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.     

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.     

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.     

Targeted, PCR-based gene disruption in cyanobacteria: inactivation of the polyhydroxyalkanoic acid synthase genes in Synechocystis sp. PCC6803

A PCR-based method is described for the efficient construction of targeted gene disruptions and gene fusions in the cyanobacterium Synechocystis sp. PCC6803. In a simple two-step PCR approach, a gene conversion cassette was synthesized targeting the polyhydroxyalkanoic acid (PHA) synthase genes. Upon transformation, PHA production in Synechocystis under normal as well as high production culture conditions was undetectable. The application of this method to the genetic inactivation of the phaE-C _Syn gene cluster demonstrates its potential for genetic engineering of cyanobacteria and the study of functional genomics in Synechocystis. Received: 3 March 2000 / Received revision: 1 June 2000 / Accepted: 9 June 2000     

Cyanobacterial multi-copy chromosomes and their replication

ABSTRACT

While the model bacteria Escherichia coli and Bacillus subtilis harbor single chromosomes, which is known as monoploidy, some freshwater cyanobacteria contain multiple chromosome copies per cell throughout their cell cycle, which is known as polyploidy. In the model cyanobacteria Synechococcus elongatus PCC 7942 and Synechocystis sp. PCC 6803, chromosome copy number (ploidy) is regulated in response to growth phase and environmental factors. In S. elongatus 7942, chromosome replication is asynchronous both among cells and chromosomes. Comparative analysis of S. elongatus 7942 and S. sp. 6803 revealed a variety of DNA replication mechanisms. In this review, the current knowledge of ploidy and DNA replication mechanisms in cyanobacteria is summarized together with information on the features common with plant chloroplasts. It is worth noting that the occurrence of polyploidy and its regulation are correlated with certain cyanobacterial lifestyles and are shared between some cyanobacteria and chloroplasts.     

Enhancement of pyruvate production byTorulopsis glabrata through supplement of oxaloacetate as carbon source

The capability of utilizing a TCA cycle intermediates as the sole carbon source by the multi-vitamin auxotrophic yeastTorulopsis glabrata CCTCC M202019 was demonstrated with plate count method. It is indicated thatT. glabrata could grew on a medium with one of the TCA cycle intermediates as the sole carbon source, but more colonies were observed when glucose, acetate and one of the TCA cycle intermediates coexisted in the medium. Among the intermediates of the TCA cycle examined in this study, cell growth was improved by supplementing oxaloacetate. Further investigation showed that the presence of acetate was necessary when oxaloacetate was supplemented. By supplementing with 10 g/L of oxaloacetate in pyruvate batch fermentation, dry cell weight increased from 11.8 g/L to 13.6 g/L, and pyruvate productivity was enhanced from 0.96 gL⁻¹h⁻¹ to 1.19 gL⁻¹h⁻¹ after cultivation of 56 h. The yield of pyruvate to glucose was also improved from 0.63 g/g to 0.66 g/g. These results indicate that under vitamins limitation, the productivity and yield of pyruvate could be enhancedvia an increase of cell growth by the supplementation of oxaloacetate.     

Photosynthetic generation of heterologous terpenoids in cyanobacteria

The work aims to convert the secondary slow metabolism of the terpenoid biosynthetic pathway into a primary activity in cyanobacteria and to generate heterologous products using these photosynthetic microorganisms as cell factories. Case study is the production of the 10-carbon monoterpene β-phellandrene (PHL) in Synechocystis sp. PCC 6803 (Synechocystis). Barriers to this objective include the slow catalytic activity of the terpenoid metabolism enzymes that limit rates and yield of product synthesis and accumulation. “Fusion constructs as protein overexpression vectors” were applied in the overexpression of the geranyl diphosphate synthase (GPPS) and β-phellandrene synthase (PHLS) genes, causing accumulation of GPPS up to 4% and PHLS up to 10% of the total cellular protein. Such GPPS and PHLS protein overexpression compensated for their slow catalytic activity and enabled transformant Synechocystis to constitutively generate 24 mg of PHL per g biomass (2.4% PHL:biomass, w-w), a substantial improvement over earlier yields. The work showed that a systematic overexpression, at the protein level, of the terpenoid biosynthetic pathway genes is a promising approach to achieving high yields of prenyl product biosynthesis, on the way to exploiting the cellular terpenoid metabolism for commodity product generation.     

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.     

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.     

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.     

PHOTOTACTIC MOTILITY OF SYNECHOCYSTIS SP. UNIWG (CYANOBACTERIA) FROM BRACKISH ENVIRONMENT1

Although type IV pilus has been implicated in the phototactic motility of some unicellular cyanobacteria, its regulatory mechanism and the effect of environmental factors on motility are still unknown. Equally important is the ability of cyanobacterial cells to anchor themselves to an environment that is conducive for survival. We compared the motility of a newly isolated unicellular brackish cyanobacterium, Synechocystis sp. UNIWG, with the morphologically and phylogenetically similar freshwater cyanobacterium Synechocystis sp. PCC6803 under different environmental conditions. The phototactic motility of Synechocystis sp. UNIWG on semisolid BG‐11 medium with various concentrations of nitrogen source was significantly faster than that of Synechocystis PCC6803. Interestingly, the cell surface of Synechocystis sp. UNIWG showed the presence of rigid spicules when grown in liquid BG‐11, a phenomenon that was absent in Synechocystis PCC6803. Negative staining of Synechocystis sp. UNIWG revealed the presence of two distinct pilus morphotypes, which resembled type IV pili and thin pili of Synechocystis PCC6803. This finding suggested a similar pattern of phototactic motility in both strains. However, the rigid spicules on Synechocystis sp. UNIWG seem to be more of a hindrance during type IV motility. It was determined that the spicules were degraded when the cells moved, such as under prolonged darkness and/or depletion of nitrogen source, indicating that the function of the spicules is to attach the cell to an environment that is conducive for its survival. Thus, Synechocystis sp. UNIWG shows phototaxis regulation that is more complex than Synechocystis PCC6803.     

Directed inactivation of the psbl gene does not affect Photosystem II in the cyanobacterium Synechocystis sp. PCC 6803

PsbI is a small, integral membrane protein component of photosystem II (PSII), a pigment-protein complex in cyanobacteria, algae and higher plants. To understand the function of this protein, we have isolated the psbI gene from the unicellular cyanobacterium Synechocystis sp. PCC 6803 and determined its nucleotide sequence. Using an antibiotic-resistance cartridge to disrupt and replace the psbI gene, we have created mutants of Synechocystis 6803 that lack the PsbI protein. Analysis of these mutants revealed that absence of the PsbI protein results in a 25–30% loss of PSII activity. However, other PSII polypeptides are present in near wild-type amounts, indicating that no significant destabilization of the PSII complex has occurred. These results contrast with recently reported data indicating that PsbI-deficient mutants of the eukaryotic alga Chlamydomonas reinhardtii are highly light-sensitive and have a significantly lower (80–90%) titer of the PSII complex. In Synechocystis 6803, PsbI-deficient cells appear to be slightly more photosensitive than wild-type cells, suggesting that this protein, while not essential for PSII biogenesis or function, plays a role in the optimization of PSII activity.     

Secondary metabolite from Nostoc XPORK14A inhibits photosynthesis and growth of Synechocystis PCC 6803

Screening of 55 different cyanobacterial strains revealed that an extract from Nostoc XPORK14A drastically modifies the amplitude and kinetics of chlorophyll a fluorescence induction of Synechocystis PCC 6803 cells. After 2 d exposure to the Nostoc XPORK14A extract, Synechocystis PCC 6803 cells displayed reduced net photosynthetic activity and significantly modified electron transport properties of photosystem II under both light and dark conditions. However, the maximum oxidizable amount of P700 was not strongly affected. The extract also induced strong oxidative stress in Synechocystis PCC 6803 cells in both light and darkness. We identified the secondary metabolite of Nostoc XPORK14A causing these pronounced effects on Synechocystis cells. Mass spectrometry and nuclear magnetic resonance analyses revealed that this compound, designated as M22, has a non‐peptide structure. We propose that M22 possesses a dual‐action mechanism: firstly, by photogeneration of reactive oxygen species in the presence of light, which in turn affects the photosynthetic machinery of Synechocystis PCC 6803; and secondly, by altering the in vivo redox status of cells, possibly through inhibition of protein kinases.     

Ammonium tolerance in the cyanobacterium Synechocystis sp. strain PCC 6803 and the role of the psbA multigene family

Ammonium is one of the major nutrients for plants, and a ubiquitous intermediate in plant metabolism, but it is also known to be toxic to many organisms, in particular to plants and oxygenic photosynthetic microorganisms. Although previous studies revealed a link between ammonium toxicity and photodamage in cyanobacteria under in vivo conditions, ammonium‐induced photodamage of photosystem II (PSII) has not yet been investigated with isolated thylakoid membranes. We show here that ammonium directly accelerated photodamage of PSII in Synechocystis sp. strain PCC6803, rather than affecting the repair of photodamaged PSII. Using isolated thylakoid membranes, it could be demonstrated that ammonium‐induced photodamage of PSII primarily occurred at the oxygen evolution complex, which has a known binding site for ammonium. Wild‐type Synechocystis PCC6803 cells can tolerate relatively high concentrations of ammonium because of efficient PSII repair. Ammonium tolerance requires all three psbA genes since mutants of any of the three single psbA genes are more sensitive to ammonium than wild‐type cells. Even the poorly expressed psbA1 gene, whose expression was studied in some detail, plays a detectable role in ammonium tolerance.     

The NADP-linked glyceraldehyde-3-phosphate dehydrogenases of Anabaena variabilis and Synechocystis PCC 6803, which lack one of the cysteines found in the higher plant enzyme,are not reductively activated

Light activation of NADP-linked glyceraldehyde-3-P dehydrogenase involves reductive cleavage of a disulfide bond. We have proposed that the inactivating disulfide locks the two domains of the enzyme, preventing catalysis, and we have tentatively identified the two critical cysteine residues in the chloroplast enzyme (D. Li, F.J. Stevens, M. Schiffer and L.E. Anderson (1994) Biophys J. 67: 29–35). We reasoned that if activation of this enzyme involves these cysteines that enzymes lacking one or both should be active in the dark and insensitive to reductants. One of these cysteines is present in the enzymes from Anabaena variabilis and Synechocystis PCC 6803 but the other is not. Consistent with the proposed mechanism, glyceraldehyde-3-P dehydrogenase is not affected by DTT-treatment in extracts of either of these cyanobacteria. Fructosebisphosphatase is DTT-activated in extracts of both of these cyanobacteria and glucose-6-P dehydrogenase is inactivated in Synechocystis, as in higher plant chloroplasts. Apparently reductive modulation is possible in these cyanobacteria but glyceraldehyde-3-P dehydrogenase is not light activated.     

Mapping photoautotrophic metabolism with isotopically nonstationary (13)C flux analysis

Understanding in vivo regulation of photoautotrophic metabolism is important for identifying strategies to improve photosynthetic efficiency or re-route carbon fluxes to desirable end products. We have developed an approach to reconstruct comprehensive flux maps of photoautotrophic metabolism by computational analysis of dynamic isotope labeling measurements and have applied it to determine metabolic pathway fluxes in the cyanobacterium Synechocystis sp. PCC6803. Comparison to a theoretically predicted flux map revealed inefficiencies in photosynthesis due to oxidative pentose phosphate pathway and malic enzyme activity, despite negligible photorespiration. This approach has potential to fill important gaps in our understanding of how carbon and energy flows are systemically regulated in cyanobacteria, plants, and algae.     

Effects of fatty acid activation on photosynthetic production of fatty acid-based biofuels in Synechocystis sp. PCC6803

Background

Direct conversion of solar energy and carbon dioxide to drop in fuel molecules in a single biological system can be achieved from fatty acid-based biofuels such as fatty alcohols and alkanes. These molecules have similar properties to fossil fuels but can be produced by photosynthetic cyanobacteria.

Results

Synechocystis sp. PCC6803 mutant strains containing either overexpression or deletion of the slr1609 gene, which encodes an acyl-ACP synthetase (AAS), have been constructed. The complete segregation and deletion in all mutant strains was confirmed by PCR analysis. Blocking fatty acid activation by deleting slr1609 gene in wild-type Synechocystis sp. PCC6803 led to a doubling of the amount of free fatty acids and a decrease of alkane production by up to 90 percent. Overexpression of slr1609 gene in the wild-type Synechocystis sp. PCC6803 had no effect on the production of either free fatty acids or alkanes. Overexpression or deletion of slr1609 gene in the Synechocystis sp. PCC6803 mutant strain with the capability of making fatty alcohols by genetically introducing fatty acyl-CoA reductase respectively enhanced or reduced fatty alcohol production by 60 percent.

Conclusions

Fatty acid activation functionalized by the slr1609 gene is metabolically crucial for biosynthesis of fatty acid derivatives in Synechocystis sp. PCC6803. It is necessary but not sufficient for efficient production of alkanes. Fatty alcohol production can be significantly improved by the overexpression of slr1609 gene.     

Conjugative transfer and autonomous replication of a promiscuous IncQ plasmid in the cyanobacterium Synechocystis PCC 6803

Summary The promiscuous IncQ plasmid pKT210 (Cm^r, Sm^r) is efficiently transferred by transpecific conjugation from Escherichia coli to the facultatively heterotrophic cyanobacterium Synechocystis PCC6803 when mobilized by a helper plasmid coding for IncP transfer functions. The IncQ plasmid is stably maintained in the cyanobacterium as an autonomously replicating multicopy plasmid with no detectable structural alterations and can be recovered by transformation back to E. coli when using a mcrA mcrB host. Thus, the replicative host-range of IncQ plasmids extends beyond purple bacteria to the distinct procaryotic taxon of cyanobacteria, allowing the use of these small plasmids as convenient cloning vectors in Synechocystis PCC6803 and presumably also in cyanobacteria that are not amenable to genetic transformation. In contrast, an IncQ plasmid bearing the TRP1 gene of Saccharomyces cerevisiae failed to replicate when transferred to that yeast by transformation.     

A comparison of the major lipid classes and fatty acid composition of marine unicellular cyanobacteria with freshwater species

The fatty acid composition of two motile (strains WH 8113 and WH 8103) and one nonmotile (strain WH 7803) marine cyanobacteria has been determined and compared with two freshwater unicellular Synechocystis species (strain PCC 6308 and PCC 6803). The fatty acid composition of lipid extracts of isolated membranes from Synechocystis PCC 6803 was found to be identical to that of whole cells. All the marine strains contained myristic acid (14:0) as the major fatty acid, with only traces of polyunsaturated fatty acids. This composition is similar to Synechocystis PCC 6308. The major lipid classes of the nonmotile marine strain were identified as digalactosyl diacylglycerol, monogalactosyl diacylglycerol, phosphatidylglycerol, and sulfoquinovosyl diacylglycerol, identical to those found in other cyanobacteria.Abbreviations DGDG Digalactosyl diacylglycerol - MGDG Monogalactosyldiacylglycerol - PG Phosphatidylglycerol - SGDG sulfoquinovosyl diacylglycerol - gc gas chromatography - ms mass spectrometry