Reconstitution of oxaloacetate metabolism in the tricarboxylic acid cycle in Synechocystis sp. PCC 6803: discovery of important factors that directly affect the conversion of oxaloacetate

The tricarboxylic acid (TCA) cycle is one of the most important metabolic pathways in nature. Oxygenic photoautotrophic bacteria, cyanobacteria, have an unusual TCA cycle. The TCA cycle in cyanobacteria contains two unique enzymes that are not part of the TCA cycle in other organisms. In recent years, sustainable metabolite production from carbon dioxide using cyanobacteria has been looked at as a means to reduce the environmental burden of this gas. Among cyanobacteria, the unicellular cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis 6803) is an optimal host for sustainable metabolite production. Recently, metabolite production using the TCA cycle in Synechocystis 6803 has been carried out. Previous studies revealed that the branch point of the oxidative and reductive TCA cycles, oxaloacetate metabolism, plays a key role in metabolite production. However, the biochemical mechanisms regulating oxaloacetate metabolism in Synechocystis 6803 are poorly understood. Concentrations of oxaloacetate in Synechocystis 6803 are extremely low, such that in vivo analysis of oxaloacetate metabolism does not seem realistic. Therefore, using purified enzymes, we reconstituted oxaloacetate metabolism in Synechocystis 6803 in vitro to reveal the regulatory mechanisms involved. Reconstitution of oxaloacetate metabolism revealed that pH, Mg²⁺ and phosphoenolpyruvate are important factors affecting the conversion of oxaloacetate in the TCA cycle. Biochemical analyses of the enzymes involved in oxaloacetate metabolism in this and previous studies revealed the biochemical mechanisms underlying the effects of these factors on oxaloacetate conversion. In addition, we clarified the function of two l- malate dehydrogenase isozymes in oxaloacetate metabolism. These findings serve as a basis for various applications of the cyanobacterial TCA cycle.     

Dissection of respiratory and cyclic electron transport in Synechocystis sp. PCC 6803

Cyclic electron transport (CET) is an attractive hypothesis for regulating photosynthetic electron transport and producing the additional ATP in oxygenic phototrophs. The concept of CET has been established in the last decades, and it is proposed to function in the progenitor of oxygenic photosynthesis, cyanobacteria. The in vivo activity of CET is frequently evaluated either from the redox state of the reaction center chlorophyll in photosystem (PS) I, P700, in the absence of PSII activity or by comparing PSI and PSII activities through the P700 redox state and chlorophyll fluorescence, respectively. The evaluation of CET activity, however, is complicated especially in cyanobacteria, where CET shares the intersystem chain, including plastoquinone, cytochrome b₆/f complex, plastocyanin, and cytochrome c₆, with photosynthetic linear electron transport (LET) and respiratory electron transport (RET). Here we sought to distinguish the in vivo electron transport rates in RET and CET in the cyanobacterium Synechocystis sp. PCC 6803. The reduction rate of oxidized P700 (P700⁺) decreased to less than 10% when PSII was inhibited, indicating that PSII is the dominant electron source to PSI but P700⁺ is also reduced by electrons derived from other sources. The oxidative pentose phosphate (OPP) pathway functions as the dominant electron source for RET, which was found to be inhibited by glycolaldehyde (GA). In the condition where the OPP pathway and respiratory terminal oxidases were inhibited by GA and KCN, the P700⁺ reduction rate was less than 1% of that without any inhibitors. This study indicate that the electron transport to PSI when PSII is inhibited is dominantly derived from the OPP pathway in Synechocystis sp. PCC 6803.

     

Characterization of trophic changes and a functional oxidative pentose phosphate pathway in <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803

Cyanobacteria have a tremendous activity to adapt to environmental changes of their growth conditions. In this study, Synechocystis sp. PCC 6803 was used as a model organism to focus on the alternatives of cyanobacterial energy metabolism. Glucose oxidation in Synechocystis sp. PCC6803 was studied by inactivation of slr1843, encoding glucose-6-phosphate dehydrogenase (G6PDH), the first enzyme of the oxidative pentose phosphate pathway (OPPP). The resulting zwf strain was not capable of glucose supported heterotrophic growth. Growth under autotrophy and under mixotrophy was similar to that of the wild-type strain, even though oxygen evolution and uptake rates of the mutant were decreased in the presence of glucose. The organic acids citrate and succinate supported photoheterotrophic growth of both WT and zwf. Proteome analysis of soluble and membrane fractions allowed identification of four growth condition-dependent proteins, pentose-5-phosphate 3-epimerase (slr1622), inorganic pyrophosphatase (sll0807), hypothetical protein (slr2032) and ammonium/methylammonium permease (sll0108) revealing details of maintenance of the cellular carbon/nitrogen/phosphate balance under different modes of growth.     

NADPH fluorescence in the cyanobacterium Synechocystis sp. PCC 6803: A versatile probe for in vivo measurements of rates,yields and pools

We measured the kinetics of light-induced NADPH formation and subsequent dark consumption by monitoring in vivo its fluorescence in the cyanobacterium Synechocystis PCC 6803. Spectral data allowed the signal changes to be attributed to NAD(P)H and signal linearity vs the chlorophyll concentration was shown to be recoverable after appropriate correction. Parameters associated to reduction of NADP⁺ to NADPH by ferredoxin–NADP⁺-oxidoreductase were determined: After single excitation of photosystem I, half of the signal rise is observed in 8 ms; Evidence for a kinetic limitation which is attributed to an enzyme bottleneck is provided; After two closely separated saturating flashes eliciting two photosystem I turnovers in less than 2 ms, more than 50% of the cytoplasmic photoreductants (reduced ferredoxin and photosystem I acceptors) are diverted from NADPH formation by competing processes. Signal quantitation in absolute NADPH concentrations was performed by adding exogenous NADPH to the cell suspensions and by estimating the enhancement factor of in vivo fluorescence (between 2 and 4). The size of the visible (light-dependent) NADP (NADP⁺ + NADPH) pool was measured to be between 1.4 and 4 times the photosystem I concentration. A quantitative discrepancy is found between net oxygen evolution and NADPH consumption by the light-activated Calvin–Benson cycle. The present study shows that NADPH fluorescence is an efficient probe for studying in vivo the energetic metabolism of cyanobacteria which can be used for assessing multiple phenomena occurring over different time scales.     

Using photosystem I as a reporter protein for C analysis in a coculture containing cyanobacterium and a heterotrophic bacterium

¹³C metabolism analysis of a microbial community is often hindered by the time-consuming and complicated separation procedure for a single species. However, a “reporter protein,” produced uniquely by one cell type, retains ¹³C fingerprint information in microbial consortia. This study describes the use of photosystem I (PSI), a multi-subunit protein complex universally found in oxygenic phototrophs, as a reliable reporter protein to probe microalgal metabolism (i.e., cyanobacterium Synechocystis sp. PCC 6803) in a mixed culture with heterotrophic bacteria (i.e., Escherichia coli). We demonstrate that efficient purification of PSI and subsequent ¹³C-based amino acid analyses may decipher photomixotrophic metabolism of Synechocystis 6803 in the coculture. This study also indicates that a supplement of NaHCO₃ at high concentration could significantly improve the robustness of cyanobacterial growth against bacterial contamination.     

Reduction of photosystem I reaction center by recombinant DrgA protein in isolated thylakoid membranes of the cyanobacterium <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803

To study the function of soluble NAD(P)H:quinone oxidoreductase of the cyanobacterium Synechocystis sp. PCC 6803 encoded by drgA gene, recombinant DrgA protein carrying 12 histidine residues on the C-terminal end was expressed in Escherichia coli and purified. Recombinant DrgA is a flavoprotein that exhibits quinone reductase and nitroreductase activities with NAD(P)H as the electron donor. Using EPR spectroscopy, it was demonstrated that addition of recombinant DrgA protein and NADPH to DCMU-treated isolated thylakoid membranes of the cyanobacterium increased the dark rereduction rate of the photosystem I reaction center (P700⁺). Thus, DrgA can participate in electron transfer from NADPH to the electron transport chain of the Synechocystis sp. PCC 6803 thylakoid membrane.     

Rre37 stimulates accumulation of 2-oxoglutarate and glycogen under nitrogen starvation in Synechocystis sp. PCC 6803

Rre37 (sll1330) in a cyanobacterium Synechocystis sp. PCC 6803 acts as a regulatory protein for sugar catabolic genes during nitrogen starvation. Low glycogen accumulation in Δrre37 was due to low expression of glycogen anabolic genes. In addition to low 2-oxoglutarate accumulation, normal upregulated expression of genes encoding glutamate synthases (gltD and gltB) as well as accumulation of metabolites in glycolysis (fructose-6-phosphate, fructose-1,6-bisphosphate, and glyceraldehyde-3-phosphate) and tricarboxylic acid (TCA) cycle (oxaloacetate, fumarate, succinate, and aconitate) were abolished by rre37 knockout. Rre37 regulates 2-oxoglutarate accumulation, glycogen accumulation through expression of glycogen anabolic genes, and TCA cycle metabolites accumulation.     

Evaluation of central metabolism based on a genomic database of<Emphasis Type="Italic">Synechocystis</Emphasis> PCC6803

Cyanobacteria produce industrially important secondary metabolites such as lipopeptide, oligosaccharide, fatty acid (esp. sulfolipid),etc. Among them,Synechocystis PCC6803 is the first strain with a publicly available full genome sequence, as of 1996, and is one of the most extensively studied photosynthetic microorganisms. Using this genomic information, the central metabolism ofSynechocystis PCC6803 was reconstructed, including photosynthesis, oxidative phosphorylation, glycolysis, pyruvate metabolism, TCA cycle, carbon fixation, and transport system. Each biochemical reaction was carefully incorporated into the model, taking into consideration the metabolite formula, stoichiometry, charge balance, and thermodynamic properties using information from genomic and metabolic databases as well as biochemical literature. The metabolic flux of the model was calculated using flux balance analysis according to its cultivation with various carbon sources. The results of simulation were in accordance with experimental data, which suggests that the central metabolism model can properly estimate the behavior ofSynechocystis PCC6803. This model would aid in the understanding of the whole cell metabolism ofSynechocystis PCC6803, the first effort of its kind for photosynthetic bacteria.     

Trichodesmium erythraeum produces a higher photocurrent than other cyanobacterial species in bio-photo electrochemical cells

The increase in world energy consumption, and the worries from potential future disasters that may derive from climate change have stimulated the development of renewable energy technologies. One promising method is the utilization of whole photosynthetic cyanobacterial cells to produce photocurrent in a bio-photo electrochemical cell (BPEC). The photocurrent can be derived from either the respiratory or photosynthetic pathways, via the redox couple NADP⁺/NADPH mediating cyclic electron transport between photosystem I inside the cells, and the anode. In the past, most studies have utilized the fresh-water cyanobacterium Synechocystis sp. PCC 6803 (Syn). Here, we show that the globally important marine cyanobacterium Trichodesmium erythraeum flourishing in the subtropical oceans can provide improved currents as compared to Syn. We applied 2D-fluorescence measurements to detect the secretion of NADPH and show that the resulting photocurrent production is enhanced by increasing the electrolyte salinity, Further enhancement of the photocurrent can be obtained by the addition of electron mediators such as NAD⁺, NADP⁺, cytochrome C, vitamin B1, or potassium ferricyanide. Finally, we produce photocurrent from additional cyanobacterial species: Synechocystis sp. PCC6803, Synechococcus elongatus PCC7942, Acaryochloris marina MBIC 11017, and Spirulina, using their cultivation media as electrolytes for the BPEC.     

Incorporation of the chlorophyll d-binding light-harvesting protein from Acaryochloris marina and its localization within the photosynthetic apparatus of Synechocystis sp. PCC6803

The gene encoding a chlorophyll d-binding light-harvesting protein, pcbA from Acaryochloris marina (now called as accessory Chlorophyll Binding Protein CBPII) marked with a His-tag was transformed into the genome of Synechocystis PCC6803. Protein gel electrophoresis and western blotting confirmed that this foreign chlorophyll d-binding protein CBPII was expressed and integrated into the thylakoid membrane and bound with chlorophyll a, the only type of chlorophyll present in Synechocystis PCC 6803. Native electrophoresis suggested that CBPII interacts with photosystem II of Synechocystis PCC 6803. Surprisingly, spectral analyses showed that the phycobiliproteins were suppressed in the transformed Synechocystis pcbA⁺, with a lower ratio of phycobilins to chlorophyll a. These results suggest that there are competitive interactions between the external antenna system of phycobiliproteins and the integral antenna system of chlorophyll-bound protein complexes.     

Role of cyanobacterial phosphoketolase in energy regulation and glucose secretion under dark anaerobic and osmotic stress conditions

Primary metabolism in cyanobacteria is built on the Calvin-Benson-Bassham (CBB) cycle, oxidative pentose phosphate (OPP) pathway, Embden–Meyerhof–Parnas (EMP) pathway, and the tricarboxylic acid (TCA) cycle. Phosphoketolase (Xpk), commonly found in cyanobacteria, is an enzyme that is linked to all these pathways. However, little is known about its physiological role. Here, we show that most of the cyanobacterial Xpk surveyed are inhibited by ATP, and both copies of Xpk in nitrogen-fixing Cyanothece ATCC51142 are further activated by ADP, suggesting their role in energy regulation. Moreover, Xpk in Synechococcus elongatus PCC7942 and Cyanothece ATCC51142 show that their expressions are dusk-peaked, suggesting their roles in dark conditions. Finally, we find that Xpk in S. elongatus PCC7942 is responsible for survival using ATP produced from the glycogen-to-acetate pathway under dark, anaerobic condition. Interestingly, under this condition, xpk deletion causes glucose secretion in response to osmotic shock such as NaHCO₃, KHCO₃ and NaCl as part of incomplete glycogen degradation. These findings unveiled the role of this widespread enzyme and open the possibility for enhanced glucose secretion from cyanobacteria.     

Proteomic analysis of Synechocystis sp. PCC6803 responses to low-temperature and high light conditions

The global changes in protein expression of Synechocystis sp. PCC6803, a photosynthetic bacterium for the production of secondary metabolites as a green cell factory, were investigated by proteome separation and a subsequent tandem mass spectrometry. Two different proteome separation techniques, strong cation exchange chromatography and off-gel electrophoresis, were applied. The combination of the two proteome separation techniques enabled the comparative analysis of the differential regulation of the Synechocystis proteome in response to two different environmental factors, temperature and light. A total of 1,483 proteins were identified, which represent over 40% of the genes in Synechocystis. Our data showed that fatty acid metabolism was inhibited by (3R)-hydroxymyristol acyl carrier protein dehydrase (Sll1605) under low temperature conditions. The expression of UDP-N-acetylglucosamine acyltransferase (Sll0379) and 3-O-[3-hydroxymyristoyl] glucosamine N-acyltransferase (Slr0776), which is involved in lipopolysaccharide metabolism, was not observed under high light conditions. Under high light exposure, proteins related to iron-sulfur metabolism were detected, which may be responsible for maintaining the redox potential of the photosystem. High light under low temperature caused severe damage to the photosystem. Some of the responses to these stresses were similar to those previously reported for other photosynthetic organisms. Notably, this study revealed the followings: (i) low temperature inhibits fatty acid synthesis; (ii) high light inhibits lipopolysaccharides synthesis and stimulates the expression of iron-sulfur related proteins; and (iii) high light under low temperature induces the photorespiratory cycle. The global proteomic analysis clearly showed that stress conditions such as low temperature and/or high light induce cellular metabolisms related with the protection of their photosystems in the model microalga Synechocystis sp. PCC6803.     

Salt-induced photosystem I cyclic electron transfer restores growth on low inorganic carbon in a type 1 NAD(P)H dehydrogenase deficient mutant of Synechocystis PCC6803

The cyanobacterium Synechocystis PCC6803 induces a photosystem I cyclic electron transfer route independent of type 1 NAD(P)H dehydrogenase. The capacity to tolerate raised salinity conditions was shown to operate in a mutant lacking functional type 1 NAD(P)H dehydrogenase. The mutant showed salt-induced enhancement of photosystem I cyclic electron transfer and respiratory capacities. Moreover, this salt-adapted energetic state also restored the capacity of the mutant to grow under inorganic carbon limitation. Uptake of the latter in these conditions became almost as efficient as in the wild-type. The acquired energetic capacities, in contrast, did not allow restoration of photoheterotrophic growth in the type 1 NAD(P)H dehydrogenase mutant.     

Growth arrest of Synechocystis sp. PCC6803 by superoxide generated from heterologously expressed Rhodobacter sphaeroides chlorophyllide a reductase

The photosynthetic growth of Synechocystis sp. PCC6803 ceased upon expression of Rhodobacter sphaeroides chlorophyllide a reductase (COR). However, an increase in cytosolic superoxide dismutase level in the recombinant Synechocystis sp. PCC6803 completely reversed the growth cessation. This demonstrates that COR generates superoxide in Synechocystis sp. PCC6803. Considering the dissolved oxygen (DO) level suitable for COR, the intracellular DO of this oxygenic photosynthetic cell appears to be low enough to support COR-mediated superoxide generation. The growth arrest of Synechocystis sp. PCC6803 by COR may give an insight into the evolutionary path from bacteriochlorophyll a biosynthetic pathway to chlorophyll a, which bypasses COR reaction.     

Cellular physiology controls photoautotrophic production of 1,2-propanediol from pools of CO2 and glycogen

Synechocystis sp. PCC 6803 PG is a cyanobacterial strain capable of synthesizing 1,2-propanediol from carbon dioxide (CO₂) via a heterologous three-step pathway and a methylglyoxal synthase (MGS) originating from Escherichia coli as an initial enzyme. The production window is restricted to the late growth and stationary phase and is apparently coupled to glycogen turnover. To understand the underlying principle of the carbon partitioning between the Calvin-Benson-Bassham (CBB) cycle and glycogen in the context of 1,2-propanediol production, experiments utilizing ¹³C labeled CO₂ have been conducted. Carbon fluxes and partitioning between biomass, storage compounds, and product have been monitored under permanent illumination as well as under dark conditions. About one-quarter of the carbon incorporated into 1,2-propanediol originated from glycogen, while the rest was derived from CO₂ fixed in the CBB cycle during product formation. Furthermore, 1,2-propanediol synthesis was depending on the availability of photosynthetic active radiation and glycogen catabolism. We postulate that the regulation of the MGS from E. coli conflicts with the heterologous reactions leading to 1,2-propanediol in Synechocystis sp. PCC 6803 PG. Additionally, homology comparison of the genomic sequence to genes encoding for the methylglyoxal bypass in E. coli suggested the existence of such a pathway also in Synechocystis sp. PCC 6803. These findings are critical for all heterologous pathways coupled to the CBB cycle intermediate dihydroxyacetone phosphate via a MGS and reveal possible engineering targets for rational strain optimization.     

Overexpression of <Emphasis Type="Italic">flv3</Emphasis> improves photosynthesis in the cyanobacterium <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC6803 by enhancement of alternative electron flow

Background

To ensure reliable sources of energy and raw materials, the utilization of sustainable biomass has considerable advantages over petroleum-based energy sources. Photosynthetic algae have attracted attention as a third-generation feedstock for biofuel production, because algae cultivation does not directly compete with agricultural resources, including the requirement for productive land and fresh water. In particular, cyanobacteria are a promising biomass feedstock because of their high photosynthetic capability.

Results

In the present study, the expression of the flv3 gene, which encodes a flavodiiron protein involved in alternative electron flow (AEF) associated with NADPH-coupled O₂ photoreduction in photosystem I, was enhanced in Synechocystis sp. PCC6803. Overexpression of flv3 improved cell growth with corresponding increases in O₂ evolution, intracellular ATP level, and turnover of the Calvin cycle. The combination of in vivo¹³C-labeling of metabolites and metabolomic analysis confirmed that the photosynthetic carbon flow was enhanced in the flv3-overexpressing strain.

Conclusions

Overexpression of flv3 improved cell growth and glycogen production in the recombinant Synechocystis sp. PCC6803. Direct measurement of metabolic turnover provided conclusive evidence that CO₂ incorporation is enhanced by the flv3 overexpression. Increase in O₂ evolution and ATP accumulation indicates enhancement of the AEF. Overexpression of flv3 improves photosynthesis in the Synechocystis sp. PCC6803 by enhancement of the AEF.

     

Effect of light fluctuations on photosynthesis and metabolic flux in Synechocystis sp. PCC 6803

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 ¹³C-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.     

FLAVODIIRON2 and FLAVODIIRON4 Proteins Mediate an Oxygen-Dependent Alternative Electron Flow in Synechocystis sp. PCC 6803 under CO2-Limited Conditions

This study aims to elucidate the molecular mechanism of an alternative electron flow (AEF) functioning under suppressed (CO₂-limited) photosynthesis in the cyanobacterium Synechocystis sp. PCC 6803. Photosynthetic linear electron flow, evaluated as the quantum yield of photosystem II [Y(II)], reaches a maximum shortly after the onset of actinic illumination. Thereafter, Y(II) transiently decreases concomitantly with a decrease in the photosynthetic oxygen evolution rate and then recovers to a rate that is close to the initial maximum. These results show that CO₂ limitation suppresses photosynthesis and induces AEF. In contrast to the wild type, Synechocystis sp. PCC 6803 mutants deficient in the genes encoding FLAVODIIRON2 (FLV2) and FLV4 proteins show no recovery of Y(II) after prolonged illumination. However, Synechocystis sp. PCC 6803 mutants deficient in genes encoding proteins functioning in photorespiration show AEF activity similar to the wild type. In contrast to Synechocystis sp. PCC 6803, the cyanobacterium Synechococcus elongatus PCC 7942 has no FLV proteins with high homology to FLV2 and FLV4 in Synechocystis sp. PCC 6803. This lack of FLV2/4 may explain why AEF is not induced under CO₂-limited photosynthesis in S. elongatus PCC 7942. As the glutathione S-transferase fusion protein overexpressed in Escherichia coli exhibits NADH-dependent oxygen reduction to water, we suggest that FLV2 and FLV4 mediate oxygen-dependent AEF in Synechocystis sp. PCC 6803 when electron acceptors such as CO₂ are not available.In photosynthesis, photon energy absorbed by PSI and PSII in thylakoid membranes oxidizes the reaction center chlorophylls (Chls), P700 in PSI and P680 in PSII, and drives the photosynthetic electron transport (PET) system. In PSII, water is oxidized to oxygen as the oxidized P680 accepts electrons from water. These electrons then reduce the cytochrome b₆/f complex through plastoquinone (PQ) in the thylakoid membranes. Photooxidized P700 in PSI accepts electrons from the reduced cytochrome b₆/f complex through plastocyanin or cytochrome c₆. Electrons released in the photooxidation of P700 are used to produce NADPH through ferredoxin and ferredoxin NADP⁺ reductase. Thus, electrons flow from water to NADPH in the so-called photosynthetic linear electron flow (LEF). Importantly, LEF induces a proton gradient across the thylakoid membranes, which provides the driving force for ATP production by ATP synthases in the thylakoid membranes. NADPH and ATP serve as chemical energy donors in the photosynthetic carbon reduction cycle (Calvin cycle).It recently has been proposed that, in cyanobacteria, the photorespiratory carbon oxidation cycle (photorespiration) functions simultaneously with the Calvin cycle to recover carbon for the regeneration of ribulose-1,5-bisphosphate, one of the substrates of Rubisco (Hagemann et al., 2013). Rubisco catalyzes the primary reactions of carbon reduction as well as oxidation cycles. However, the presence of a specific carbon concentration mechanism (CCM) in cyanobacteria had been thought to prevent the operation of photorespiration. CCM maintains a high concentration of CO₂ around Rubisco so that the oxygenase activity of Rubisco is suppressed (Badger and Price, 1992). However, recent studies on mutants deficient in photorespiration enzymes have shown that photorespiration functions, particularly under CO₂-limited conditions, in cyanobacteria as it does in higher plants (Eisenhut et al., 2006, 2008).Decreased consumption of NADPH under CO₂-limited or high-light conditions causes electrons to accumulate in the PET system. As a result, the photooxidation and photoreduction cycles of the reaction center Chls in PSI and PSII become uncoupled from the production of NADPH, inducing alternative electron flow (AEF) pathways (Mullineaux, 2014). In cyanobacteria, several AEFs that differ from those in higher plants are proposed to function as electron sinks (Mullineaux, 2014). Electrons accumulated in the PET system flow to oxygen through FLAVODIIRON1 (FLV1) and FLV3 proteins in PSI and the terminal oxidase, cytochrome c oxidase complex, and cytochrome bd-quinol oxidase (Pils and Schmetterer, 2001; Berry et al., 2002; Helman et al., 2003; Nomura et al., 2006; Lea-Smith et al., 2013). Cyanobacterial FLV comprises a diiron center, a flavodoxin domain with an FMN-binding site, and a flavin reductase domain (Vicente et al., 2002). In Synechocystis sp. PCC 6803, Helman et al. (2003) identified four genes encoding FLV1 to FLV4 and showed that FLV1 and FLV3 were essential for the photoreduction of oxygen by PSI. FLV1 and FLV3 were proposed to function as a heterodimer (Allahverdiyeva et al., 2013). FLV2/4 have been proposed to function in energy dissipation associated with PSII (Zhang et al., 2012). In addition, hydrogenases convert H⁺ to H₂ with NADPH as an electron donor (Appel et al., 2000). Furthermore, Flores et al. (2005) suggested that the nitrate assimilation pathway functions in AEF when the cells live in medium containing nitrate.To elucidate the physiological functions of these AEFs, evaluation of the presence and capacity of each AEF pathway is required. Therefore, in vivo analyses of electron fluxes are essential. We had found that an electron flow uncoupled from photosynthetic oxygen evolution functioned under suppressed (CO₂-limited) photosynthesis in the cyanobacterium Synechocystis sp. PCC 6803 but not in Synechococcus elongatus PCC 7942 (Hayashi et al., 2014), indicating that an AEF operated in Synechocystis sp. PCC 6803. This AEF was induced in high-[CO₂]-grown Synechocystis sp. PCC 6803 during the transition from CO₂-saturated photosynthesis to CO₂-limited photosynthesis (Hayashi et al., 2014). In contrast, in Synechocystis sp. PCC 6803 grown at ambient CO₂ concentration, AEF was detected immediately following the transition to CO₂-limited photosynthesis (Hayashi et al., 2014), suggesting that AEF was already induced under ambient atmospheric conditions.The expression of the AEF activity observed under CO₂-limited photosynthesis required the presence of oxygen in Synechocystis sp. PCC 6803 (Hayashi et al., 2014). In Synechocystis sp. PCC 6803, FLV1/3 were proposed to catalyze the photoreduction of oxygen (Helman et al., 2003). However, Hayashi et al. (2014) found no evidence that FLV1/3 operated under CO₂-limited photosynthesis: a mutant Synechocystis sp. PCC 6803 deficient in FLV1/3 maintained almost constant electron flux under CO₂-limited photosynthesis after the transition from CO₂-saturated conditions. Thus, the postulated photoreduction of oxygen by FLV1/3 was not responsible for the electron flux observed under CO₂-limited photosynthesis in Synechocystis sp. PCC 6803.In this study, we aimed to elucidate the molecular mechanism of the oxygen-dependent AEF functioning under CO₂-limited photosynthesis in Synechocystis sp. PCC 6803. The possibility that FLV2 and FLV4 catalyze the photoreduction of oxygen under CO₂-limited photosynthesis could not be excluded, given that AEF in high-[CO₂]-grown Synechocystis sp. PCC 6803 was induced following the transition to CO₂-limited photosynthesis (Hayashi et al., 2014). Both FLV2 and FLV4 are predicted to possess oxidoreductase motifs, similar to FLV1 and FLV3 (Helman et al., 2003; Zhang et al., 2012). Furthermore, the expression of two FLV genes (flv2 and flv4) was enhanced under low-[CO₂] conditions (Zhang et al., 2009). Zhang et al. (2012) proposed that FLV2 and FLV4 did not donate electrons to oxygen on the basis of the finding that the Synechocystis sp. PCC 6803 mutants deficient in FLV1/3 showed no light-dependent oxygen uptake (Helman et al., 2003). However, Helman et al. (2003) cultivated Synechocystis sp. PCC 6803 strains deficient in FLV1 and FLV3 proteins under high-[CO₂] conditions, and we cannot exclude the possibility that the FLV2 and FLV4 proteins were not produced in the studied cells. Taken together, it seems plausible that FLV2 and FLV4 mediate oxygen-dependent AEF following the transition to CO₂-limited photosynthesis. To evaluate this possibility, we constructed Synechocystis sp. PCC 6803 mutants deficient in flv2 and flv4 and measured their oxygen evolution and Chl fluorescence simultaneously. The mutants showed suppressed LEF after transition to CO₂-limited photosynthesis, similar to S. elongatus PCC 7942. We also tested the possibility that photorespiration functions as an electron sink under CO₂-limited photosynthesis in Synechocystis sp. PCC 6803. A recent study revealed photorespiratory oxygen uptake in a flv1/3 mutant under CO₂-depleted conditions (Allahverdiyeva et al., 2011). In this study, we found that the quantum yield of photosystem II [Y(II)] of mutants deficient in genes encoding proteins that function in photorespiration was similar to that of wild-type Synechocystis sp. PCC 6803. Thus, FLV2 and FLV4 appear to function in the oxygen-dependent AEF under CO₂-limited photosynthesis in Synechocystis sp. PCC 6803. This inference is further supported by the lack of FLV2 and FLV4 homologs in the genome of S. elongatus PCC 7942 (Bersanini et al., 2014). In addition, we found oxygen-reducing activities of recombinant glutathione S-transferase (GST)-FLV4 fusion protein, similar to those of recombinant FLV3 protein (Vicente et al., 2002). In light of these results, we discuss the molecular mechanism of the oxygen-dependent AEF under CO₂-limited photosynthesis and the physiological function of FLV proteins in Synechocystis sp. PCC 6803.