Interplay between photosynthetic electron flux and organic carbon sinks in sucrose-excreting Synechocystis sp. PCC 6803 revealed by omics approaches.

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 CO2 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 CO2 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-11 and NDH-2 and the terminal respiratory oxidases Cyd and Cox, which transfer electrons to O2. 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 CO2 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.

Accounting for photosystem I photoinhibition sheds new light on seasonal acclimation strategies of boreal conifers.

The photosynthetic acclimation of boreal evergreen conifers is controlled by regulatory and photoprotective mechanisms that allow conifers to cope with extreme environmental changes. However, the underlying dynamics of photosystem II (PSII) and photosystem I (PSI) remain unresolved. Here, we investigated the dynamics of PSII and PSI during the spring recovery of photosynthesis in Pinus sylvestris and Picea abies using a combination of chlorophyll-a fluorescence, P700 difference absorbance measurements, and quantification of key thylakoid protein abundances. In particular, we derived a new set of PSI quantum yield equations, correcting for the effects of PSI photoinhibition. Using the corrected equations, we found that the seasonal dynamics of PSII and PSI photochemical yields remained largely in balance, despite substantial seasonal changes in the stoichiometry of PSII and PSI core complexes driven by PSI photoinhibition. Similarly, the previously reported seasonal upregulation of cyclic electron flow was no longer evident, after accounting for PSI photoinhibition. Overall, our results emphasize the importance of considering the dynamics of PSII and PSI to elucidate the seasonal acclimation of photosynthesis in overwintering evergreens. Beyond the scope of conifers, our corrected PSI quantum yields expand the toolkit for future studies aimed at elucidating the dynamic regulation of PSI.

PSI Photoinhibition and Changing CO2 Levels Initiate Retrograde Signals to Modify Nuclear Gene Expression.

Photosystem I (PSI) is a critical component of the photosynthetic machinery in plants. Under conditions of environmental stress, PSI becomes photoinhibited, leading to a redox imbalance in the chloroplast. PSI photoinhibition is caused by an increase in electron pressure within PSI, which damages the iron-sulfur clusters. In this study, we investigated the susceptibility of PSI to photoinhibition in plants at different concentrations of CO2, followed by global gene expression analyses of the differentially treated plants. PSI photoinhibition was induced using a specific illumination protocol that inhibited PSI with minimal effects on PSII. Unexpectedly, the varying CO2 levels combined with the PSI-PI treatment neither increased nor decreased the likelihood of PSI photodamage. All PSI photoinhibition treatments, independent of CO2 levels, upregulated genes generally involved in plant responses to excess iron and downregulated genes involved in iron deficiency. PSI photoinhibition also induced genes encoding photosynthetic proteins that act as electron acceptors from PSI. We propose that PSI photoinhibition causes a release of iron from damaged iron-sulfur clusters, which initiates a retrograde signal from the chloroplast to the nucleus to modify gene expression. In addition, the deprivation of CO2 from the air initiated a signal that induced flavonoid biosynthesis genes, probably via jasmonate production.

Photosynthetic and transcriptome responses to fluctuating light in Arabidopsis thylakoid ion transport triple mutant.

Fluctuating light intensity challenges fluent photosynthetic electron transport in plants, inducing photoprotection while diminishing carbon assimilation and growth, and also influencing photosynthetic signaling for regulation of gene expression. Here, we employed in vivo chlorophyll-a fluorescence and P700 difference absorption measurements to demonstrate the enhancement of photoprotective energy dissipation of both photosystems in wild-type Arabidopsis thaliana after 6 h exposure to fluctuating light as compared with constant light conditions. This acclimation response to fluctuating light was hampered in a triple mutant lacking the thylakoid ion transport proteins KEA3, VCCN1, and CLCe, leading to photoinhibition of photosystem I. Transcriptome analysis revealed upregulation of genes involved in biotic stress and defense responses in both genotypes after exposure to fluctuating as compared with constant light, yet these responses were demonstrated to be largely upregulated in triple mutant already under constant light conditions compared with wild type. The current study illustrates the rapid acclimation of plants to fluctuating light, including photosynthetic, transcriptomic, and metabolic adjustments, and highlights the connection among thylakoid ion transport, photosynthetic energy balance, and cell signaling.

Enhanced function of non-photoinhibited photosystem II complexes upon PSII photoinhibition.

Light induced photosystem (PS)II photoinhibition inactivates and irreversibly damages the reaction center protein(s) but the light harvesting complexes continue the collection of light energy. Here we addressed the consequences of such a situation on thylakoid light harvesting and electron transfer reactions. For this purpose, Arabidopsis thaliana leaves were subjected to investigation of the function and regulation of the photosynthetic machinery after a distinct portion of PSII centers had experienced photoinhibition in the presence and absence of Lincomycin (Lin), a commonly used agent to block the repair of damaged PSII centers. In the absence of Lin, photoinhibition increased the relative excitation of PSII and decreased NPQ, together enhancing the electron transfer from still functional PSII centers to PSI. In contrast, in the presence of Lin, PSII photoinhibition increased the relative excitation of PSI and led to strong oxidation of the electron transfer chain. We hypothesize that plants are able to minimize the detrimental effects of high-light illumination on PSII by modulating the energy and electron transfer, but lose such a capability if the repair cycle is arrested. It is further hypothesized that dynamic regulation of the LHCII system has a pivotal role in the control of excitation energy transfer upon PSII damage and repair cycle to maintain the photosynthesis safe and efficient.

Flv3A facilitates O2 photoreduction and affects H2 photoproduction independently of Flv1A in diazotrophic Anabaena filaments.

The model heterocyst-forming filamentous cyanobacterium Anabaena sp. PCC 7120 (Anabaena) is a typical example of a multicellular organism capable of simultaneously performing oxygenic photosynthesis in vegetative cells and O2 -sensitive N2 -fixation inside heterocysts. The flavodiiron proteins have been shown to participate in photoprotection of photosynthesis by driving excess electrons to O2 (a Mehler-like reaction). Here, we performed a phenotypic and biophysical characterization of Anabaena mutants impaired in vegetative-specific Flv1A and Flv3A in order to address their physiological relevance in the bioenergetic processes occurring in diazotrophic Anabaena under variable CO2 conditions. We demonstrate that both Flv1A and Flv3A are required for proper induction of the Mehler-like reaction upon a sudden increase in light intensity, which is likely important for the activation of carbon-concentrating mechanisms and CO2 fixation. Under ambient CO2 diazotrophic conditions, Flv3A is responsible for moderate O2 photoreduction, independently of Flv1A, but only in the presence of Flv2 and Flv4. Strikingly, the lack of Flv3A resulted in strong downregulation of the heterocyst-specific uptake hydrogenase, which led to enhanced H2 photoproduction under both oxic and micro-oxic conditions. These results reveal a novel regulatory network between the Mehler-like reaction and the diazotrophic metabolism, which is of great interest for future biotechnological applications.

The Arabidopsis thylakoid chloride channel ClCe regulates ATP availability for light-harvesting complex II protein phosphorylation.

Coping with changes in light intensity is challenging for plants, but well-designed mechanisms allow them to acclimate to most unpredicted situations. The thylakoid K+/H+ antiporter KEA3 and the voltage-dependent Cl- channel VCCN1 play important roles in light acclimation by fine-tuning electron transport and photoprotection. Good evidence exists that the thylakoid Cl- channel ClCe is involved in the regulation of photosynthesis and state transitions in conditions of low light. However, a detailed mechanistic understanding of this effect is lacking. Here we report that the ClCe loss-of-function in Arabidopsis thaliana results in lower levels of phosphorylated light-harvesting complex II (LHCII) proteins as well as lower levels of the photosystem I-LHCII complexes relative to wild type (WT) in low light conditions. The phosphorylation of the photosystem II core D1/D2 proteins was less affected either in low or high light conditions. In low light conditions, the steady-state levels of ATP synthase conductivity and of the total proton flux available for ATP synthesis were lower in ClCe loss-of-function mutants, but comparable to WT at standard and high light intensity. As a long-term acclimation strategy, expression of the ClCe gene was upregulated in WT plants grown in light-limiting conditions, but not in WT plants grown in standard light even when exposed for up to 8 h to low light. Taken together, these results suggest a role of ClCe in the regulation of the ATP synthase activity which under low light conditions impacts LHCII protein phosphorylation and state transitions.

Plants acclimate to Photosystem I photoinhibition by readjusting the photosynthetic machinery.

Photosynthetic light reactions require strict regulation under dynamic environmental conditions. Still, depending on environmental constraints, photoinhibition of Photosystem (PSII) or PSI occurs frequently. Repair of photodamaged PSI, in sharp contrast to that of PSII, is extremely slow and leads to a functional imbalance between the photosystems. Slow PSI recovery prompted us to take advantage of the PSI-specific photoinhibition treatment and investigate whether the imbalance between functional PSII and PSI leads to acclimation of photosynthesis to PSI-limited conditions, either by short-term or long-term acclimation mechanisms as tested immediately after the photoinhibition treatment or after 24 h recovery in growth conditions, respectively. Short-term acclimation mechanisms were induced directly upon inhibition, including thylakoid protein phosphorylation that redirects excitation energy to PSI as well as changes in the feedback regulation of photosynthesis, which relaxed photosynthetic control and excitation energy quenching. Longer-term acclimation comprised reprogramming of the stromal redox system and an increase in ATP synthase and Cytochrome b6 f abundance. Acclimation to PSI-limited conditions restored the CO2 assimilation capacity of plants without major PSI repair. Response to PSI inhibition demonstrates that plants efficiently acclimate to changes occurring in the photosynthetic apparatus, which is likely a crucial component in plant acclimation to adverse environmental conditions.

Gene expression and organization of thylakoid protein complexes in the PSII-less mutant of Synechocystis sp. PCC 6803.

Photosystems I and II (PSI and PSII) are the integral components of the photosynthetic electron transport chain that utilize light to provide chemical energy for CO2 fixation. In this study, we investigated how the deficiency of PSII affects the gene expression, accumulation, and organization of thylakoid protein complexes as well as physiological characteristics of Synechocystis sp. PCC 6803 by combining biochemical, biophysical, and transcriptomic approaches. RNA-seq analysis showed upregulated expression of genes encoding the PSII core proteins, and downregulation of genes associated with interaction between light-harvesting phycobilisomes and PSI. Two-dimensional separation of thylakoid protein complexes confirmed the lack of PSII complexes, yet unassembled PSII subunits were detected. The content of PsaB representing PSI was lower, while the content of cytochrome b6f complexes was higher in the PSII-less strain as compared with control (CS). Application of oxygraph measurements revealed higher rates of dark respiration and lower PSI activity in the mutant. The latter likely resulted from the detected decrease in the accumulation of PSI, PSI monomerization, increased proportion of energetically decoupled phycobilisomes in PSII-less cultures, and low abundance of phycocyanin. Merging the functional consequences of PSII depletion with differential protein and transcript accumulation in the mutant, in comparison to CS, identified signal transduction from the photosynthetic apparatus to the genome level.

Patterning of the Autotrophic, Mixotrophic, and Heterotrophic Proteomes of Oxygen-Evolving Cyanobacterium Synechocystis sp. PCC 6803.

Proteomes of an oxygenic photosynthetic cyanobacterium, Synechocystis sp. PCC 6803, were analyzed under photoautotrophic (low and high CO2, assigned as ATLC and ATHC), photomixotrophic (MT), and light-activated heterotrophic (LAH) conditions. Allocation of proteome mass fraction to seven sub-proteomes and differential expression of individual proteins were analyzed, paying particular attention to photosynthesis and carbon metabolism-centered sub-proteomes affected by the quality and quantity of the carbon source and light regime upon growth. A distinct common feature of the ATHC, MT, and LAH cultures was low abundance of inducible carbon-concentrating mechanisms and photorespiration-related enzymes, independent of the inorganic or organic carbon source. On the other hand, these cells accumulated a respiratory NAD(P)H dehydrogenase I (NDH-11) complex in the thylakoid membrane (TM). Additionally, in glucose-supplemented cultures, a distinct NDH-2 protein, NdbA, accumulated in the TM, while the plasma membrane-localized NdbC and terminal oxidase decreased in abundance in comparison to both AT conditions. Photosynthetic complexes were uniquely depleted under the LAH condition but accumulated under the ATHC condition. The MT proteome displayed several heterotrophic features typical of the LAH proteome, particularly including the high abundance of ribosome as well as amino acid and protein biosynthesis machinery-related components. It is also noteworthy that the two equally light-exposed ATHC and MT cultures allocated similar mass fractions of the total proteome to the seven distinct sub-proteomes. Unique trophic condition-specific expression patterns were likewise observed among individual proteins, including the accumulation of phosphate transporters and polyphosphate polymers storing energy surplus in highly energetic bonds under the MT condition and accumulation under the LAH condition of an enzyme catalyzing cyanophycin biosynthesis. It is concluded that the rigor of cell growth in the MT condition results, to a great extent, by combining photosynthetic activity with high intracellular inorganic carbon conditions created upon glucose breakdown and release of CO2, besides the direct utilization of glucose-derived carbon skeletons for growth. This combination provides the MT cultures with excellent conditions for growth that often exceeds that of mere ATHC.

Role of serine/threonine protein kinase STN7 in the formation of two distinct photosystem I supercomplexes in Physcomitrium patens.

Reversible thylakoid protein phosphorylation provides most flowering plants with dynamic acclimation to short-term changes in environmental light conditions. Here, through generating Serine/Threonine protein kinase 7 (STN7)-depleted mutants in the moss Physcomitrella (Physcomitrium patens), we identified phosphorylation targets of STN7 kinase and their roles in short- and long-term acclimation of the moss to changing light conditions. Biochemical and mass spectrometry analyses revealed STN7-dependent phosphorylation of N-terminal Thr in specific Light-Harvesting Complex II (LHCII) trimer subunits (LHCBM2 and LHCBM4/8) and provided evidence that phospho-LHCBM accumulation is responsible for the assembly of two distinct Photosystem I (PSI) supercomplexes (SCs), both of which are largely absent in STN7-depleted mutants. Besides the canonical state transition complex (PSI-LHCI-LHCII), we isolated the larger moss-specific PSI-Large (PSI-LHCI-LHCB9-LHCII) from stroma-exposed thylakoids. Unlike PSI-LHCI-LHCII, PSI-Large did not demonstrate short-term dynamics for balancing the distribution of excitation energy between PSII and PSI. Instead, PSI-Large contributed to a more stable increase in PSI antenna size in Physcomitrella, except under prolonged high irradiance. Additionally, the STN7-depleted mutants revealed altered light-dependent phosphorylation of a monomeric antenna protein, LHCB6, whose phosphorylation displayed a complex regulation by multiple kinases. Collectively, the unique phosphorylation plasticity and dynamics of Physcomitrella monomeric LHCB6 and trimeric LHCBM isoforms, together with the presence of PSI SCs with different antenna sizes and responsiveness to light changes, reflect the evolutionary position of mosses between green algae and vascular plants, yet with clear moss-specific features emphasizing their adaptation to terrestrial low-light environments.

A kaleidoscope of photosynthetic antenna proteins and their emerging roles.

Photosynthetic light-harvesting antennae are pigment-binding proteins that perform one of the most fundamental tasks on Earth, capturing light and transferring energy that enables life in our biosphere. Adaptation to different light environments led to the evolution of an astonishing diversity of light-harvesting systems. At the same time, several strategies have been developed to optimize the light energy input into photosynthetic membranes in response to fluctuating conditions. The basic feature of these prompt responses is the dynamic nature of antenna complexes, whose function readily adapts to the light available. High-resolution microscopy and spectroscopic studies on membrane dynamics demonstrate the crosstalk between antennae and other thylakoid membrane components. With the increased understanding of light-harvesting mechanisms and their regulation, efforts are focusing on the development of sustainable processes for effective conversion of sunlight into functional bio-products. The major challenge in this approach lies in the application of fundamental discoveries in light-harvesting systems for the improvement of plant or algal photosynthesis. Here, we underline some of the latest fundamental discoveries on the molecular mechanisms and regulation of light harvesting that can potentially be exploited for the optimization of photosynthesis.

True oxygen reduction capacity during photosynthetic electron transfer in thylakoids and intact leaves.

Reactive oxygen species (ROS) are generated in electron transport processes of living organisms in oxygenic environments. Chloroplasts are plant bioenergetics hubs where imbalances between photosynthetic inputs and outputs drive ROS generation upon changing environmental conditions. Plants have harnessed various site-specific thylakoid membrane ROS products into environmental sensory signals. Our current understanding of ROS production in thylakoids suggests that oxygen (O2) reduction takes place at numerous components of the photosynthetic electron transfer chain (PETC). To refine models of site-specific O2 reduction capacity of various PETC components in isolated thylakoids of Arabidopsis thaliana, we quantified the stoichiometry of oxygen production and consumption reactions associated with hydrogen peroxide (H2O2) accumulation using membrane inlet mass spectrometry and specific inhibitors. Combined with P700 spectroscopy and electron paramagnetic resonance spin trapping, we demonstrate that electron flow to photosystem I (PSI) is essential for H2O2 accumulation during the photosynthetic linear electron transport process. Further leaf disc measurements provided clues that H2O2 from PETC has a potential of increasing mitochondrial respiration and CO2 release. Based on gas exchange analyses in control, site-specific inhibitor-, methyl viologen-, and catalase-treated thylakoids, we provide compelling evidence of no contribution of plastoquinone pool or cytochrome b6f to chloroplastic H2O2 accumulation. The putative production of H2O2 in any PETC location other than PSI is rapidly quenched and therefore cannot function in H2O2 translocation to another cellular location or in signaling.

Hydrocarbon Desaturation in Cyanobacterial Thylakoid Membranes Is Linked With Acclimation to Suboptimal Growth Temperatures.

The ability to produce medium chain length aliphatic hydrocarbons is strictly conserved in all photosynthetic cyanobacteria, but the molecular function and biological significance of these compounds still remain poorly understood. This study gives a detailed view to the changes in intracellular hydrocarbon chain saturation in response to different growth temperatures and osmotic stress, and the associated physiological effects in the model cyanobacterium Synechocystis sp. PCC 6803. We show that the ratio between the representative hydrocarbons, saturated heptadecane and desaturated heptadecene, is reduced upon transition from 38°C toward 15°C, while the total content is not much altered. In parallel, it appears that in the hydrocarbon-deficient ∆ado (aldehyde deformylating oxygenase) mutant, phenotypic and metabolic changes become more evident under suboptimal temperatures. These include hindered growth, accumulation of polyhydroxybutyrate, altered pigment profile, restricted phycobilisome movement, and ultimately reduced CO2 uptake and oxygen evolution in the ∆ado strain as compared to Synechocystis wild type. The hydrocarbons are present in relatively low amounts and expected to interact with other nonpolar cellular components, including the hydrophobic part of the membrane lipids. We hypothesize that the function of the aliphatic chains is specifically associated with local fluidity effects of the thylakoid membrane, which may be required for the optimal movement of the integral components of the photosynthetic machinery. The findings support earlier studies and expand our understanding of the biological role of aliphatic hydrocarbons in acclimation to low temperature in cyanobacteria and link the proposed role in the thylakoid membrane to changes in photosynthetic performance, central carbon metabolism, and cell growth, which need to be effectively fine-tuned under alternating conditions in nature.

Photosystem I Inhibition, Protection and Signalling: Knowns and Unknowns.

Photosynthesis is the process that harnesses, converts and stores light energy in the form of chemical energy in bonds of organic compounds. Oxygenic photosynthetic organisms (i.e., plants, algae and cyanobacteria) employ an efficient apparatus to split water and transport electrons to high-energy electron acceptors. The photosynthetic system must be finely balanced between energy harvesting and energy utilisation, in order to limit generation of dangerous compounds that can damage the integrity of cells. Insight into how the photosynthetic components are protected, regulated, damaged, and repaired during changing environmental conditions is crucial for improving photosynthetic efficiency in crop species. Photosystem I (PSI) is an integral component of the photosynthetic system located at the juncture between energy-harnessing and energy consumption through metabolism. Although the main site of photoinhibition is the photosystem II (PSII), PSI is also known to be inactivated by photosynthetic energy imbalance, with slower reactivation compared to PSII; however, several outstanding questions remain about the mechanisms of damage and repair, and about the impact of PSI photoinhibition on signalling and metabolism. In this review, we address the knowns and unknowns about PSI activity, inhibition, protection, and repair in plants. We also discuss the role of PSI in retrograde signalling pathways and highlight putative signals triggered by the functional status of the PSI pool.

Characterization of the Free and Membrane-Associated Fractions of the Thylakoid Lumen Proteome in Arabidopsis thaliana.

The thylakoid lumen houses proteins that are vital for photosynthetic electron transport, including water-splitting at photosystem (PS) II and shuttling of electrons from cytochrome b6f to PSI. Other lumen proteins maintain photosynthetic activity through biogenesis and turnover of PSII complexes. Although all lumen proteins are soluble, these known details have highlighted interactions of some lumen proteins with thylakoid membranes or thylakoid-intrinsic proteins. Meanwhile, the functional details of most lumen proteins, as well as their distribution between the soluble and membrane-associated lumen fractions, remain unknown. The current study isolated the soluble free lumen (FL) and membrane-associated lumen (MAL) fractions from Arabidopsis thaliana, and used gel- and mass spectrometry-based proteomics methods to analyze the contents of each proteome. These results identified 60 lumenal proteins, and clearly distinguished the difference between the FL and MAL proteomes. The most abundant proteins in the FL fraction were involved in PSII assembly and repair, while the MAL proteome was enriched in proteins that support the oxygen-evolving complex (OEC). Novel proteins, including a new PsbP domain-containing isoform, as well as several novel post-translational modifications and N-termini, are reported, and bi-dimensional separation of the lumen proteome identified several protein oligomers in the thylakoid lumen.

Comparison of alternative integration sites in the chromosome and the native plasmids of the cyanobacterium Synechocystis sp. PCC 6803 in respect to expression efficiency and copy number.

BACKGROUND: Synechocystis sp. PCC 6803 provides a well-established reference point to cyanobacterial metabolic engineering as part of basic photosynthesis research, as well as in the development of next-generation biotechnological production systems. This study focused on expanding the current knowledge on genomic integration of expression constructs in Synechocystis, targeting a range of novel sites in the chromosome and in the native plasmids, together with established loci used in literature. The key objective was to obtain quantitative information on site-specific expression in reference to replicon copy numbers, which has been speculated but never compared side by side in this host. RESULTS: An optimized sYFP2 expression cassette was successfully integrated in two novel sites in Synechocystis chromosome (slr0944; sll0058) and in all four endogenous megaplasmids (pSYSM/slr5037-slr5038; pSYSX/slr6037; pSYSA/slr7023; pSYSG/slr8030) that have not been previously evaluated for the purpose. Fluorescent analysis of the segregated strains revealed that the expression levels between the megaplasmids and chromosomal constructs were very similar, and reinforced the view that highest expression in Synechocystis can be obtained using RSF1010-derived replicative vectors or the native small plasmid pCA2.4 evaluated in comparison. Parallel replicon copy number analysis by RT-qPCR showed that the expression from the alternative loci is largely determined by the gene dosage in Synechocystis, thereby confirming the dependence formerly proposed based on literature. CONCLUSIONS: This study brings together nine different integrative loci in the genome of Synechocystis to demonstrate quantitative differences between target sites in the chromosome, the native plasmids, and a RSF1010-based replicative expression vector. To date, this is the most comprehensive comparison of alternative integrative sites in Synechocystis, and provides the first direct reference between expression efficiency and replicon gene dosage in the context. In the light of existing literature, the findings support the view that the small native plasmids can be notably more difficult to target than the chromosome or the megaplasmids, and that the RSF1010-derived vectors may be surprisingly well maintained under non-selective culture conditions in this cyanobacterial host. Altogether, the work broadens our views on genomic integration and the rational use of different integrative loci versus replicative plasmids, when aiming at expressing heterologous genes in Synechocystis.

Global proteomic response of unicellular cyanobacterium Synechocystis sp. PCC 6803 to fluctuating light upon CO2 step-down.

Photosynthetic cyanobacteria are exposed to rapid changes in light intensity in their natural habitats, as well as in photobioreactors. To understand the effects of such fluctuations on Synechocystis sp. PCC 6803, the global proteome of cells grown under a fluctuating light condition (low background light interrupted with high light pulses) was compared to the proteome of cells grown under constant light with concomitant acclimation of cells to low CO2 level. The untargeted global proteome of Synechocystis sp. PCC 6803 was analyzed by data-dependent acquisition (DDA), which relies on the high mass accuracy and sensitivity of orbitrap-based tandem mass spectrometry. In addition, a targeted selected reaction monitoring (SRM) approach was applied to monitor the proteomic changes in a strain lacking flavodiiron proteins Flv1 and Flv3. This strain is characterized by impaired growth and photosynthetic activity under fluctuating light. An obvious reprogramming of cell metabolism was observed in this study and was compared to a previous transcriptional analysis performed under the same fluctuating light regime. Cyanobacterial responses to fluctuating light correlated at mRNA and protein levels to some extent, but discrepancies indicate that several proteins are post-transcriptionally regulated (affecting observed protein abundances). The data suggest that Synechocystis sp. PCC 6803 maintain higher nitrogen assimilation, serving as an electron valve, for long-term acclimation to fluctuating light upon CO2 step-down. Although Flv1 and Flv3 are known to be crucial for the cells at the onset of illumination, the flavodiiron proteins, as well as components of carbon assimilation pathways, were less abundant under fluctuating light.

NordAqua, a Nordic Center of Excellence to develop an algae-based photosynthetic production platform.

NordAqua is a multidisciplinary Nordic Center of Excellence funded by NordForsk Bioeconomy program (2017-2022). The research center promotes Blue Bioeconomy and endeavours to reform the use of natural resources in a environmentally sustainable way. In this short communication, we summarize particular outcomes of the consortium. The key research progress of NordAqua includes (1) improving of photosynthetisis, (2) developing novel photosynthetic cell factories that function in a "solar-driven direct CO2 capture to target bioproducts" mode, (3) promoting the diversity of Nordic cyanobacteria and algae as an abundant and resilient alternative for less sustainable forest biomass and for innovative production of biochemicals, and (4) improving the bio-based wastewater purification and nutrient recycling technologies to provide new tools for integrative circular economy platforms.