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Utilizing ammonium in wastewater is a prospective way to reduce costs for bioproduction by photosynthetic organisms. A model cyanobacterium Synechocystis sp. PCC 6803 takes advantage of tolerance to ammonium compared to other microalgae. However, in this study, we report that Synechocystis growth was inhibited when cultured in a medium containing ammonium. This may be due to the pH decreasing below 6 caused by consuming ammonium. Transcriptomic analysis by RNA-seq revealed that the expression of the genes for proteases, chaperones, and antioxidant-scavenging enzymes was induced, but photosynthetic components were repressed. Although these regulations are similar to the previous studies on acidic stress in nitrate-containing culture, the expression of genes such as sigD, slr0042, slr0373, slr0374, and slr1501 was different, indicating that these phenomena are not simply identical to the known responses to acidic stress. The expression of the genes for photosynthesis, gluconeogenesis, and nitrogen assimilation was repressed, and glycolysis and the tricarboxylic acid cycle were induced. Despite the up-regulation of the carbon catabolism and down-regulation of nitrogen assimilation, the 2-oxoglutarate content in the ammonium-grown cells was lower than that in the nitrate-grown cells, and the contents of the major amino acids, such as Glu, Ala, Asp, and Gly were decreased, while the minor amino acids were the same or increased, especially Arg, Lys, Val, and Ile. These results demonstrated that the acidic stress induced by the consumption of ammonium ions differs from the sudden pH drop, and the Synechocystis cell manages amino acid levels to endure carbon limitation under the stress., (Copyright © 2024 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Although most cyanobacteria grow in visible light (VL; λ = 400-700 nm), some cyanobacteria can also use far-red light (FRL; λ = 700-800 nm) for oxygenic photosynthesis by performing far-red light photoacclimation. These two types of cyanobacteria can be found in the same environment. However, how they respond to each other remains unknown. Here, we reveal that coculture stresses FRL-using Chlorogloeopsis fritschii PCC 9212 and VL-using Synechocystis sp. PCC 6803. No significant growth difference was found in Synechocystis sp. PCC 6803 between the coculture and the monoculture. Conversely, the growth of Chlorogloeopsis fritschii PCC 9212 was suppressed in VL under coculture. According to transcriptomic analysis, Chlorogloeopsis fritschii PCC 9212 in coculture shows low transcript levels of metabolic activities and high transcript levels of ion transporters, with the differences being more noticeable in VL than in FRL. The transcript levels of stress responses in coculture were likewise higher than in monoculture in Synechocystis sp. PCC 6803 under FRL. The low transcript level of metabolic activities in coculture or the inhibition of cyanobacterial growth indicates a possible negative interaction between these two cyanobacterial strains.IMPORTANCEThe interaction between two cyanobacterial species is the primary focus of this study. One species harvests visible light, while the other can harvest far-red and visible light. Prior research on cyanobacteria interaction concentrated on its interactions with algal, coral, and fungal species. Interactions between cyanobacterial species were, nevertheless, rarely discussed. Thus, we characterized the interaction between two cyanobacterial species, one capable of photosynthesis using far-red light and the other not. Through experimental and bioinformatic approaches, we demonstrate that when one cyanobacterium thrives under optimal light conditions, it stresses the remaining cyanobacterial species. We contribute to an ecological understanding of these two kinds of cyanobacteria distribution patterns. Cyanobacteria that utilize far-red light probably disperse in environments with limited visible light to avoid competition with other cyanobacteria. From a biotechnological standpoint, this study suggests that the simultaneous cultivation of two cyanobacterial species in large-scale cultivation facilities may reduce the overall biomass yield., Competing Interests: The authors declare no conflict of interest.

The high performance of biomass and metabolite biosynthesis by photosynthetic microorganisms is directly influenced by the cultivation system employed. Photobioreactors (PBRs) stand out as controlled and fundamental systems for increasing the production of biocompounds. However, the high costs associated with these systems hinder their viability. Thus, a more practical and economical approach is necessary. Accordingly, this study aimed to design and evaluate low-cost flat-panel photobioreactors on a laboratory scale for the cultivation of photosynthetic microorganisms, using economical materials and instruments. Additionally, internal optimization of the low-cost system was aimed to maximize growth and biomass production. The PBRs were designed and built with uniform dimensions, employing 4 mm translucent glass and agitation through compressors. The internally optimized system (PBR-OII) was equipped with perforated acrylic plates used as static mixers. To evaluate the performance of the low-cost PBR-OII, a comparison was made with the control photobioreactor (PBR-CI), of the same geometry but without internal optimization, using a culture of Synechocystis sp. CACIAM 05 culture. The results showed that the PBR-OII achieved maximum biomass yield and productivity of 6.82 mg/mL and 250 mg/L/day, respectively, values superior to the PBR-CI (1.87 mg/mL and 62 mg/L/day). Additionally, the chlorophyll concentration in the PBR-OII system was 28.89 ± 3.44 µg/mL, while in the control system, the maximum reached was 23.12 ± 1.85 µg/mL. Therefore, low-cost photobioreactors have demonstrated to be an essential tool for significantly increasing biomass production, supporting research, and reducing costs associated with the process, enabling their implementation on a laboratory scale.

Cyanobacteria play a key role in primary production in both oceans and fresh waters and hold great potential for sustainable production of a large number of commodities. During their life, cyanobacteria cells need to acclimate to a multitude of challenges, including shifts in intensity and quality of incident light. Despite our increasing understanding of metabolic regulation under various light regimes, detailed insight into fitness advantages and limitations under shifting light quality remains underexplored. Here, we study photo-physiological acclimation in the cyanobacterium Synechocystis sp. PCC 6803 throughout the photosynthetically active radiation (PAR) range. Using light emitting diodes (LEDs) with qualitatively different narrow spectra, we describe wavelength dependence of light capture, electron transport and energy transduction to main cellular pools. In addition, we describe processes that fine-tune light capture, such as state transitions, or the efficiency of energy transfer from phycobilisomes to photosystems (PS). We show that growth was the most limited under blue light due to inefficient light harvesting, and that many cellular processes are tightly linked to the redox state of the plastoquinone (PQ) pool, which was the most reduced under red light. The PSI-to-PSII ratio was low under blue photons, however, it was not the main growth-limiting factor, since it was even more reduced under violet and near far-red lights, where Synechocystis grew faster compared to blue light. Our results provide insight into the spectral dependence of phototrophic growth and can provide the foundation for future studies of molecular mechanisms underlying light acclimation in cyanobacteria, leading to light optimization in controlled cultivations., (© The Author(s) 2024. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.)

Alka(e)nes are produced by many living organisms and exhibit diverse physiological roles, reflecting a high functional versatility. Alka(e)nes serve as waterproof wax in plants, communicating pheromones for insects, and microbial signaling molecules in some bacteria. Although alka(e)nes have been found in cyanobacteria and algal chloroplasts, their importance for photosynthetic membranes has remained elusive. In this study, we investigated the consequences of the absence of alka(e)nes on membrane lipid composition and photosynthesis using the cyanobacterium Synechocystis PCC6803 as a model organism. By following the dynamics of membrane lipids and the photosynthetic performance in strains defected and altered in alka(e)ne biosynthesis, we show that drastic changes in the glycerolipid contents occur in the absence of alka(e)nes, including a decrease in the membrane carotenoid content, a decrease in some digalactosyldiacylglycerol (DGDG) species and a parallel increase in monogalactosyldiacylglycerol (MGDG) species. These changes are associated with a higher susceptibility of photosynthesis and growth to high light in alka(e)ne-deficient strains. All these phenotypes are reversed by expressing an algal photoenzyme producing alka(e)nes from fatty acids. Therefore, alkenes, despite their low abundance, are an essential component of the lipid composition of membranes. The profound remodeling of lipid composition that results from their absence suggests that they play an important role in one or more membrane properties in cyanobacteria. Moreover, the lipid compensatory mechanism observed is not sufficient to restore normal functioning of the photosynthetic membranes, particularly under high-light intensity. We conclude that alka(e)nes play a crucial role in maintaining the lipid homeostasis of thylakoid membranes, thereby contributing to the proper functioning of photosynthesis, particularly under elevated light intensities., Competing Interests: Conflict of interest statement. The authors declare that there is no potential conflict of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

The metabolism of phototrophic cyanobacteria is an integral part of global biogeochemical cycles, and the capability of cyanobacteria to assimilate atmospheric CO2 into organic carbon has manifold potential applications for a sustainable biotechnology. To elucidate the properties of cyanobacterial metabolism and growth, computational reconstructions of genome-scale metabolic networks play an increasingly important role. Here, we present an updated reconstruction of the metabolic network of the cyanobacterium Synechocystis sp. PCC 6803 and its quantitative evaluation using flux balance analysis (FBA). To overcome limitations of conventional FBA, and to allow for the integration of experimental analyses, we develop a novel approach to describe light absorption and light utilization within the framework of FBA. Our approach incorporates photoinhibition and a variable quantum yield into the constraint-based description of light-limited phototrophic growth. We show that the resulting model is capable of predicting quantitative properties of cyanobacterial growth, including photosynthetic oxygen evolution and the ATP/NADPH ratio required for growth and cellular maintenance. Our approach retains the computational and conceptual simplicity of FBA and is readily applicable to other phototrophic microorganisms., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Höper et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Glycosyltransferases (GTs) are enzymes that transfer sugars to various targets. They play important roles in diverse biological processes, including photosynthesis, cell motility, exopolysaccharide biosynthesis, and lipid metabolism; however, their involvement in regulating carbon metabolism in Synechocystis sp. PCC 6803 has not been reported. We identified a novel GT protein, Slr1064, involved in carbon metabolism. The effect of slr1064 deletion on the growth of Synechocystis cells and functional mechanisms of Slr1064 on carbon metabolism were thoroughly investigated through physiological, biochemistry, proteomic, and metabolic analyses. We found that this GT, which is mainly distributed in the membrane compartment, is essential for the growth of Synechocystis under heterotrophic and mixotrophic conditions, but not under autotrophic conditions. The deletion of slr1064 hampers the turnover rate of Gap2 under mixotrophic conditions and disrupts the assembly of the PRK/GAPDH/CP12 complex under dark culture conditions. Additionally, UDP-GlcNAc, the pivotal metabolite responsible for the O-GlcNAc modification of GAPDH, is downregulated in the Δslr1064. Our work provides new insights into the role of GTs in carbon metabolism in Synechocystis and elucidate the mechanism by which carbon metabolism is regulated in this important model organism., (© 2024 The Authors. New Phytologist © 2024 New Phytologist Foundation.)

Polyhydroxyalkanoates (PHAs) are biopolymers produced by microorganisms under nutrient limiting conditions and in the presence of excess carbon source. PHAs have gained popularity as a sustainable alternative to traditional plastics. However, large scale production of PHAs is economically challenging due to the relatively high costs of organic carbon. Alternative options include using organisms capable of phototrophic or mixotrophic growth. This study aimed at the production of poly(3-hydroxybutyrate) P(3HB), a type of PHA, at pilot scale using the freshwater cyanobacterium Synechocystis sp. PCC6803. First, to identify optimal conditions for P(3HB) production from Synechocystis sp. PCC6803, different supplemental carbon source concentrations and salinity levels were tested at laboratory scale. The addition of 4 g/L acetate with no added NaCl led to P(3HB) accumulation of 10.7 % dry cell weight on the 28th day of cultivation. Although acetate additions were replicated in an outdoor 400 L serpentine photobioreactor, P(3HB) content was lower, implying uncontrolled conditions impact on biopolymer production efficiency. An optimized P(3HB) extraction methodology was developed to remove pigments, and the biopolymer was characterized and subjected to 3D printing (fused deposition modelling) to confirm its processability. This study thus successfully led to the large-scale production of P(3HB) using sustainable and environmentally friendly cyanobacterial fermentation., Competing Interests: Declaration of Competing Interest None., (Crown Copyright © 2024. Published by Elsevier B.V. All rights reserved.)

Some cyanobacteria can grow photoautotrophically or photomixotrophically by using simultaneously CO 2 and glucose. The switch between these trophic modes and the role of glycogen, their main carbon storage macromolecule, was investigated. We analysed the effect of glucose addition on the physiology, metabolic and photosynthetic state of Synechocystis sp. PCC 6803 and mutants lacking phosphoglucomutase and ADP-glucose pyrophosphorylase, with limitations in glycogen synthesis. Glycogen acted as a metabolic buffer: glucose addition increased growth and glycogen reserves in the wild-type (WT), but arrested growth in the glycogen synthesis mutants. Already 30 min after glucose addition, metabolites from the Calvin-Benson-Bassham cycle and the oxidative pentose phosphate shunt increased threefold more in the glycogen synthesis mutants than the WT. These alterations substantially affected the photosynthetic performance of the glycogen synthesis mutants, as O 2 evolution and CO 2 uptake were both impaired. We conclude that glycogen synthesis is essential during transitions to photomixotrophy to avoid metabolic imbalance that induces inhibition of electron transfer from PSII and subsequently accumulation of reactive oxygen species, loss of PSII core proteins, and cell death. Our study lays foundations for optimising photomixotrophy-based biotechnologies through understanding the coordination of the crosstalk between photosynthetic electron transport and metabolism., (© 2024 The Authors. New Phytologist © 2024 New Phytologist Foundation.)

Background: Advancing the engineering of photosynthesis-based prokaryotic cell factories is important for sustainable chemical production and requires a deep understanding of the interplay between bioenergetic and metabolic pathways. Rearrangements in photosynthetic electron flow to increase the efficient use of the light energy for carbon fixation must be balanced with a strong carbon sink to avoid photoinhibition. In the cyanobacterium Synechocystis sp. PCC 6803, the flavodiiron protein Flv3 functions as an alternative electron acceptor of photosystem I and represents an interesting engineering target for reorganizing electron flow in attempts to enhance photosynthetic CO 2 fixation and increase production yield., Results: We have shown that inactivation of Flv3 in engineered sucrose-excreting Synechocystis (S02:Δflv3) induces a transition from photoautotrophic sucrose production to mixotrophic growth sustained by sucrose re-uptake and the formation of intracellular carbon sinks such as glycogen and polyhydroxybutyrate. The growth of S02:Δflv3 exceeds that of the sucrose-producing strain (S02) and demonstrates unforeseen proteomic and metabolomic changes over the course of the nine-day cultivation. In the absence of Flv3, a down-regulation of proteins related to photosynthetic light reactions and CO 2 assimilation occurred concomitantly with up-regulation of those related to glycolytic pathways, before any differences in sucrose production between S02 and S02:Δflv3 strains were observed. Over time, increased sucrose degradation in S02:Δflv3 led to the upregulation of respiratory pathway components, such as the plastoquinone reductase complexes NDH-1 1 and NDH-2 and the terminal respiratory oxidases Cyd and Cox, which transfer electrons to O 2 . While glycolytic metabolism is significantly up-regulated in S02:Δflv3 to provide energy for the cell, the accumulation of intracellular storage compounds and the increase in respiration serve as indirect sinks for photosynthetic electrons., Conclusions: Our results show that the presence of strong carbon sink in the engineered sucrose-producing Synechocystis S02 strain, operating under high light, high CO 2 and salt stress, cannot compensate for the lack of Flv3 by directly balancing the light transducing source and carbon fixing sink reactions. Instead, the cells immediately sense the imbalance, leading to extensive reprogramming of cellular bioenergetic, metabolic and ion transport pathways that favor mixotrophic growth rather than enhancing photoautotrophic sucrose production., (© 2024. The Author(s).)

The cyanobacterial response to pharmaceuticals is less frequently investigated compared to green algae. Pharmaceuticals can influence not only the growth rate of cyanobacteria culture, but can also cause changes at the cellular level. The effect of diclofenac (DCF) as one of the for cyanobacteria has been rarely tested, and DCF has never been applied with cellular biomarkers. The aim of this work was to test the response of two unicellular cyanobacteria (Synechocystis salina and Microcystis aeruginosa) toward DCF (100 mg L -1 ) under photoautotrophic growth conditions. Such endpoints were analyzed as cells number, DCF uptake, the change in concentrations of photosynthetic pigments, the production of toxins, and chlorophyll a in vivo fluorescence. It was noted that during a 96 h exposure, cell proliferation was not impacted. Nevertheless, a biochemical response was observed. The increased production of microcystin was noted for M. aeruginosa. Due to the negligible absorption of DCF into cells, it is possible that the biochemical changes are induced by an external signal. The application of non-standard biomarkers demonstrates the effect of DCF on microorganism metabolism without a corresponding effect on biomass. The high resistance of cyanobacteria to DCF and the stimulating effect of DCF on the secretion of toxins raise concerns for environment biodiversity., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Magda Caban reports financial support was provided by National Science Centre Poland. Magda Caban reports a relationship with National Science Centre Poland that includes: funding grants. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The low-molecular-weight PsbM and PsbT proteins of Photosystem II (PS II) are both located at the monomer-monomer interface of the mature PS II dimer. Since the extrinsic proteins are associated with the final step of assembly of an active PS II monomer and, in the case of PsbO, are known to impact the stability of the PS II dimer, we have investigated the potential cooperativity between the PsbM and PsbT subunits and the PsbO, PsbU and PsbV extrinsic proteins. Blue-native polyacrylamide electrophoresis and western blotting detected stable PS II monomers in the ∆PsbM:∆PsbO and ∆PsbT:∆PsbO mutants that retained sufficient oxygen-evolving activity to support reduced photoautotrophic growth. In contrast, the ∆PsbM:∆PsbU and ∆PsbT:∆PsbU mutants assembled dimeric PS II at levels comparable to wild type and supported photoautotrophic growth at rates similar to those obtained with the corresponding ∆PsbM and ∆PsbT cells. Removal of PsbV was more detrimental than removal of PsbO. Only limited levels of dimeric PS II were observed in the ∆PsbM:∆PsbV mutant and the overall reduced level of assembled PS II in this mutant resulted in diminished rates of photoautotrophic growth and PS II activity below those obtained in the ∆PsbM:∆PsbO and ∆PsbT:∆PsbO strains. In addition, the ∆PsbT:∆PsbV mutant did not assemble active PS II centers although inactive monomers could be detected. The inability of the ∆PsbT:∆PsbV mutant to grow photoautotrophically, or to evolve oxygen, suggested a stable oxygen-evolving complex could not assemble in this mutant., (© 2024. The Author(s).)

The stoichiometry of photosystem II (PSII) and photosystem I (PSI) varies between photoautotrophic organisms. The cyanobacterium Synechocystis sp. PCC 6803 maintains two- to fivefold more PSI than PSII reaction center complexes, and we sought to modify this stoichiometry by changing the promoter region of the psaAB operon. We thus generated mutants with varied psaAB expression, ranging from ~3% to almost 200% of the wild-type transcript level, but all showing a reduction in PSI levels, relative to wild type, suggesting a role of the psaAB promoter region in translational regulation. Mutants with 25%-70% of wild-type PSI levels were photoautotrophic, with whole-chain oxygen evolution rates on a per-cell basis comparable to that of wild type. In contrast, mutant strains with <10% of the wild-type level of PSI were obligate photoheterotrophs. Variable fluorescence yields of all mutants were much higher than those of wild type, indicating that the PSI content is localized differently than in wild type, with less transfer of PSII-absorbed energy to PSI. Strains with less PSI saturate at a higher light intensity, enhancing productivity at higher light intensities. This is similar to what is found in mutants with reduced antennae. With 3-(3,4-dichlorophenyl)-1,1-dimethylurea present, P700 + re-reduction kinetics in the mutants were slower than in wild type, consistent with the notion that there is less cyclic electron transport if less PSI is present. Overall, strains with a reduction in PSI content displayed surprisingly vigorous growth and linear electron transport., Importance: Consequences of reduction in photosystem I content were investigated in the cyanobacterium Synechocystis sp. PCC 6803 where photosystem I far exceeds the number of photosystem II complexes. Strains with less photosystem I displayed less cyclic electron transport, grew more slowly at lower light intensity and needed more light for saturation but were surprisingly normal in their whole-chain electron transport rates, implying that a significant fraction of photosystem I is dispensable for linear electron transport in cyanobacteria. These strains with reduced photosystem I levels may have biotechnological relevance as they grow well at higher light intensities., Competing Interests: The authors declare no conflict of interest.

In nature, photosynthetic organisms are exposed to fluctuating light, and their physiological systems must adapt to this fluctuation. To maintain homeostasis, these organisms have a light fluctuation photoprotective mechanism, which functions in both photosystems and metabolism. Although the photoprotective mechanisms functioning in the photosystem have been studied, it is unclear how metabolism responds to light fluctuations within a few seconds. In the present study, we investigated the metabolic response of Synechocystis sp. PCC 6803 to light fluctuations using 13 C-metabolic flux analysis. The light intensity and duty ratio were adjusted such that the total number of photons or the light intensity during the low-light phase was equal. Light fluctuations affected cell growth and photosynthetic activity under the experimental conditions. However, metabolic flux distributions and cofactor production rates were not affected by the light fluctuations. Furthermore, the estimated ATP and NADPH production rates in the photosystems suggest that NADPH-consuming electron dissipation occurs under fluctuating light conditions. Although we focused on the water-water cycle as the electron dissipation path, no growth effect was observed in an flv3-disrupted strain under fluctuating light, suggesting that another path contributes to electron dissipation under these conditions., (© 2023 American Institute of Chemical Engineers.)

Background: Phenylpropanoids represent a diverse class of industrially important secondary metabolites, synthesized in plants from phenylalanine and tyrosine. Cyanobacteria have a great potential for sustainable production of phenylpropanoids directly from CO 2 , due to their photosynthetic lifestyle with a fast growth compared to plants and the ease of generating genetically engineered strains. This study focuses on photosynthetic production of the starting compounds of the phenylpropanoid pathway, trans-cinnamic acid and p-coumaric acid, in the unicellular cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis)., Results: A selected set of phenylalanine ammonia lyase (PAL) enzymes from different organisms was overexpressed in Synechocystis, and the productivities of the resulting strains compared. To further improve the titer of target compounds, we evaluated the use of stronger expression cassettes for increasing PAL protein levels, as well as knock-out of the laccase gene slr1573, as this was previously reported to prevent degradation of the target compounds in the cell. Finally, to investigate the effect of growth conditions on the production of trans-cinnamic and p-coumaric acids from Synechocystis, cultivation conditions promoting rapid, high density growth were tested. Comparing the different PALs, the highest specific titer was achieved for the strain AtC, expressing PAL from Arabidopsis thaliana. A subsequent increase of protein level did not improve the productivity. Production of target compounds in strains where the slr1573 laccase had been knocked out was found to be lower compared to strains with wild type background, and the Δslr1573 strains exhibited a strong phenotype of slower growth rate and lower pigment content. Application of a high-density cultivation system for the growth of production strains allowed reaching the highest total titers of trans-cinnamic and p-coumaric acids reported so far, at around 0.8 and 0.4 g L -1 , respectively, after 4 days., Conclusions: Production of trans-cinnamic acid, unlike that of p-coumaric acid, is not limited by the protein level of heterologously expressed PAL in Synechocystis. High density cultivation led to higher titres of both products, while knocking out slr1573 did not have a positive effect on production. This work contributes to capability of exploiting the primary metabolism of cyanobacteria for sustainable production of plant phenylpropanoids., (© 2022. The Author(s).)

Background: Cyanobacteria are engineered via heterologous biosynthetic pathways to produce value-added chemicals via photosynthesis. Various chemicals have been successfully produced in engineered cyanobacteria. Chemical inducer-dependent promoters are used to induce the expression of target biosynthetic pathway genes. A chemical inducer is not ideal for large-scale reactions owing to its high cost; therefore, it is important to develop scaling-up methods to avoid their use. In this study, we designed a green light-inducible alcohol production system using the CcaS/CcaR green light gene expression system in the cyanobacterium Synechocystis sp. PCC 6803 (PCC 6803)., Results: To establish the green light-inducible production of isobutanol and 3-methyl-1-butanol (3MB) in PCC 6803, keto-acid decarboxylase (kdc) and alcohol dehydrogenase (adh) were expressed under the control of the CcaS/CcaR system. Increases in the transcription level were induced by irradiation with red and green light without severe effects on host cell growth. We found that the production of isobutanol and 3MB from carbon dioxide (CO 2 ) was induced under red and green light illumination and was substantially repressed under red light illumination alone. Finally, production titers of isobutanol and 3MB reached 238 mg L -1 and 75 mg L -1 , respectively, in 5 days under red and green light illumination, and these values are comparable to those reported in previous studies using chemical inducers., Conclusion: A green light-induced alcohol production system was successfully integrated into cyanobacteria to produce value-added chemicals without using expensive chemical inducers. The green light-regulated production of isobutanol and 3MB from CO 2 is eco-friendly and cost-effective. This study demonstrates that light regulation is a potential tool for producing chemicals and increases the feasibility of cyanobacterial bioprocesses., (© 2022. The Author(s).)

The detection and identification of heavy metal contaminants are becoming increasingly important as environmental pollution causes an ever-increasing health hazard in the last decades. Bacterial heavy metal reporters, which constitute an environmentally friendly and cheap approach, offer great help in this process. Although their application has great potential in the detection of heavy metal contamination, their sensitivity still needs to be improved. In this study, we describe a simple molecular biology approach to improve the sensitivity of bacterial heavy metal biosensors. The constructs are luxAB marker genes regulated by the promoters of heavy metal exporter genes. We constructed a mutant strain lacking the cluster of genes responsible for heavy metal transport and hence achieved increased intracellular heavy metal content of the Synechocystis PCC6803 cyanobacterium. Taking advantage of this increased intracellular heavy metal concentration the Ni2+; Co2+ and Zn2+ detection limits of the constructs were three to tenfold decreased compared to the sensitivity of the same constructs in the wild-type cyanobacterium., Competing Interests: The authors have declared that no competing interests exist.

In anaerobic digestion of agro-industrial effluents and livestock wastes, concentrations of ammoniacal nitrogen above 800 mg L -1 are reported to lead to the eutrophication of water bodies. Through the metabolic versatility of microalgae, this nitrogen source can be used and removed, producing carotenoids, phycobiliproteins, polyhydroxyalkanoates, and fatty acids of industrial interest. The challenge of making it feasible is the toxicity of ammoniacal nitrogen to microalgae. Therefore, three strategies were evaluated. The first one was to find species of cyanobacteria with high ammoniacal nitrogen removal efficiency comparing Arthrospira platensis, Synechocystis D202, and Spirulina labyrinthiformis cultivations. The most promising species was cultivated in the second strategy, where cell acclimatization and increasing of the inoculum were evaluated. The cultivation condition that culminated in the best efficiency of ammoniacal nitrogen removal was combined with the third strategy, which consisted of conducting the fed-batch bioprocess. In the batch mode, ammoniacal nitrogen was supplied only once in one fed and was present in high initial concentrations. In fed-batch, multiple feedings with low concentrations of ammoniacal nitrogen were used to decrease the inhibitory effect of ammoniacal nitrogen. Arthrospira platensis showed high potential for ammoniacal nitrogen removal. Using the highest initial cell concentration of Arthrospira platensis cultivated by fed-batch, an increase in the consumption of NH 3 to 165.1 ± 1.8 mg L -1 and an ammoniacal nitrogen removal efficiency close to 90% were observed. Under this condition, 180.52 ± 11.67 mg g -1 of phycocyanin was attained. Also, the fed-batch cultivations have the potential to reduce the biomass cost production by 33% in comparison to batch experiments., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Although engineered cyanobacteria for the production of lipids and fatty acids (FAs) are intelligently used as sustainable biofuel resources, intracellularly overproduced FAs disturb cellular homeostasis and eventually generate lethal toxicity. In order to improve their production by enhancing FFAs secretion into a medium, we constructed three engineered Synechocystis 6803 strains including KA (a mutant lacking the aas gene), KAOL (KA overexpressing lipA , encoding lipase A in membrane lipid hydrolysis), and KAOGR (KA overexpressing quadruple glpD / rbcLXS , related to the CBB cycle). Certain contents of intracellular lipids and secreted FFAs of all engineered strains were higher than those of the wild type. Remarkably, the KAOL strain attained the highest level of secreted FFAs by about 21.9%w/DCW at day 5 of normal BG 11 cultivation, with a higher growth rate and shorter doubling time. TEM images provided crucial evidence on the morphological changes of the KAOL strain, which accumulated abundant droplets on regions of thylakoid membranes throughout the cell when compared with wild type. On the other hand, BG 11 -N condition significantly induced contents of both intracellular lipids and secreted FFAs of the KAOL strain up to 37.2 and 24.5%w/DCW, respectively, within 5 days. Then, for the first time, we shone a spotlight onto the overexpression of lipA in the aas mutant of Synechocystis as another potential strategy to achieve higher FFAs secretion with sustainable growth.

NADP+, the phosphorylated form of nicotinamide adenine dinucleotide (NAD), plays an essential role in many cellular processes. NAD kinase (NADK), which is conserved in all living organisms, catalyzes the phosphorylation of NAD+ to NADP+. However, the physiological role of phosphorylation of NAD+ to NADP+ in the cyanobacterium Synechocystis remains unclear. In this study, we report that slr0400, an NADK-encoding gene in Synechocystis, functions as a growth repressor under light-activated heterotrophic growth conditions and light and dark cycle conditions in the presence of glucose. We show, via characterization of NAD(P)(H) content and enzyme activity, that NAD+ accumulation in slr0400-deficient mutant results in the unsuppressed activity of glycolysis and tricarboxylic acid (TCA) cycle enzymes. In determining whether Slr0400 functions as a typical NADK, we found that constitutive expression of slr0400 in an Arabidopsis nadk2-mutant background complements the pale-green phenotype. Moreover, to determine the physiological background behind the growth advantage of mutants lacking slr04000, we investigated the photobleaching phenotype of slr0400-deficient mutant under high-light conditions. Photosynthetic analysis found in the slr0400-deficient mutant resulted from malfunctions in the Photosystem II (PSII) photosynthetic machinery. Overall, our results suggest that NADP(H)/NAD(H) maintenance by slr0400 plays a significant role in modulating glycolysis and the TCA cycle to repress the growth rate and maintain the photosynthetic capacity., (© The Author(s) 2021. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

A carbon-free energy supply is essential to sustain our future. Biophotovoltaics (BPV) provides a promising solution for hydrogen supply by directly coupling light-driven water splitting to hydrogen formation using oxygenic photoautotrophic cyanobacteria. However, BPV is currently limited by its low photon-to-current efficiency, and current experimental setups at a miniaturized scale hinder the rational investigation of the process and thus system optimization. In this article, we developed and optimized a new technical-scale (~250 ml working volume) BPV platform with defined and controllable operating parameters. Factors that interfered with reproducible and stable current output signals were identified and adapted. We found that the classical BG11 medium, used for the cultivation of cyanobacteria and also in many BPV studies, caused severe interferences in the bioelectrochemical experiments. An optimized nBG11 medium guaranteed a low and stable background current in the BPV reactor, regardless of the presence of light and/or mediators. As proof-of-principle, a very high long-term light-dependent current output (peak current of over 20 µA) was demonstrated in the new set-up over 12 days with living Synechocystis sp. PCC6803 cells and validated with appropriate controls. These results report the first reliable BPV platform generating reproducible photocurrent while still allowing quantitative investigation, rational optimization, and scale-up of BPV processes., (© 2021 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals LLC.)

Despite the high level of symmetry between the PsaA and PsaB polypeptides in Photosystem I, some amino acids pairs are strikingly different, such as PsaA-Gly693 and PsaB-Trp673, which are located near a cluster of 11 water molecules between the A 1A and A 1B quinones and the F X iron-sulfur cluster. In this work, we changed PsaB-Trp673 to PsaB-Phe673 in Synechocystis sp. PCC 6803. The variant contains ~ 85% of wild-type (WT) levels of Photosystem I but is unable to grow photoautotrophically. Both time-resolved and steady-state optical measurements show that in the PsaB-W673F variant less than 50% of the electrons reach the terminal iron-sulfur clusters F A and F B ; the majority of the electrons recombine from A 1A - and A 1B - . However, in those reaction centers which pass electrons forward the transfer is heterogeneous: a minor population shows electron transfer rates from A 1A - and A 1B - to F X slightly slower than that of the WT, whereas a major population shows forward electron transfer rates to F X slowed to the ~ 10 µs time range. Competition between relatively similar forward and backward rates of electron transfer from the quinones to the F X cluster account for the relatively low yield of long-lived charge separation in the PsaB-W673F variant. A higher water content and its increased mobility observed in MD simulations in the interquinone cavity of the PsaB-W673F variant shifts the pK of PsaB-Asp575 and allows its deprotonation in situ. The heterogeneity found may be rooted in protonation state of PsaB-Asp575, which controls whether electron transfer can proceed beyond the phylloquinone cofactors., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Increased tolerance to light stress in cyanobacteria is a desirable feature for their applications. Here, we obtained a high light tolerant (Tol) strain of Synechocystis sp. PCC6803 through an adaptive laboratory evolution, in which the cells were repeatedly sub-cultured for 52 days under high light stress conditions (7000 to 9000 μmol m -2 s -1 ). Although the growth of the parental strain almost stopped when exposed to 9000 μmol m -2 s -1 , no growth inhibition was observed in the Tol strain. Excitation-energy flow was affected because of photosystem II damage in the parental strain under high light conditions, whereas the damage was alleviated and normal energy flow was maintained in the Tol strain. The transcriptome data indicated an increase in isiA expression in the Tol strain under high light conditions. Whole genome sequence analysis and reverse engineering revealed two mutations in hik26 and slr1916 involved in high light stress tolerance in the Tol strain.

Cyanobacterial type I NADH dehydrogenase (NDH-1) is involved in various bioenergetic reactions including respiration, cyclic electron transport (CET), and CO 2 uptake. The role of NDH-1 is usually minor under normal growth conditions and becomes important under stress conditions. However, in our previous study, flux balance analysis (FBA) simulation predicted that the drive of NDH-1 as CET pathway with a photosystem (PS) I/PSII excitation ratio around 1.0 contributes to achieving an optimal specific growth rate. In this study, to experimentally elucidate the predicted functions of NDH-1, first, we measured the PSI/PSII excitation ratios of Synechocystis sp. PCC 6803 grown under four types of spectral light conditions. The specific growth rate was the highest and PSI/PSII excitation ratio was with 0.88 under the single-peak light at 630 nm (Red1). Considering this measured excitation ratios, FBA simulation predicted that NDH-1-dependent electron transport was the major pathway under Red1. Moreover, in actual culture, an NDH-1 deletion strain had slower growth rate than that of wild type only under Red1 light condition. Therefore, we experimentally demonstrated that NDH-1 plays an important role under optimal light conditions such as Red1 light, where Synechocystis exhibits the highest specific growth rate and PSI/PSII excitation ratio of around 1.0., (Copyright © 2020 Elsevier B.V. All rights reserved.)

We investigated variations in cell growth and ATP Sulfurylase (ATPS) activity when two cyanobacterial strains-Synechocystis sp. PCC6803 and Synechococcus sp. WH7803-were grown in conventional media, and media with low ammonium, low sulfate and a high CO 2 /low O 2 atmosphere. In both organisms, a transition and adaptation to the reconstructed environmental media resulted in a decrease in ATPS activity. This variation appears to be decoupled from growth rate, suggesting the enzyme is not rate-limiting in S assimilation and raising questions about the role of ATPS redox regulation in cell physiology and throughout Earth history.

Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942 exhibit dissimilar tolerance to Cr(VI) with a tenfold difference in their EC 50 value for Cr(VI). This contrasting tolerance was attributed to the difference in the ability to transport Cr(VI) and to detoxify ROS. The present study used biochemical assays and chlorophyll fluorescence to investigate the effect of growth with Cr(VI) on photosynthesis in the two cyanobacteria. In absence of Cr(VI), all the measured parameters viz ., rates of CO 2 fixation, PSII and PSI activities were higher in Synechocystis in comparison to Synechococcus , suggesting intrinsic differences in their photosynthesis. Growth in the presence of Cr(VI) reduced the pigment content and photosystems' activities in both cyanobacteria. It was further observed that photosynthetic functions were more adversely affected in Synechocystis in comparison to Synechococcus, in spite of exposure to tenfold lower Cr(VI) concentration. The effective quantumyield of PSII and PSI obtained by chlorophyll fluorescence measurements increased in the presence of Cr(VI) in Synechococcus whereas it decreased in Synechocystis . However, the overall CO 2 fixation remained unchanged. These results indicated that, in addition to the intrinsic difference in photosynthetic rates, the two cyanobacteria exhibit differential modulation of photosynthetic machinery upon Cr(VI) exposure and Synechococcus could adapt better it's photosystems to counter the oxidative stress.

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 PpsbA2. The resulting strain, termed PPT1 (ΔpirC-REphaAB), 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.

There arose one of the most important ecological transitions in Earth's history approximately 750 million years ago during the middle Neoproterozoic Era (1000 to 541 million years ago, Ma). Biomarker evidence suggests that around this time there was a rapid shift from a predominantly bacterial-dominated world to more complex ecosystems governed by eukaryotic primary productivity. The resulting 'Rise of the algae' led to dramatically altered food webs that were much more efficient in terms of nutrient and energy transfer. Yet, what triggered this ecological shift? In this study we examined the theory that it was the alleviation of phosphorus (P) deficiency that gave eukaryotic alga the prime opportunity to flourish. We performed laboratory experiments on the cyanobacterium Synechocystis salina and the eukaryotic algae Tetraselmis suecica and examined their ability to compete for phosphorus. Both these organisms co-occur in modern European coastal waters and are not known to have any allelopathic capabilities. The strains were cultured in mono and mixed cultures in chemostats across a range of dissolved inorganic phosphorus (DIP) concentrations to reflect modern and ancient oceanic conditions of 2 μM P and 0.2 μM P, respectively. Our results show that the cyanobacteria outcompete the algae at the low input (0.2 μM P) treatment, yet the eukaryotic algae were not completely excluded and remained a constant background component in the mixed-culture experiments. Also, despite their relatively large cell size, the algae T. suecica had a high affinity for DIP. With DIP input concentrations resembling modern-day levels (2 μM), the eukaryotic algae could effectively compete against the cyanobacteria in terms of total biomass production. These results suggest that the availability of phosphorus could have influenced the global expansion of eukaryotic algae. However, P limitation does not seem to explain the complete absence of eukaryotic algae in the biomarker record before ca. 750 Ma., Competing Interests: The authors have declared that no competing interests exist.

Motivation: Determining intracellular metabolic flux through isotope labeling techniques such as 13C metabolic flux analysis (13C-MFA) incurs significant cost and effort. Previous studies have shown transcriptomic data coupled with constraint-based metabolic modeling can determine intracellular fluxes that correlate highly with 13C-MFA measured fluxes and can achieve higher accuracy than constraint-based metabolic modeling alone. These studies, however, used validation data limited to E. coli and S. cerevisiae grown on glucose, with significantly similar flux distribution for central metabolism. It is unclear whether those results apply to more diverse metabolisms, and therefore further, extensive validation is needed., Results: In this paper, we formed a dataset of transcriptomic data coupled with corresponding 13C-MFA flux data for 21 experimental conditions in different unicellular organisms grown on varying carbon substrates and conditions. Three computational flux-balance analysis (FBA) methods were comparatively assessed. The results show when uptake rates of carbon sources and key metabolites are known, transcriptomic data provides no significant advantage over constraint-based metabolic modeling (average correlation coefficients, transcriptomic E-Flux2 0.725 and SPOT 0.650 vs non-transcriptomic pFBA 0.768). When uptake rates are unknown, however, predictions obtained utilizing transcriptomic data are generally good and significantly better than those obtained using constraint-based metabolic modeling alone (E-Flux2 0.385 and SPOT 0.583 vs pFBA 0.237). Thus, transcriptomic data coupled with constraint-based metabolic modeling is a promising method to obtain intracellular flux estimates in microorganisms, particularly in cases where uptake rates of key metabolites cannot be easily determined, such as for growth in complex media or in vivo conditions., Competing Interests: The authors have declared that no competing interests exist.

Microbes are essential to the global ecosystem, but undesirable microbial growth causes issues ranging from food spoilage and infectious diseases to harmful cyanobacterial blooms. The use of chemicals to control microbial growth has achieved significant success, while specific roles for a majority of essential genes in growth control remain unexplored. Here, we show the growth inhibition of cyanobacterial species by targeting an essential enzyme for the biosynthesis of branched-chain amino acids. Specifically, we report the biochemical, genetic, and structural characterization of dihydroxyacid dehydratase from the model cyanobacterium Synechocystis sp. PCC 6803 (SnDHAD). Our studies suggest that SnDHAD is an oxygen-stable enzyme containing a [2Fe-2S] cluster. Furthermore, we demonstrate that SnDHAD is selectively inhibited in vitro and in vivo by the natural product aspterric acid, which also inhibits the growth of representative bloom-forming Microcystis and Anabaena strains but has minimal effects on microbial pathogens with [4Fe-4S] containing DHADs. This study suggests DHADs as a promising target for the precise growth control of microbes and highlights the exploration of other untargeted essential genes for microbial management.

The photosynthetic apparatus and metabolic enzymes of cyanobacteria are subject to various controls, such as transcriptional regulation and post-translational modifications, to ensure that the entire cellular system functions optimally. In particular, phosphorylation plays key roles in many cellular controls such as enzyme activity, signal transduction, and photosynthetic apparatus restructuring. Therefore, elucidating the governing functions of phosphorylation is crucial to understanding the regulatory mechanisms underlying metabolism and photosynthesis. In this study, we determined protein content and phosphorylation levels to reveal the regulation of intracellular metabolism and photosynthesis in Synechocystis sp. PCC 6803; for this, we obtained quantitative data of proteins and their phosphorylated forms involved in photosynthesis and metabolism under various growth conditions (photoautotrophic, mixotrophic, heterotrophic, dark, and nitrogen-deprived conditions) using targeted proteomic and phosphoproteomic analyses with nano-liquid chromatography-triple quadrupole mass spectrometry. The results indicated that in addition to the regulation of protein expression, the regulation of phosphorylation levels of cyanobacterial photosynthetic apparatus and metabolic enzymes was pivotal for adapting to changing environmental conditions. Furthermore, reduced protein levels of CpcC and altered phosphorylation levels of CpcB, ApcA, OCP, and PsbV contributed to the cellular response of the photosynthesis apparatus to nitrogen deficiency.

Cyanobacteria can form biofilms in nature, which have ecological roles and high potential for practical applications. In order to study them we need biofilm models that contain healthy cells and can withstand physical manipulations needed for structural studies. At present, combined studies on the structural and physiological features of axenic cyanobacterial biofilms are limited, mostly due to the shortage of suitable model systems. Here, we present a simple method to establish biofilms using the cyanobacterium Synechocystis PCC6803 under standard laboratory conditions to be directly used for photosynthetic activity measurements and scanning electron microscopy (SEM). We found that glass microfiber filters (GMF) with somewhat coarse surface features provided a suitable skeleton to form Synechocystis PCC6803 biofilms. Being very fragile, untreated GMFs were unable to withstand the processing steps needed for SEM. Therefore, we used polyhydroxybutyrate coating to stabilize the filters. We found that up to five coats resulted in GMF stabilization and made possible to obtain high resolution SEM images of the structure of the surface-attached cells and the extensive exopolysaccharide and pili network, which are essential features of biofilm formation. By using pulse-amplitude modulated variable chlorophyll fluorescence imaging, it was also demonstrated that the biofilms contain photosynthetically active cells. Therefore, the Synechocystis PCC6803 biofilms formed on coated GMFs can be used for both structural and functional investigations. The model presented here is easy to replicate and has a potential for high-throughput studies., Competing Interests: The authors have declared that no competing interests exist.

Synechocystis sp. PCC 6803, a cyanobacterium widely used for basic research, is often cultivated in a synthetic medium, BG-11, in the presence of 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES) or 2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid buffer. Owing to the high cost of HEPES buffer (96.9% of the total cost of BG-11 medium), the biotechnological application of BG-11 is limited. In this study, we cultured Synechocystis sp. PCC 6803 cells in BG-11 medium without HEPES buffer and examined the effects on the primary metabolism. Synechocystis sp. PCC 6803 cells could grow in BG-11 medium without HEPES buffer after adjusting for nitrogen sources and light intensity; the production rate reached 0.54 g cell dry weight·L -1 ·day -1 , exceeding that of commercial cyanobacteria and Synechocystis sp. PCC 6803 cells cultivated under other conditions. The exclusion of HEPES buffer markedly altered the metabolites in the central carbon metabolism; particularly, the levels of compatible solutes, such as sucrose, glucosylglycerol, and glutamate were increased. Although the accumulation of sucrose and glucosylglycerol under high salt conditions is antagonistic to each other, these metabolites accumulated simultaneously in cells grown in the cost-effective medium. Because these metabolites are used in industrial feedstocks, our results reveal the importance of medium composition for the production of metabolites using cyanobacteria., (© 2020 Wiley Periodicals, Inc.)

Protein lysine methylation is a prevalent post-translational modification (PTM) and plays critical roles in all domains of life. However, its extent and function in photosynthetic organisms are still largely unknown. Cyanobacteria are a large group of prokaryotes that carry out oxygenic photosynthesis and are applied extensively in studies of photosynthetic mechanisms and environmental adaptation. Here we integrated propionylation of monomethylated proteins, enrichment of the modified peptides, and mass spectrometry (MS) analysis to identify monomethylated proteins in Synechocystis sp. PCC 6803 (Synechocystis). Overall, we identified 376 monomethylation sites in 270 proteins, with numerous monomethylated proteins participating in photosynthesis and carbon metabolism. We subsequently demonstrated that CpcM, a previously identified asparagine methyltransferase in Synechocystis, could catalyze lysine monomethylation of the potential aspartate aminotransferase Sll0480 both in vivo and in vitro and regulate the enzyme activity of Sll0480. The loss of CpcM led to decreases in the maximum quantum yield in primary photosystem II (PSII) and the efficiency of energy transfer during the photosynthetic reaction in Synechocystis. We report the first lysine monomethylome in a photosynthetic organism and present a critical database for functional analyses of monomethylation in cyanobacteria. The large number of monomethylated proteins and the identification of CpcM as the lysine methyltransferase in cyanobacteria suggest that reversible methylation may influence the metabolic process and photosynthesis in both cyanobacteria and plants., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

In this study, FeSO 4 supplementation ranging from 0 to 4.5 mM, and MgSO 4 supplementation ranging from 0 to 5.1 mM were investigated to observe the effect on the population dynamics, biochemical composition and fatty acid content of mixed microalgae grown in Anaerobic Liquid Digestate (ALD). Overall, 3.1 mM FeSO 4 addition into ALD increased the total protein content 60% and led to highest biomass (1.56 g L -1 ) and chlorophyll-a amount (18.7 mg L -1 ) produced. Meanwhile, 0.4 mM MgSO 4 addition increased the total carotenoid amount 2.2 folds and slightly increased the biomass amount. According to the microbial community analysis, Diphylleia rotans, Synechocystis PCC-6803 and Chlorella sorokiniana were identified as mostly detected species after confirmation with 4 different markers. The abundance of Chlorella sorokiniana and Synechocystis PCC-6803 increased almost 2 folds both in iron and magnesium addition. On the other hand, the dominancy of Diphylleia rotans was not affected by iron addition while drastically decreased (95%) with magnesium addition. This study helps to understand how the dynamics of symbiotic life changes if macro elements are added to the ALD and reveal that microalgae can adapt to adverse environmental conditions by fostering the diversity with a positive effect on high value product.

Cyanobacteria form a heterogeneous bacterial group with diverse lifestyles, acclimation strategies, and differences in the presence of circadian clock proteins. In Synechococcus elongatus PCC 7942, a unique posttranslational KaiABC oscillator drives circadian rhythms. ATPase activity of KaiC correlates with the period of the clock and mediates temperature compensation. Synechocystis sp. strain PCC 6803 expresses additional Kai proteins, of which KaiB3 and KaiC3 proteins were suggested to fine-tune the standard KaiAB1C1 oscillator. In the present study, we therefore characterized the enzymatic activity of KaiC3 as a representative of nonstandard KaiC homologs in vitro KaiC3 displayed ATPase activity lower than that of the Synechococcus elongatus PCC 7942 KaiC protein. ATP hydrolysis was temperature dependent. Hence, KaiC3 is missing a defining feature of the model cyanobacterial circadian oscillator. Yeast two-hybrid analysis showed that KaiC3 interacts with KaiB3, KaiC1, and KaiB1. Further, KaiB3 and KaiB1 reduced in vitro ATP hydrolysis by KaiC3. Spot assays showed that chemoheterotrophic growth in constant darkness is completely abolished after deletion of Δ kaiAB1C1 and reduced in the absence of kaiC3 We therefore suggest a role for adaptation to darkness for KaiC3 as well as a cross talk between the KaiC1- and KaiC3-based systems. IMPORTANCE The circadian clock influences the cyanobacterial metabolism, and deeper understanding of its regulation will be important for metabolic optimizations in the context of industrial applications. Due to the heterogeneity of cyanobacteria, characterization of clock systems in organisms apart from the circadian model Synechococcus elongatus PCC 7942 is required. Synechocystis sp. strain PCC 6803 represents a major cyanobacterial model organism and harbors phylogenetically diverged homologs of the clock proteins, which are present in various other noncyanobacterial prokaryotes. By our in vitro studies we unravel the interplay of the multiple Synechocystis Kai proteins and characterize enzymatic activities of the nonstandard clock homolog KaiC3. We show that the deletion of kaiC3 affects growth in constant darkness, suggesting its involvement in the regulation of nonphotosynthetic metabolic pathways., (Copyright © 2020 Wiegard et al.)

Bio-based production technologies may complement or replace petroleum-based production of chemicals, but they face a number of technical challenges, including product toxicity and/or water insolubility. Plants and microorganisms naturally biosynthesize chemicals that often are converted into derivatives with reduced toxicity or enhanced solubility. Inspired by this principle, we propose a bioderivatization strategy for biotechnological chemicals production, defined as purposeful biochemical derivatization of intended target molecules. As proof of principle, the effects of hydrophobic (e.g., esterification) and hydrophilic (e.g., glycosylation) bioderivatization strategies on the biosynthesis of a relatively toxic and poorly soluble chemical, 1-octanol, were evaluated in Escherichia coli and Synechocystis sp. PCC 6803. The 1-octanol pathway was first optimized to reach product titers at which the host displayed symptoms of toxicity. Solvent overlay used to capture volatile products partially masked product toxicity. Regardless of whether solvent overlay was used, most strains with bioderivatization had a higher molar product titer and product yield, as well as improved cellular growth and glucose consumption, compared with strains without bioderivatization. The positive effect on bioproduction was observed with both the hydrophobic and hydrophilic strategies. Interestingly, in several combinations of genotype/induction strength, bioderivatization had a positive effect on productivity without any apparent effect on growth. We attribute this to enhanced product solubility in the aqueous or solvent fraction of the bioreactor liquid phase (depending on the derivative and medium used), with consequent enhanced product removal. Overall, under most conditions, a benefit of bioproduction was observed, and the bioderivatization strategy could be considered for other similar chemicals as well., Competing Interests: Competing interest statement: The authors are inventors on the UK patent application entitled “'Bio-based production of toxic chemicals.” The subject is a method of producing a derivative of 1-octanol, as well as microorganisms and expression vectors for use in said method., (Copyright © 2020 the Author(s). Published by PNAS.)

Cyanobacteria acclimate and adapt to changing light conditions by controlling the energy transfer between photosystem I (PSI) and II (PSII) and pigment composition. Photosynthesis is driven by balancing the excitation between PSI and PSII. To predict the detailed electron transfer flux of cyanobacteria, we refined the photosynthesis-related reactions in our previously reconstructed genome-scale model. Two photosynthetic bacteria, Arthrospira and Synechocystis, were used as models. They were grown under various spectral light conditions and flux balance analysis (FBA) was performed using photon uptake fluxes into PSI and PSII, which were converted from each light spectrum by considering the photoacclimation of pigments and the distribution ratio of phycobilisome to PSI and PSII. In Arthrospira, the FBA was verified with experimental data using six types of light-emitting diodes (White, Blue, Green, Yellow, Red1, and Red2). FBA predicted the cell growth of Synechocystis for the LEDs, excepting Red2. In an FBA simulation, cells used respiratory terminal oxidases and two NADH dehydrogenases (NDH-1 and NDH-2) to balance the PSI and PSII excitations depending on the light conditions. FBA simulation with our refined model functionally implicated NDH-1 and NDH-2 as a component of cyclic electron transport in the varied light environments.

In light of the climate change challenge, the advantageous trait of using solar energy and carbon dioxide to produce organic molecules has granted cyanobacteria deserved interest as hosts for metabolic engineering. Importantly, these organisms do not directly compete with agricultural resources. Aromatic amino acids and derived phenylpropanoids are of high importance because they are used by the pharmaceutical, food, cosmetic, and agricultural industries as precursors of active ingredients. Amino acids are traditionally produced by extraction from protein hydrolysates, chemical synthesis or fermentation pathways using heterotrophic microorganisms. In this work we demonstrate for the first time the efficient overproduction of phenylalanine and tyrosine from CO 2 in a Synechocystis sp. PCC 6803 strain heterologously expressing the feedback-inhibition-resistant AroG and TyrA enzymes from E. coli. Production titers reached 904 ± 53 mg/gDW (580 ± 34 mg/L) of phenylalanine and 64 ± 3.7 mg/gDW (41 ± 2.3 mg/L) of tyrosine after 10 days of photoautotrophic growth. We estimate that the production of the two amino acids corresponds to 56% of the total fixed carbon. Phenylalanine and tyrosine are the precursors for phenylpropanoids, thus, we tested the functionality of several phenylpropanoid biosynthetic enzymes in the generated cyanobacterium strains and successfully achieved the production of 470 ± 70 mg/gDW (207 mg/L) of p-coumaric acid, 267 ± 31 mg/gDW (114 mg/L) of cinnamic acid and 47.4 ± 13.9 mg/gDW (12.6 mg/L) of caffeic acid after 6 days of photoautotrophic growth. All compounds were secreted to the growth medium. Our work enlarges the repertoire and yield of heterologous chemicals produced by Synechocystis and contributes to extend the molecular knowledge about this cyanobacterium., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

To meet the increasing global demand of biodiesel over the next decades, alternative methods for producing one of the key constituents of biodiesel (e.g. fatty acid methyl esters (FAMEs)) are needed. Algal biodiesel has been a long-term target compromised by excessive costs for harvesting and processing. In this work, we engineered cyanobacteria to convert carbon dioxide into excreted FAME, without requiring methanol as a methyl donor. To produce FAME, acyl-ACP, a product of the fatty acid biosynthesis pathway, was first converted into free fatty acid (FFA) by a thioesterase, namely 'UcFatB1 from Umbellularia californica. Next, by employing a juvenile hormone acid O-methyltransferase (DmJHAMT) from Drosophila melanogaster and S-adenosylmethionine (SAM) as a methyl donor, FFAs were converted into corresponding FAMEs. The esters were naturally secreted extracellularly, allowing simple product separation by solvent overlay as opposed to conventional algae biodiesel production where the algae biomass must first be harvested and processed for transesterification of extracted triacylglycerols (TAGs). By optimizing both the promoter and RBS elements, up to 120 mg/L of FAMEs were produced in 10 days. Quantification of key proteins and metabolites, together with constructs over-expressing SAM synthetase (MetK), indicated that 'UcFatB1, MetK, and DmJHAMT were the main factors limiting pathway flux. In order to solve the latter limitation, two reconstructed ancestral sequences of DmJHAMT were also tried, resulting in strains showing a broader methyl ester chain-length profile in comparison to the native DmJHAMT. Altogether, this work demonstrates a promising pathway for direct sunlight-driven conversion of CO 2 into excreted FAME., (Copyright © 2019 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Synechocystis sp. has remarkable mixotrophic capabilities, as well as an efficient exploitation of nitrogen and phosphorus, that may be applied in wastewater treatment based on cyanobacteria. To better investigate the exploitation of algal mixotrophy in bioremediation, this species was used in axenic respirometric tests to ascertain the effect of high light and non limiting CO 2 supply on the overall regulation of mixotrophy, resulting in an inhibition of the exploitation of organic carbon. The same species was then cultured in real, unsterilized effluent obtained from the acidogenic fermentation of sludge, which contains a high concentration of nutrients (approximately 600, 90 and 6000 mg L -1 of N, P and COD, respectively) and it is often inhibiting for many microalgal species. On the contrary, Synechocystis sp., showed a remarkable growth and a removal up to 96% of phosphorus, 66% of nitrogen and of 68% of COD in such a complex waste stream.

Motile strains of the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 readily aggregate into flocs, or floating multicellular assemblages, when grown in liquid culture. As described here, we used confocal imaging to probe the structure of these flocs, and we developed a quantitative assay for floc formation based on fluorescence imaging of 6-well plates. The flocs are formed from strands of linked cells, sometimes packed into dense clusters but also containing voids with very few cells. Cells within the dense clusters show signs of nutrient stress, as judged by the subcellular distribution of green fluorescent protein (GFP)-tagged Vipp1 protein. We analyzed the effects on flocculation of a series of mutations that alter piliation and motility, including Δ hfq , Δ pilB1 , Δ pilT1 , and Δ ushA mutations and deletion mutations affecting major and minor pilins. The extent of flocculation is increased in the hyperpiliated Δ pilT1 mutant, but active cycles of pilus extension and retraction are not required for flocculation. Deletion of PilA1, the major subunit of type IV pili, has no effect on flocculation; however, flocculation is lost in mutants lacking an operon coding for the minor pilins PilA9 to -11. Therefore, minor pilins appear crucial for flocculation. We show that flocculation is a tightly regulated process that is promoted by blue light perception by the cyanobacteriochrome Cph2. Floc formation also seems to be a highly cooperative process. A proportion of nonflocculating Δ hfq cells can be incorporated into wild-type flocs, but the presence of a high proportion of Δ hfq cells disrupts the large-scale architecture of the floc. IMPORTANCE Some bacteria form flocs, which are multicellular floating assemblages of many thousands of cells. Flocs have been relatively little studied compared to surface-adherent biofilms, but flocculation could play many physiological roles, be a crucial factor in marine carbon burial, and enable more efficient biotechnological cell harvesting. We studied floc formation and architecture in the model cyanobacterium Synechocystis sp. strain PCC 6803, using mutants to identify specific cell surface structures required for floc formation. We show that floc formation is regulated by blue and green light perceived by the photoreceptor Cph2. The flocs have a characteristic structure based on strands of linked cells aggregating into dense clusters. Cells within the dense clusters show signs of nutrient stress, pointing to a disadvantage of floc formation., (Copyright © 2019 Conradi et al.)

The biogenesis of the cyanobacterial photosystem II (PSII) complex requires a number of auxiliary assembly factors that improve efficiency of the process but their precise function is not well understood. To assess a possible synergic action of the Ycf48 and Ycf39 factors acting in early steps of the biogenesis via interaction with the nascent D1 subunit of PSII, we constructed and characterised a double mutant of the cyanobacterium Synechocystis PCC 6803 lacking both these proteins. In addition, we also deleted the ycf39 gene in the double mutant lacking Ycf48 and Pam68, the latter being a ribosomal factor promoting insertion of chlorophyll (Chl) into the CP47 subunit of PSII. The resulting double ΔYcf48/ΔYcf39 and triple ΔYcf48/ΔPam68/ΔYcf39 mutants were deficient in PSII and total Chl, and in contrast to the source mutants, they lost the capacity for autotrophy. Interestingly, autotrophic growth was restored in both of the new multiple mutants by enhancing Chl biosynthesis using a specific ferrochelatase inhibitor. Taking together with the weak radioactive labelling of the D1 protein, these findings can be explained by inhibition of the D1 synthesis caused by the lack and/or incorrect binding of Chl molecules. The results emphasise the key importance of the sufficient Chl supply for the PSII biogenesis and also support the existence of a so far enigmatic regulatory mechanism leading to the reduced overall Chl biosynthesis/accumulation when the PSII assembly is impaired.

Oxygenic photosynthesis relies on accessory factors to promote the assembly and maintenance of the photosynthetic apparatus in the thylakoid membranes. The highly conserved membrane-bound rubredoxin-like protein RubA has previously been implicated in the accumulation of both PSI and PSII, but its mode of action remains unclear. Here, we show that RubA in the cyanobacterium Synechocystis sp PCC 6803 is required for photoautotrophic growth in fluctuating light and acts early in PSII biogenesis by promoting the formation of the heterodimeric D1/D2 reaction center complex, the site of primary photochemistry. We find that RubA, like the accessory factor Ycf48, is a component of the initial D1 assembly module as well as larger PSII assembly intermediates and that the redox-responsive rubredoxin-like domain is located on the cytoplasmic surface of PSII complexes. Fusion of RubA to Ycf48 still permits normal PSII assembly, suggesting a spatiotemporal proximity of both proteins during their action. RubA is also important for the accumulation of PSI, but this is an indirect effect stemming from the downregulation of light-dependent chlorophyll biosynthesis induced by PSII deficiency. Overall, our data support the involvement of RubA in the redox control of PSII biogenesis., (© 2019 American Society of Plant Biologists. All rights reserved.)

Cyanobacteria are a model photoautotroph and a chassis for the sustainable production of fuels and chemicals. Knowledge of photoautotrophic metabolism in the natural environment of day/night cycles is lacking, yet has implications for improved yield from plants, algae and cyanobacteria. Here, a thorough approach to characterizing diverse metabolites-including carbohydrates, lipids, amino acids, pigments, cofactors, nucleic acids and polysaccharides-in the model cyanobacterium Synechocystis sp. PCC 6803 (S. 6803) under sinusoidal diurnal light:dark cycles was developed and applied. A custom photobioreactor and multi-platform mass spectrometry workflow enabled metabolite profiling every 30-120 min across a 24-h diurnal sinusoidal LD ('sinLD') cycle peaking at 1600 μmol photons m -2 sec -1 . We report widespread oscillations across the sinLD cycle with 90%, 94% and 40% of the identified polar/semi-polar, non-polar and polymeric metabolites displaying statistically significant oscillations, respectively. Microbial growth displayed distinct lag, biomass accumulation and cell division phases of growth. During the lag phase, amino acids and nucleic acids accumulated to high levels per cell followed by decreased levels during the biomass accumulation phase, presumably due to protein and DNA synthesis. Insoluble carbohydrates displayed sharp oscillations per cell at the day-to-night transition. Potential bottlenecks in central carbon metabolism are highlighted. Together, this report provides a comprehensive view of photosynthetic metabolite behavior with high temporal resolution, offering insight into the impact of growth synchronization to light cycles via circadian rhythms. Incorporation into computational modeling and metabolic engineering efforts promises to improve industrially relevant strain design., (© 2019 The Authors The Plant Journal © 2019 John Wiley & Sons Ltd.)

During the culturing of cyanobacteria, heterotrophic bacteria can compete for nutrients, compromise the quality of the harvested biomass, or cause culture crashes. We systematically investigated the effects of depleting inorganic phosphate (Pi) on the growth of the cyanobacterium Synechocystis sp. PCC 6803, its community of heterotrophic bacteria, and the biomass's chemical composition. On the one hand, depleting Pi had minimal impact on total biomass, extracellular polymeric substances (ESP), soluble microbial products (SMP), and most types of intracellular organic polymers production. On the other hand, depleting Pi led to markedly less lipid content, less heterotrophic biomass, and a shift in the heterotrophic community from Burkholderiales to Sphingobacteriales and Saprospirales. The causes of the large impacts were that Synechocystis was much better at scavenging a very low Pi concentration and lowering the Pi available to the heterotrophs. This work lays a foundation for controlling the accumulation of heterotrophs and reducing their deleterious effects in cyanobacteria culturing.

NdbA, one of the three type 2 NAD(P)H dehydrogenases (NDH-2) in Synechocystis sp. PCC 6803 (hereafter Synechocystis) was here localized to the thylakoid membrane (TM), unique for the three NDH-2s, and investigated with respect to photosynthetic and cellular redox metabolism. For this purpose, a deletion mutant (ΔndbA) and a complementation strain overexpressing NdbA (ΔndbA::ndbA) were constructed. It is demonstrated that NdbA is expressed at very low level in the wild-type (WT) Synechocystis under photoautotrophic (PA) growth whilst substantially higher expression occurs under light-activated heterotrophic growth (LAHG). The absence of NdbA resulted in non-optimal growth of Synechocystis under LAHG and concomitantly enhanced the expression of photoprotection-related flavodiiron proteins and carbon acquisition-related proteins as well as various transporters, but downregulated a few iron homeostasis-related proteins. NdbA overexpression, on the other hand, promoted photosynthetic pigmentation and functionality of photosystem I under LAHG conditions while distinct photoprotective and carbon concentrating proteins were downregulated. NdbA overexpression also exerted an effect on the expression of many signaling and gene regulation proteins. It is concluded that the amount and function of NdbA in the TM has a capacity to modulate the redox signaling of gene expression, but apparently has a major physiological role in maintaining iron homeostasis under LAHG conditions. LC-MS/MS data are available via ProteomeXchange with identifier PXD011671., (� The Author(s) 2019. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.)

Pyridine nucleotides (NAD(P)(H)) are electron carriers that are the driving forces in various metabolic pathways. Phosphorylation of NAD(H) to NADP(H) is performed by the enzyme NAD kinase (NADK). Synechocystis sp. PCC 6803 harbors two genes (sll1415 and slr0400) that encode proteins with NADK homology. When genetic mutants for sll1415 and slr0400 (Δ1415 and Δ0400, respectively) were cultured under photoheterotrophic growth conditions only the Δ1415 cells showed a growth defect. In wild-type cells, the sll1415 transcript accumulated after the cells were transferred to photoheterotrophic conditions. Furthermore, NAD(P)(H) measurements demonstrated that a dynamic metabolic conversion was implemented during the adaptation from photoautotrophic to photoheterotrophic conditions. Electron microscopy observation and biochemistry quantification demonstrated the accumulation of glycogen in the Δ1415 cells under photoheterotrophic conditions at 96 h. Quantitative real-time reverse transcription PCR (qRT-PCR) demonstrated the accumulation of mRNAs that encoded glycogen biosynthesis-related enzymes in photoheterotrophic Δ1415 cells. At 96 h, enzyme activity measurement in the photoheterotrophic Δ1415 cells demonstrated that the activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were decreased, but the activities of glucose dehydrogenase were increased. Furthermore, metabolomics analysis demonstrated that the Δ1415 cells showed increased glucose-6-phosphate and 6-phosphogluconate content at 96 h. Therefore, sll1415 has a significant function in the oxidative pentose phosphate (OPP) pathway for catabolism of glucose under photoheterotrophic conditions. Additionally, it is presumed that the slr0400 had a different role in glucose catabolism during growth. These results suggest that the two Synechocystis sp. PCC 6803 NADKs (Sll1415 and Slr0400) have distinct functions in photoheterotrophic cyanobacterial metabolism., (© 2019 The Authors The Plant Journal © 2019 John Wiley & Sons Ltd.)

Carbonic anhydrases (CAs) are involved in CO 2 uptake and conversion, a fundamental process in photosynthetic organisms. Nevertheless, the mechanism underlying the regulation of CO 2 uptake and intracellular conversion in cyanobacteria is largely unknown. We report the characterization of a previously unrecognized thylakoid-located CA Slr0051 (EcaB) from the cyanobacterium Synechocystis sp. PCC 6803, which possesses CA activity to regulate CO 2 uptake. Inactivation of ecaB stimulated CO 2 hydration in the thylakoids, suppressed by the classical CA inhibitor acetazolamide. Absence of ecaB increased the reduced state of the photosynthetic electron transport system, lowered the rate of photosynthetic O 2 evolution at high light (HL) and pH, and decreased the cellular affinity for extracellular inorganic carbon. Furthermore, EcaB was upregulated in cells grown at limiting CO 2 concentration or HL in tandem with CupA. EcaB is mainly located in the thylakoid membranes where it interacts with CupA and CupB involved in CO 2 uptake by converting it to bicarbonate. We propose that modulation of the EcaB level and activity in response to CO 2 changes, illumination or pH reversibly regulates its conversion to HCO 3 by the two CO 2 -uptake systems (CupA, CupB), dissipating the excess HCO 3 - and alleviating photoinhibition, and thereby optimizes photosynthesis, especially under HL and alkaline conditions., (© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.)

Phototrophic biofilms are key to nutrient cycling in natural environments and bioremediation technologies, but few studies describe biofilm formation by pure (axenic) cultures of a phototrophic microbe. The cyanobacterium Synechocystis sp. strain PCC 6803 (here Synechocystis ) is a model microorganism for the study of oxygenic photosynthesis and biofuel production. We report here that wild-type (WT) Synechocystis caused extensive biofilm formation in a 2,000-liter outdoor nonaxenic photobioreactor under conditions attributed to nutrient limitation. We developed a biofilm assay and found that axenic Synechocystis forms biofilms of cells and extracellular material but only when cells are induced by an environmental signal, such as a reduction in the concentration of growth medium BG11. Mutants lacking cell surface structures, namely type IV pili and the S-layer, do not form biofilms. To further characterize the molecular mechanisms of cell-cell binding by Synechocystis , we also developed a rapid (8-h) axenic aggregation assay. Mutants lacking type IV pili were unable to aggregate, but mutants lacking a homolog to Wza, a protein required for type 1 exopolysaccharide export in Escherichia coli , had a superbinding phenotype. In WT cultures, 1.2× BG11 medium induced aggregation to the same degree as 0.8× BG11 medium. Overall, our data support that Wza-dependent exopolysaccharide is essential to maintain stable, uniform suspensions of WT Synechocystis cells in unmodified growth medium and that this mechanism is counteracted in a pilus-dependent manner under altered BG11 concentrations. IMPORTANCE Microbes can exist as suspensions of individual cells in liquids and also commonly form multicellular communities attached to surfaces. Surface-attached communities, called biofilms, can confer antibiotic resistance to pathogenic bacteria during infections and establish food webs for global nutrient cycling in the environment. Phototrophic biofilm formation is one of the earliest phenotypes visible in the fossil record, dating back over 3 billion years. Despite the importance and ubiquity of phototrophic biofilms, most of what we know about the molecular mechanisms, genetic regulation, and environmental signals of biofilm formation comes from studies of heterotrophic bacteria. We aim to help bridge this knowledge gap by developing new assays for Synechocystis , a phototrophic cyanobacterium used to study oxygenic photosynthesis and biofuel production. With the aid of these new assays, we contribute to the development of Synechocystis as a model organism for the study of axenic phototrophic biofilm formation., (Copyright © 2019 American Society for Microbiology.)