Reconstruction and verification of a genome-scale metabolic model for <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC6803

In terms of generating sustainable energy resources, the prospect of producing energy and other useful materials using cyanobacteria has been attracting increasing attention since these processes require only carbon dioxide and solar energy. To establish production processes with a high productivity, in silico models to predict the metabolic activity of cyanobacteria are highly desired. In this study, we reconstructed a genome-scale metabolic model of the cyanobacterium Synechocystis sp. PCC6803, which included 465 metabolites and 493 metabolic reactions. Using this model, we performed constraint-based metabolic simulations to obtain metabolic flux profiles under various environmental conditions. We evaluated the simulated results by comparing these with experimental results from ¹³C-tracer metabolic flux analyses, which were obtained under heterotrophic and mixotrophic conditions. There was a good agreement of simulation and experimental results under both conditions. Furthermore, using our model, we evaluated the production of ethanol by Synechocystis sp. PCC6803, which enabled us to estimate quantitatively how its productivity depends on the environmental conditions. The genome-scale metabolic model provides useful information for the evaluation of the metabolic capabilities, and prediction of the metabolic characteristics, of Synechocystis sp. PCC6803.     

Flux balance analysis of photoautotrophic metabolism

Photosynthesis is the principal process responsible for fixation of inorganic carbon dioxide into organic molecules with sunlight as the energy source. Potentially, many chemicals could be inexpensively produced by photosynthetic organisms. Mathematical modeling of photoautotrophic metabolism is therefore important to evaluate maximum theoretical product yields and to deeply understand the interactions between biochemical energy, carbon fixation, and assimilation pathways. Flux balance analysis based on linear programming is applied to photoautotrophic metabolism. The stoichiometric network of a model photosynthetic prokaryote, Synechocystis sp. PCC 6803, has been reconstructed from genomic data and biochemical literature and coupled with a model of the photophosphorylation processes. Flux map topologies for the hetero-, auto-, and mixotrophic modes of metabolism under conditions of optimal growth were determined and compared. The roles of important metabolic reactions such as the glyoxylate shunt and the transhydrogenase reaction were analyzed. We also theoretically evaluated the effect of gene deletions or additions on biomass yield and metabolic flux distributions.     

Simultaneous increases in the levels of compatible solutes by cost-effective cultivation of Synechocystis sp. PCC 6803

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.     

聚球藻7942混养培养中碳代谢与能量利用

为了考察聚球藻7942在混养条件下的能量利用效率,分别以葡萄糖和乙酸为碳源开展了聚球藻7942的混养培养研究,并在此基础上利用代谢通量分析方法对聚球藻7942混养条件下的碳代谢和能量利用进行了探讨。结果表明:葡萄糖和乙酸均能促进藻细胞生长,且乙酸促进藻细胞生长的作用更为明显;葡萄糖利用可明显增加藻细胞糖酵解途径中碳代谢流量,而乙酸利用则导致糖酵解途径中碳代谢流量减小,两种有机碳源均增加了柠檬酸循环中碳代谢流量;有机碳源导致藻细胞光化学效率下降,而葡萄糖较之乙酸降低藻细胞光化学效率更为明显。虽然混养条件下光能的贡献率要小于光自养,但基于能量的细胞得率和能量转换率均高于光自养,光自养和以葡萄糖、乙酸为碳源的混养中基于ATP生成的能量转换效率分别为6.81%、7.43%和8.77%。     

Photoheterotrophic Fluxome in Synechocystis sp. Strain PCC 6803 and Its Implications for Cyanobacterial Bioenergetics

This study investigated metabolic responses in Synechocystis sp. strain PCC 6803 to photosynthetic impairment. We used 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU; a photosystem II inhibitor) to block O₂ evolution and ATP/NADPH generation by linear electron flow. Based on ¹³C-metabolic flux analysis (¹³C-MFA) and RNA sequencing, we have found that Synechocystis sp. PCC 6803 employs a unique photoheterotrophic metabolism. First, glucose catabolism forms a cyclic route that includes the oxidative pentose phosphate (OPP) pathway and the glucose-6-phosphate isomerase (PGI) reaction. Glucose-6-phosphate is extensively degraded by the OPP pathway for NADPH production and is replenished by the reversed PGI reaction. Second, the Calvin cycle is not fully functional, but RubisCO continues to fix CO₂ and synthesize 3-phosphoglycerate. Third, the relative flux through the complete tricarboxylic acid (TCA) cycle and succinate dehydrogenase is small under heterotrophic conditions, indicating that the newly discovered cyanobacterial TCA cycle (via the γ-aminobutyric acid pathway or α-ketoglutarate decarboxylase/succinic semialdehyde dehydrogenase) plays a minimal role in energy metabolism. Fourth, NAD(P)H oxidation and the cyclic electron flow (CEF) around photosystem I are the two main ATP sources, and the CEF accounts for at least 40% of total ATP generation from photoheterotrophic metabolism (without considering maintenance loss). This study not only demonstrates a new topology for carbohydrate oxidation but also provides quantitative insights into metabolic bioenergetics in cyanobacteria.     

Carbon metabolism and energy conversion of Synechococcus sp. PCC 7942 under mixotrophic conditions: comparison with photoautotrophic condition

To investigate the carbon metabolism and energy conversion efficiency of the cyanobacterium Synechococcus sp. PCC 7942 under mixotrophic conditions, we studied its growth characteristics in mixotrophic cultures with glucose and with acetate, respectively, and further discussed the carbon metabolism and energy utilization based on metabolic flux analysis. Results showed that both glucose and acetate could enhance the growth of Synechococcus sp. PCC 7942. The metabolic flux through the glycolytic pathway, tricarboxylic acid cycle, and mitochondrial oxidative phosphorylation was affected by the two organic substrates. Additionally, the cellular composition was also modulated by glucose and acetate. Under mixotrophic conditions, glucose exerts more significant impact on the diminishment of photochemical efficiency. Although the contribution of light energy was smaller, the cell yields based on total energy in mixotrophic cultures were higher compared with that of photoautotrophic one. On the basis of chlorophyll fluorescence analysis, the actual energy conversion efficiencies based on ATP synthesis in the photoautotrophic, glucose-mixotrophic, and acetate-mixotrophic cultures were evaluated to be 4.59%, 5.86%, and 6.60%, respectively.     

Effect of Glucose Utilization on Nitrite Excretion by the Unicellular Cyanobacterium Synechocystis sp. Strain PCC 6803

Up to 1 mM nitrite was excreted by Synechocystis strain 6803 cells growing under mixotrophic or photoheterotrophic conditions. This excretion is not due to a lower ratio of nitrite and nitrate reductase activities in the presence of glucose but seems to be related to a shortage of reduced ferredoxin, their electron donor, as a result of a decrease in noncyclic photosynthetic flow observed under these circumstances. Because about 60% of the reduced nitrate is excreted, the potential utilization of cyanobacteria for removal of nitrate from contaminated waters containing high concentrations of organic compounds is questioned.     

集胞藻6803的混合培养——光照强度和葡萄糖的影响

利用摇瓶研究了混合营养条件下单细胞蓝藻集胞藻6803(Synechocystissp.PCC6803)的生长特性,以及葡萄糖和光照强度对集胞藻6803生长的影响。实验结果表明,在葡萄糖消耗完之前,集胞藻6803的混合营养型生长处于对数生长期,且葡萄糖浓度及光照强度都对集胞藻6803的混合营养型生长有显著影响:在初始葡萄糖浓度097～480g/L范围内,同一光照强度培养下藻细胞的比生长速率随葡萄糖浓度的增大而降低;而在光照强度15～55μE·m^-2·s^-1范围内,初始葡萄糖浓度相同条件下藻细胞的比生长速率及对葡萄糖的藻体得率都随光照强度的增强而增大,但当光照强度在55～96μE·m^-2·s^-1时,集胞藻6803混合培养的比生长速率基本不变,出现了光饱和现象。     

Reconstruction and analysis of genome-scale metabolic model of a photosynthetic bacterium

Background  

Synechocystis sp. PCC6803 is a cyanobacterium considered as a candidate photo-biological production platform - an attractive cell factory capable of using CO₂ and light as carbon and energy source, respectively. In order to enable efficient use of metabolic potential of Synechocystis sp. PCC6803, it is of importance to develop tools for uncovering stoichiometric and regulatory principles in the Synechocystis metabolic network.     

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.     

Nutrient acquisition and limitation for the photoautotrophic growth of Synechocystis sp. PCC6803 as a renewable biomass source

Photoautotrophic microorganisms (cyanobacteria and algae) offer high promise as a source of biomass for renewable energy due to their rapid growth rates and high biomass yields. To provide a framework for evaluating the feasibility of growing phototrophic microorganisms with high biomass production rates, we operated a bench‐scale photobioreactor using Synechocystis sp. PCC6803 and with light conditions imitating actual day–night light irradiance (LI). During the time of peak LI, PCC6803's specific growth rate (1.7 day⁻¹) and the nitrate uptake rate (0.46 g N/g DW day) were high compared to past reports. Analysis employing the stoichiometry of photosynthesis of PCC6803 and ionic speciation showed that bicarbonate and phosphate were driven to very low concentrations for the high‐LI conditions. In particular, the systematic evaluation of rate‐limiting factors identified when the CO₂–C_i supply rate needed to be increased to mitigate HCO depletion and a large pH increase. It also showed that the traditional BG‐11 medium needs to be augmented with phosphate to avoid severe P depletion. This work exploits quantitative understanding the stoichiometry and kinetics of cyanobacteria for the high‐rate production of a renewable biomass. Biotechnol. Bioeng. 2011;108: 277–285. © 2010 Wiley Periodicals, Inc.     

Genome-scale stoichiometry analysis to elucidate the innate capability of the cyanobacterium Synechocystis for electricity generation

Synechocystis sp. PCC 6803 has been considered as a promising biocatalyst for electricity generation in recent microbial fuel cell research. However, the innate maximum current production potential and underlying metabolic pathways supporting the high current output are still unknown. This is mainly due to the fact that the high-current production cell phenotype results from the interaction among hundreds of reactions in the metabolism and it is impossible for reductionist methods to characterize the pathway selection in such a metabolic state. In this study, we employed computational metabolic techniques, flux balance analysis, and flux variability analysis, to exploit the maximum current outputs of Synechocystis sp. PCC 6803, in five electron transfer cases, namely, ferredoxin- and plastoquinol-dependent electron transfers under photoautotrophic cultivation, and NADH-dependent mediated electron transfer under photoautotrophic, heterotrophic, and mixotrophic conditions. In these five modes, the maximum current outputs were computed as 0.198, 0.7918, 0.198, 0.4652, and 0.4424 A gDW^?1, respectively. Comparison of the five operational modes suggests that plastoquinol-/c-type cytochrome-targeted electricity generation had an advantage of liberating the highest current output achievable for Synechocystis sp. PCC 6803. On the other hand, the analysis indicates that the currency metabolite, NADH-, dependent electricity generation can rely on a number of reactions from different pathways, and is thus more robust against environmental perturbations.     

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.     

Increased Bioplastic Production with an RNA Polymerase Sigma Factor SigE during Nitrogen Starvation in Synechocystis sp. PCC 6803

Because cyanobacteria directly harvest CO₂ and light energy, their carbon metabolism is important for both basic and applied sciences. Here, we show that overexpression of the sigma factor sigE in Synechocystis sp. PCC 6803 widely changes sugar catabolism and increases production of the biodegradable polyester polyhydroxybutyrate (PHB) during nitrogen starvation. sigE overexpression elevates the levels of proteins implicated in glycogen catabolism, the oxidative pentose phosphate pathway, and polyhydroxyalkanoate biosynthesis. PHB accumulation is enhanced by sigE overexpression under nitrogen-limited conditions, yet the molecular weights of PHBs synthesized by the parental glucose-tolerant and sigE overexpression strain are similar. Although gene expression induced by nitrogen starvation is changed and other metabolites (such as GDP-mannose and citrate) accumulate under sigE overexpression, genetic engineering of this sigma factor altered the metabolic pathway from glycogen to PHB during nitrogen starvation.     

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.     

Flux Balance Analysis of Cyanobacterial Metabolism: The Metabolic Network of Synechocystis sp. PCC 6803

Cyanobacteria are versatile unicellular phototrophic microorganisms that are highly abundant in many environments. Owing to their capability to utilize solar energy and atmospheric carbon dioxide for growth, cyanobacteria are increasingly recognized as a prolific resource for the synthesis of valuable chemicals and various biofuels. To fully harness the metabolic capabilities of cyanobacteria necessitates an in-depth understanding of the metabolic interconversions taking place during phototrophic growth, as provided by genome-scale reconstructions of microbial organisms. Here we present an extended reconstruction and analysis of the metabolic network of the unicellular cyanobacterium Synechocystis sp. PCC 6803. Building upon several recent reconstructions of cyanobacterial metabolism, unclear reaction steps are experimentally validated and the functional consequences of unknown or dissenting pathway topologies are discussed. The updated model integrates novel results with respect to the cyanobacterial TCA cycle, an alleged glyoxylate shunt, and the role of photorespiration in cellular growth. Going beyond conventional flux-balance analysis, we extend the computational analysis to diurnal light/dark cycles of cyanobacterial metabolism.     

Characteristics of mixotrophic growth of Synechocystis sp. in an enclosed photobioreactor

Synechocystis sp. PCC 6803 was grown in a 2.5 l enclosed photobioreactor on medium with or without glucose. The incident light intensities ranged from 1.5 klux to 7 klux. The highest average specific growth rates of mixotrophic culture and photoautotrophic culture were, respectively, 1.3 h^–1 at a light intensity of 7 klux on 3.2 g l^–1 glucose and 0.3 h^–1 at both light intensities of 5 klux and 7 klux. The highest cell density 2.5 g l ^–1 was obtained at both of light intensities 5 klux and 7 klux on 3.2 g glucose l^–1. Glucose consumption decreased with decreasing light intensity. The energy yields of mixotrophic cultures were 4 to 6 times higher than that of photoautotrophic cultures. Light favored mixotrophic growth of Synechocystis sp. PCC 6803, especially at higher light intensities (5–7 klux).     

Comparison of the Photosynthetic Yield of Cyanobacteria and Green Algae: Different Methods Give Different Answers

The societal importance of renewable carbon-based commodities and energy carriers has elicited a particular interest for high performance phototrophic microorganisms. Selection of optimal strains is often based on direct comparison under laboratory conditions of maximal growth rate or additional valued features such as lipid content. Instead of reporting growth rate in culture, estimation of photosynthetic efficiency (quantum yield of PSII) by pulse-amplitude modulated (PAM) fluorimetry is an often applied alternative method. Here we compared the quantum yield of PSII and the photonic yield on biomass for the green alga Chlorella sorokiniana 211-8K and the cyanobacterium Synechocystis sp. PCC 6803. Our data demonstrate that the PAM technique inherently underestimates the photosynthetic efficiency of cyanobacteria by rendering a high F₀ and a low F_M, specifically after the commonly practiced dark pre-incubation before a yield measurement. Yet when comparing the calculated biomass yield on light in continuous culture experiments, we obtained nearly equal values for both species. Using mutants of Synechocystis sp. PCC 6803, we analyzed the factors that compromise its PAM-based quantum yield measurements. We will discuss the role of dark respiratory activity, fluorescence emission from the phycobilisomes, and the Mehler-like reaction. Based on the above observations we recommend that PAM measurements in cyanobacteria are interpreted only qualitatively.     

Relationship Between the Growth of Synechocystis sp. PCC 6803 on Medium with Glucose and the Photosynthetic Energy Transformation

Measurements of chlorophyll fluorescence kinetics from dark-starved cells, light-grown cells and mixotrophic cells of Synechocystis sp. PCC 6803 were obtained using a pulse amplitude modulation (PAM) fluorometer. Photosystem Ⅱ photochemical efficiency Ⅱand the extent of reduction of Q－A in the three kinds of cells described above were compared. The millisecond delayed light emission (MDLE) of light-grown cells and mixotrophic cells were also detected. On the basis of the analysis of fluorescence kinetic parameters, comparison of the slow phase of MDLE and the influence of inhibitors of photosynthetic electron transport3-(3,4-dichlorophenyl)-1,1-dimethylurea(DCMU), 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) on the mixotrophic growth of Synechocystis sp. PCC 6803, it was concluded that the reasons for higher growth rate under mixotrophic than that under photoautotrophic might be that glucose promoted the photoautotrophic growth of mixotrephic cells and the donation of eletrons to the plastoquinone pool from the respiratory substance and the transform of energy was promoted by photosynthetic system, which provided the energy needed by anabolism of cells caused by the glucose added to the medium. 