26 research outputs found
Light Driven CO2 Fixation by Using Cyanobacterial Photosystem I and NADPH-Dependent Formate Dehydrogenase
Light Driven CO<sub>2</sub> Fixation by Using Cyanobacterial Photosystem I and NADPH-Dependent Formate Dehydrogenase
<div><p>The ultimate goal of this research is to construct a new direct CO<sub>2</sub> fixation system using photosystems in living algae. Here, we report light-driven formate production from CO<sub>2</sub> by using cyanobacterial photosystem I (PS I). Formate, a chemical hydrogen carrier and important industrial material, can be produced from CO<sub>2</sub> by using the reducing power and the catalytic function of formate dehydrogenase (FDH). We created a bacterial FDH mutant that experimentally switched the cofactor specificity from NADH to NADPH, and combined it with an <i>in vitro</i>-reconstituted cyanobacterial light-driven NADPH production system consisting of PS I, ferredoxin (Fd), and ferredoxin-NADP<sup>+</sup>-reductase (FNR). Consequently, light-dependent formate production under a CO<sub>2</sub> atmosphere was successfully achieved. In addition, we introduced the NADPH-dependent FDH mutant into heterocysts of the cyanobacterium <i>Anabaena</i> sp. PCC 7120 and demonstrated an increased formate concentration in the cells. These results provide a new possibility for photo-biological CO<sub>2</sub> fixation.</p> </div
Formate production by pAM505-fdh exoconjugant cyanobacteria.
<p>The wild-type and exoconjugant <i>Anabaena</i> cultures (BG11, OD<sub>700</sub> ~3) were inoculated into BG11<sub>0</sub> and cultivated for 36 h under a light of 15 µmol photon m<sup>-2</sup> s<sup>-1</sup> and under air atmosphere. The media were then degassed and bubbled with argon + 10% CO<sub>2</sub> for 12 h under a light of 150 µmol photon m<sup>-2</sup> s<sup>-1</sup>. The cells anaerobically harvested were homogenized in 1N HCl. The resulting lysate was analyzed by ion-chromatography and the chromatograms of <i>Anabaena</i>-WT (A) and <i>Anabaena</i>-fdh (B) are shown. The lysate of <i>Anabaena</i>-fdh was also treated with <i>Candida</i><i>boidinii</i> FDH (Sigma-Aldrich, Missouri, USA) and NAD<sup>+</sup> overnight and was then analyzed (C). The chromatograms of formate solutions without and with the treatment with <i>Candida</i><i>boidinii</i> FDH and NAD<sup>+</sup> are shown as controls (D and E, respectively).</p
Expression of PsFDH[QN] in <i>Anabaena</i>-fdh.
<p><i>Anabaena</i>-fdh cells were cultured in non-inducible ((-), BG11) or inducible ((+), BG11<sub>0</sub>) medium and their whole lysates (C), lysate supernatants (S) and lysate precipitates (P) were analyzed by Western blotting using anti-Histag antibody HRP conjugate. Chemiluminescent signals were detected at approximately 44 kDa as expected.</p
Formate production activities of NADPH-FDHs.
<p>The reaction solution contained 1.67 mg/mL of FDH, 1 mM NAD(P) H, 0.05% Triton X-100 and 1.1–1.3 mg/ml dissolved CO<sub>2</sub>. The concentration of formate was determined by a HPLC equipped with a Shim-pack SCR-102H column and a conductivity detector, CDD-10A<sub>VP</sub> (Shimadzu, Kyoto, Japan). Open circles, closed circles, open triangles and closed triangles represent data of combinations of WTs with NADPH, mutants with NADPH, WTs with NADH, and mutants with NADH, respectively. All measurements were performed in triplicate.</p
Substrate dependence and light sensitivity of PsFDH(QN).
<p>The formate production in the presence of 1 mM NADPH (circles) were compared with that in the presence of 10 mM NADPH (diamonds), that under visible light at an intensity of 1000 µmol photon m<sup>-2</sup> s<sup>-1</sup> (squares), and that in which NaHCO<sub>3</sub> was used instead of CO<sub>2</sub> (crosses). These experiments were performed as mentioned in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071581#pone-0071581-g002" target="_blank">Figure 2</a>, except as described above.</p
<i>In vitro</i> light-driven CO<sub>2</sub> fixation by PS I and NADPH-FDH.
<p>The reaction solutions contained PS I (100 µg chlorophyll/mL), 10 mM ascorbate, 10 µM PC, 2 µM Fd, 0.5 µM FNR, 1 mM NADP<sup>+</sup>, and 4 mM NADPH, 2.5 mg/mL (~56 µM) PsFDH(QN), 0.05% Triton X-100 and 10% sucrose in PBS. The solution were degassed and then charging with 100% CO<sub>2</sub> for 1 min. The reaction was initiated by illuminating with visible light (<420 nm) at an intensity of 1000 µmol photon m<sup>-2</sup> s<sup>-1</sup>, and monitored by sampling at regular intervals without exposure to O<sub>2</sub> (closed square). The control reactions were performed without the illumination (open circles) and in the presence of 10% oxygen gas (open triangles).</p