electron transport of ΔhoxEFUYH, and a knock-out strain with dysfunctional NDH-1 (ΔndhD1/ΔndhD2) of the cyanobacterium Synechocystis sp. PCC 6803. Photohydrogen production was prolonged in ΔndhD1/ΔndhD2 due to diminished hydrogen uptake. Electrons from hydrogen oxidation must follow a different route into the photosynthetic electron transport in this mutant compared to wild type cells. Furthermore, respiration was reduced in ΔhoxEFUYH and the ΔndhD1/ΔndhD2 localization of the hydrogenase to the membrane was impaired. These data indicate that electron transfer from the hydrogenase to the NDH-1 complex is either direct, by the binding of the hydrogenase to the complex, or indirect, via an additional mediator

Evidence for Electron Transfer from the Bidirectional Hydrogenase to the Photosynthetic Complex I (NDH-1) in the Cyanobacterium Synechocystis sp. PCC 6803

Gefördert durch den Publikationsfonds der Universität Kasse

Synechocystis sp. PCC 6803 Requires the Bidirectional Hydrogenase to Metabolize Glucose and Arginine Under Oxic Conditions

Gefördert durch den Publikationsfonds der Universität Kasse

Triterpene levels in engineered <i>R</i>. <i>capsulatus</i> SB1003 (Rc) and <i>Synechocystis</i> sp. PCC 6803 (Syn) strains.

<p>Triterpene levels in engineered <i>R</i>. <i>capsulatus</i> SB1003 (Rc) and <i>Synechocystis</i> sp. PCC 6803 (Syn) strains.</p

LC-MS detection of triterpenoids produced in engineered <i>R</i>. <i>capsulatus</i> SB1003 (Rc) and <i>Synechocystis</i> sp. PCC 6803 (Syn) strains.

<p>Cells of the respective strains (indicated in chromatograms and color-coded with red for Rc-, green for Syn-strains) were subjected to extraction and analysis after expression of <i>A</i>. <i>thaliana</i> triterpenoid biosynthesis genes (SQS1, squalene synthase; SQE1, squalene epoxidase; CAS1, cycloartenol synthase; LUP1, lupeol synthase; MRN1, marneral synthase). Triterpenoid identity was verified by comparison of retention times, as well as MS and MS/MS spectra to commercial or biological references (<b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189816#pone.0189816.s005" target="_blank">S2 Fig</a></b>). <b>(A)</b> Signals in EICs of m/z 411.399 at RT 14.9 min correspond to squalene (C₃₀H₅₀), signals in EICs of m/z 427.393 at RT 11.8 min correspond to 2,3-oxidosqualene (C₃₀H₅₀O). <b>(B)</b> Signals in EICs of m/z 427.393 at RT 10.9 min correspond to cycloartenol (C₃₀H₅₀O). <b>(C)</b> Signals in EICs of m/z 409.383 at RT 9.9 min correspond to lupeol (C₃₀H₅₀O-H₂O). Signals in EICs of m/z 427.393 (and 409.383) at RT 6.7 min correspond to lupX (tentatively identified as lupanediol (C₃₀H₅₂O₂-H₂O)). In both EICs, signals at RT 11.7 min correspond to 2,3-oxidosqualene (C₃₀H₅₀O). <b>(D)</b> Signals in EICs of m/z 429.409 at RT 10.7 min correspond to marnerol (C₃₀H₅₂O), signals in EICs of m/z 445.404 at RT 7.1 min correspond to hydroxymarnerol (C₃₀H₅₂O₂). Signals in EICs of m/z 429.409 at RT 11.8 min correspond to the (M+2) isotopic peak of 2,3-oxidosqualene (C₃₀H₅₂O). Peaks in EICs of m/z 445.404 also detected in Syn<i>_</i>Δ<i>shc</i>-SQE1 (control) also correspond to 2,3-oxidosqualene-derived signals. As a reference, chromatograms of samples from <i>S</i>. <i>cerevisiae</i> GIL77 (Sc), carrying pYES/DEST-52 with <i>MRN1</i> or as empty vector control (EVC), are shown. Shown chromatograms are representative for replicate measurements from at least three independent cultivations. The corresponding quantitative data are summarized in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189816#pone.0189816.t002" target="_blank">Table 2</a></b>.</p

Triterpene pathway design.

<p><b>(A)</b> Pathways for targeted biosynthesis of triterpenes in <i>R</i>. <i>capsulatus</i> and <i>Synechocystis</i> by implementation of <i>A</i>. <i>thaliana</i> biosynthesis modules. In both organisms, the common triterpene precursor FPP is provided by the native 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway. To establish the first step of the triterpene-specific precursor module in <i>Synechocystis</i>, a knock-out mutant of the <i>shc</i> gene encoding the squalene-hopene cyclase of the intrinsic hopanoid biosynthesis was employed [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189816#pone.0189816.ref037" target="_blank">37</a>]. In <i>R</i>. <i>capsulatus</i>, heterologous expression of SQS1 was implemented to form squalene. For monooxygenation of squalene to the central precursor 2,3-oxidosqualene, SQE1 was heterologously expressed in both host strains. Subsequently, the cyclization module was designed to convert the linear precursor 2,3-oxidosqualene into plant sterols and further cyclic triterpenes. To cover different triterpene scaffolds, the OSC enzymes CAS1, LUP1, THAS1, and MRN1 from <i>A</i>. <i>thaliana</i> were expressed in each host to synthesize cycloartenol (sterol), lupeol (exhibiting the often occurring pentacyclic scaffold) as well as thalianol and marneral (representing more unusual tri- or monocyclic structures). Respective substrate folding is indicated (CBC, chair-boat-chair; CCC, chair-chair-chair; CB, chair-boat). <b>(B)</b> Schematic representation of expression constructs used for triterpenoid biosynthesis in <i>R</i>. <i>capsulatus</i> and <i>Synechocystis</i>. The respective genes were arranged as synthetic operons, using native ammonium (P_<i>nif</i>) and cobalt (P_<i>coaT</i>) regulated promoters for transcription activation. FPP, farnesyl pyrophosphate; SQS, squalene synthase; SQE, squalene epoxidase; OSC, oxidosqualene cyclase.</p