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Background PHB (poly-hydroxy-butyrate) represents a promising bioplastic alternative with good biodegradation properties. Furthermore, PHB can be produced in a completely carbon–neutral fashion in the natural producer cyanobacterium Synechocystis sp. PCC 6803. This strain has been used as model system in past attempts to boost the intracellular production of PHB above ~ 15% per cell-dry-weight (CDW). Results We have created a new strain that lacks the regulatory protein PirC (product of sll0944 ), which exhibits a higher activity of the phosphoglycerate mutase resulting in increased PHB pools under nutrient limiting conditions. To further improve the intracellular PHB content, two genes involved in PHB metabolism, phaA and phaB, from the known producer strain Cupriavidus necator , were introduced under the control of the strong promotor P psbA2 . The resulting strain, termed PPT1 (Δ pirC -RE phaAB ), produced high amounts of PHB under continuous light as well under a day-night regime. When grown in nitrogen and phosphorus depleted medium, the cells produced up to 63% per CDW. Upon the addition of acetate, the content was further increased to 81% per CDW. The produced polymer consists of pure PHB, which is highly isotactic. Conclusion The amounts of PHB achieved with PPT1 are the highest ever reported in any known cyanobacterium and demonstrate the potential of cyanobacteria for a sustainable, industrial production of PHB.

Main conclusion : The Ycf46 mutant of Synechocystis showed growth inhibition under low dissolved CO sub( 2 ) conditions, suggesting a role for the Ycf46 protein in the process of photosynthetic CO sub( 2 ) uptake and utilization. Abstract: Hypothetical chloroplast open reading frame Ycf46 proteins are highly conserved in all cyanobacterial lineages and most algal chloroplast genomes, but their exact function is still unknown. In the cyanobacterium Synechocystis sp. PCC 6803, the Ycf46 encoding gene slr0374 is part of an operon (with slr0373 and slr0376) and responds to many environmental stresses. Transcript levels of the slr0373, slr0374 and slr0376 genes were increased under a low concentration of dissolved inorganic carbon (C sub(i)). Compared with the wild type, the mutant lacking slr0374 showed growth arrest under C sub(i)-deficient conditions but not under iron-deficient or low-light conditions. In addition, the mutant grew more slowly than the wild type under pH 6.0 conditions in which CO sub(2) was the dominant C sub(i) source, indicating the mutant cells had weak CO sub(2) uptake and/or utilization ability. Supplying a high concentration of CO sub(2) (5 %, v/v) to the mutant restored its phenotype to the wild type level. The photosynthetic activity of the mutant was inhibited to a lesser extent by a carbonic anhydrase inhibitor than that of the wild type, which specifically blocked CO sub(2) uptake. Inactivation of slr0374 decreased expression of the ecaB gene and reduced carbonic anhydrase activity. A subcellular localization assay indicated that the Ycf46 protein was soluble. By co-immunoprecipitation assay using Slr0374 as a bait-protein, potential interacting proteins in the size range of 30 kDa were identified. These results suggest that the Ycf46 protein plays a role in the regulation of photosynthesis in cyanobacteria, especially in CO sub(2) uptake and utilization.

Culturing cyanobacteria in a highly alkaline environment is a possible strategy for controlling contamination by other organisms. Synechocystis PCC 6803 cells were grown in continuous cultures to assess their growth performance at different pH values. Light conversion efficiency linearly decreased with the increase in pH and ranged between 12.5 % (PAR) at pH 7.5 (optimal) and decreased to 8.9 % at pH 11.0. Photosynthetic activity, assessed by measuring both chlorophyll fluorescence and photosynthesis rate, was not much affected going from pH 7.5 to 11.0, while productivity, growth yield, and biomass yield on light energy declined by 32, 28, and 26 % respectively at pH 11.0. Biochemical composition of the biomass did not change much within pH 7 and 10, while when grown at pH 11.0, carbohydrate content increased by 33 % while lipid content decreased by about the same amount. Protein content remained almost constant (average 65.8 % of dry weight). Cultures maintained at pH above 11.0 could grow free of contaminants (protozoa and other competing microalgae belonging to the species of Poterioochromonas).

Several studies have described that cyanobacteria use blue light less efficiently for photosynthesis than most eukaryotic phototrophs, but comprehensive studies of this phenomenon are lacking. Here, we study the effect of blue (450 nm), orange (625 nm), and red (660 nm) light on growth of the model cyanobacterium Synechocystis sp. PCC 6803, the green alga Chlorella sorokiniana and other cyanobacteria containing phycocyanin or phycoerythrin. Our results demonstrate that specific growth rates of the cyanobacteria were similar in orange and red light, but much lower in blue light. Conversely, specific growth rates of the green alga C. sorokiniana were similar in blue and red light, but lower in orange light. Oxygen production rates of Synechocystis sp. PCC 6803 were five-fold lower in blue than in orange and red light at low light intensities but approached the same saturation level in all three colors at high light intensities. Measurements of 77 K fluorescence emission demonstrated a lower ratio of photosystem I to photosystem II (PSI:PSII ratio) and relatively more phycobilisomes associated with PSII (state 1) in blue light than in orange and red light. These results support the hypothesis that blue light, which is not absorbed by phycobilisomes, creates an imbalance between the two photosystems of cyanobacteria with an energy excess at PSI and a deficiency at the PSII-side of the photosynthetic electron transfer chain. Our results help to explain why phycobilisome-containing cyanobacteria use blue light less efficiently than species with chlorophyll-based light-harvesting antennae such as Prochlorococcus, green algae and terrestrial plants.

Cyanobacteria have been proven to be cheaper and more effective for the removal of metallic elements in aqueous solutions. In this study, the living cyanobacteria Synechocystis sp. PCC6803 was used to adsorb Cd(II) and its extracellular polymeric substances (EPS) were investigated in the adsorption process. The initial stage of adsorption of Cd(II) was a rapid process, and then increase slowly accompanied with the increases of biomass. The final adsorption percentage could achieve 86% when the Cd(II) concentration was 0.5 mg/L. It proved that Synechocystis sp. PCC6803 has a good adsorption capacity for heavy metal ions. EPS was extracted to investigate the secretion of which was dynamic and the maximum extracellular polysaccharides and proteins were 134.2 and 100.9 mg/g, respectively. Furthermore, the real-time PCR (RT-PCR) results of genes ( slr0977 and exoD ) involved in EPS synthesis and secretion indicated that the EPS production was firstly increased and then decreased slightly. Transmission electron microscope (TEM) observation revealed that heavy metal ions were absorbed into EPS layer. Fourier transform infrared spectrum (FT-IR) analysis showed that EPS was rich in functional groups which could combine with heavy metal ions, such as –OH and –NH groups. All the results obtained show that the secretion of EPS by cyanobacteria was one of the ways to resist heavy metal stress. And it shows a trend of rising first and then decreasing, the change regulation of which was consistent with adsorptive behavior.

Cyanobacterial flavodiiron proteins (FDPs; A-type flavoprotein, Flv) comprise, besides the β-lactamase–like and flavodoxin domains typical for all FDPs, an extra NAD(P)H:flavin oxidoreductase module and thus differ from FDPs in other Bacteria and Archaea. Synechocystis sp. PCC 6803 has four genes encoding the FDPs. Flv1 and Flv3 function as an NAD(P)H:oxygen oxidoreductase, donating electrons directly to O ₂ without production of reactive oxygen species. Here we show that the Flv1 and Flv3 proteins are crucial for cyanobacteria under fluctuating light, a typical light condition in aquatic environments. Under constant-light conditions, regardless of light intensity, the Flv1 and Flv3 proteins are dispensable. In contrast, under fluctuating light conditions, the growth and photosynthesis of the Δ flv1(A) and/or Δ flv3(A) mutants of Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120 become arrested, resulting in cell death in the most severe cases. This reaction is mainly caused by malfunction of photosystem I and oxidative damage induced by reactive oxygen species generated during abrupt short-term increases in light intensity. Unlike higher plants that lack the FDPs and use the Proton Gradient Regulation 5 to safeguard photosystem I, the cyanobacterial homolog of Proton Gradient Regulation 5 is shown not to be crucial for growth under fluctuating light. Instead, the unique Flv1/Flv3 heterodimer maintains the redox balance of the electron transfer chain in cyanobacteria and provides protection for photosystem I under fluctuating growth light. Evolution of unique cyanobacterial FDPs is discussed as a prerequisite for the development of oxygenic photosynthesis.

One strategy for CO mitigation is using photosynthetic microorganisms to sequester CO under high concentrations, such as in flue gases. While elevated CO levels generally promote growth, excessively high levels inhibit growth through uncertain mechanisms. This study investigated the physiology of the cyanobacterium Synechocystis sp. PCC 6803 under very high CO concentrations and yet stable pH around 7.5. The growth rate of the wild type (WT) at 200 µmol photons m  s and a gas phase containing 30% CO was 2.7-fold lower compared to 4% CO . Using a CRISPR interference mutant library, we identified genes that, when repressed, either enhanced or impaired growth under 30% or 4% CO . Repression of genes involved in light harvesting (cpc and apc), photochemical electron transfer (cytM, psbJ, and petE), and several genes with little or unknown functions promoted growth under 30% CO , while repression of key regulators of photosynthesis (pmgA) and CO capture and fixation (ccmR, cp12, and yfr1) increased growth inhibition under 30% CO . Experiments confirmed that WT cells were more susceptible to light inhibition under 30% than under 4% CO and that a light-harvesting-impaired ΔcpcG mutant showed improved growth under 30% CO compared to the WT. These findings suggest that enhanced fitness under very high CO involves modifications in light harvesting, electron transfer, and carbon metabolism, and that the native regulatory machinery is insufficient, and in some cases obstructive, for optimal growth under 30% CO . This genetic profiling provides potential targets for engineering cyanobacteria with improved photosynthetic efficiency and stress resilience for biotechnological applications. KEY POINTS: • Synechocystis growth was inhibited under very high CO . • Inhibition of growth under very high CO was light dependent. • Repression of photosynthesis genes improved growth under very high CO .

Fluctuating light (FL) poses a challenge to cyanobacteria by disrupting photosynthesis and damaging photosystems. Although key FL tolerance components are known, their genetic enhancement remains unexplored. We evolve Synechocystis PCC 6803 under two FL regimes (one lethal to the starter strain, LT) in order to identify previously unknown adaptive alleles. Our analysis reveals 44 mutations, 28 of which impact proteins/RNAs. Mutations in Pam68 (PSII assembly) and Sll0518, present in all strains, enhance non-lethal FL tolerance in LT. Mutated Pam68 increased PSII abundance and activity. A gain-of-function mutation in RpaB (regulator of phycobilisome association B) significantly increases tolerance to both lethal FL and high-light conditions. This is associated with an increased PSI/PSII ratio and downregulation of light harvesting. In summary, our results suggest that adaptive laboratory evolution can simultaneously identify FL tolerance factors and their advantageous alleles. The identified point mutations rewire multiple protective responses by as yet unknown molecular mechanisms. Adaptive laboratory evolution enables cyanobacteria to tolerate fluctuating light by acquiring beneficial mutations. Key mutations improve photosystem performance or reduce light harvesting, enhancing survival under stressful light conditions.

Pyridine nucleotide transhydrogenase (PntAB) is an integral membrane protein complex participating in the regulation of NAD(P)+:NAD(P)H redox homeostasis in various prokaryotic and eukaryotic organisms. In the present study we addressed the function and biological role of PntAB in oxygenic photosynthetic cyanobacteria capable of both autotrophic and heterotrophic growth, with support from structural three-dimensional (3D)-modeling. The pntA gene encoding the α subunit of heteromultimeric PntAB in Synechocystis sp. PCC 6803 was inactivated, followed by phenotypic and biophysical characterization of the ΔpntA mutant under autotrophic and mixotrophic conditions. Disruption of pntA resulted in phenotypic growth defects observed under low light intensities in the presence of glucose, whereas under autotrophic conditions the mutant did not differ from the wild-type strain. Biophysical characterization and protein-level analysis of the ΔpntA mutant revealed that the phenotypic defects were accompanied by significant malfunction and damage of the photosynthetic machinery. Our observations link the activity of PntAB in Synechocystis directly to mixotrophic growth, implicating that under these conditions PntAB functions to balance the NADH: NADPH equilibrium specifically in the direction of NADPH. The results also emphasize the importance of NAD(P)+:NAD(P)H redox homeostasis and associated ATP:ADP equilibrium for maintaining the integrity of the photosynthetic apparatus under low-light glycolytic metabolism.

In photosynthetic organisms, photons are captured by light-harvesting antenna complexes, and energy is transferred to reaction centers where photochemical reactions take place. We describe here the isolation and characterization of a fully functional megacomplex composed of a phycobilisome antenna complex and photosystems I and II from the cyanobacterium Synechocystis PCC 6803. A combination of in vivo protein cross-linking, mass spectrometry, and time-resolved spectroscopy indicates that the megacomplex is organized to facilitate energy transfer but not intercomplex electron transfer, which requires diffusible intermediates and the cytochrome b 6 f complex. The organization provides a basis for understanding how phycobilisomes transfer excitation energy to reaction centers and how the energy balance of two photosystems is achieved, allowing the organism to adapt to varying ecophysiological conditions.