Comparative statistical component analysis of transgenic, cyanophycin-producing potatoes in greenhouse and field trials.

Potatoes are a promising system for industrial production of the biopolymer cyanophycin as a second compound in addition to starch. To assess the efficiency in the field, we analysed the stability of the system, specifically its sensitivity to environmental factors. Field and greenhouse trials with transgenic potatoes (two independent events) were carried out for three years. The influence of environmental factors was measured and target compounds in the transgenic plants (cyanophycin, amino acids) were analysed for differences to control plants. Furthermore, non-target parameters (starch content, number, weight and size of tubers) were analysed for equivalence with control plants. The huge amount of data received was handled using modern statistical approaches to model the correlation between influencing environmental factors (year of cultivation, nitrogen fertilization, origin of plants, greenhouse or field cultivation) and key components (starch, amino acids, cyanophycin) and agronomic characteristics. General linear models were used for modelling, and standard effect sizes were applied to compare conventional and genetically modified plants. Altogether, the field trials prove that significant cyanophycin production is possible without reduction of starch content. Non-target compound composition seems to be equivalent under varying environmental conditions. Additionally, a quick test to measure cyanophycin content gives similar results compared to the extensive enzymatic test. This work facilitates the commercial cultivation of cyanophycin potatoes.

CphA2 is a novel type of cyanophycin synthetase in N2-fixing cyanobacteria.

Most cyanobacteria use a single type of cyanophycin synthetase, CphA1, to synthesize the nitrogen-rich polymer cyanophycin. The genomes of many N2-fixing cyanobacteria contain an additional gene that encodes a second type of cyanophycin synthetase, CphA2. The potential function of this enzyme has been debated due to its reduced size and the lack of one of the two ATP-binding sites that are present in CphA1. Here, we analysed CphA2 from Anabaena variabilis ATCC 29413 and Cyanothece sp. PCC 7425. We found that CphA2 polymerized the dipeptide ß-aspartyl-arginine to form cyanophycin. Thus, CphA2 represents a novel type of cyanophycin synthetase. A cphA2 disruption mutant of A. variabilis was generated. Growth of this mutant was impaired under high-light conditions and nitrogen deprivation, suggesting that CphA2 plays an important role in nitrogen metabolism under N2-fixing conditions. Electron micrographs revealed that the mutant had fewer cyanophycin granules, but no alteration in the distribution of granules in its cells was observed. Localization of CphA2 by immunogold electron microscopy demonstrated that the enzyme is attached to cyanophycin granules. Expression of CphA1 and CphA2 was examined in Anabaena WT and cphA mutant cells. Whilst the CphA1 level increased upon nitrogen deprivation, the CphA2 level remained nearly constant.

Identification of the light-independent phosphoserine pathway as an additional source of serine in the cyanobacterium Synechocystis sp. PCC 6803.

L-serine is one of the proteinogenic amino acids and participates in several essential processes in all organisms. In plants, the light-dependent photorespiratory and the light-independent phosphoserine pathways contribute to serine biosynthesis. In cyanobacteria, the light-dependent photorespiratory pathway for serine synthesis is well characterized, but the phosphoserine pathway has not been identified. Here, we investigated three candidate genes for enzymes of the phosphoserine pathway in Synechocystis sp. PCC 6803. Only the gene for the D-3-phosphoglycerate dehydrogenase is correctly annotated in the genome database, whereas the 3-phosphoserine transaminase and 3-phosphoserine phosphatase (PSP) proteins are incorrectly annotated and were identified here. All enzymes were obtained as recombinant proteins and showed the activities necessary to catalyse the three-step phosphoserine pathway. The genes coding for the phosphoserine pathway were found in most cyanobacterial genomes listed in CyanoBase. The pathway seems to be essential for cyanobacteria, because it was impossible to mutate the gene coding for PSP in Synechocystis sp. PCC 6803 or in Synechococcus elongatus PCC 7942. A model approach indicates a 30-60% contribution of the phosphoserine pathway to the overall serine pool. Hence, this study verified that cyanobacteria, similar to plants, use the phosphoserine pathway in addition to photorespiration for serine biosynthesis.

All1371 is a polyphosphate-dependent glucokinase in Anabaena sp. PCC 7120.

The polyphosphate glucokinases can phosphorylate glucose to glucose 6-phosphate using polyphosphate as the substrate. ORF all1371 encodes a putative polyphosphate glucokinase in the filamentous heterocyst-forming cyanobacterium Anabaena sp. PCC 7120. Here, ORF all1371 was heterologously expressed in Escherichia coli, and its purified product was characterized. Enzyme activity assays revealed that All1371 is an active polyphosphate glucokinase that can phosphorylate both glucose and mannose in the presence of divalent cations in vitro. Unlike many other polyphosphate glucokinases, for which nucleoside triphosphates (e.g. ATP or GTP) act as phosphoryl group donors, All1371 required polyphosphate to confer its enzymic activity. The enzymic reaction catalysed by All1371 followed classical Michaelis-Menten kinetics, with kcat = 48.2 s(-1) at pH 7.5 and 28 °C and KM = 1.76 µM and 0.118 mM for polyphosphate and glucose, respectively. Its reaction mechanism was identified as a particular multi-substrate mechanism called the 'bi-bi ping-pong mechanism'. Bioinformatic analyses revealed numerous polyphosphate-dependent glucokinases in heterocyst-forming cyanobacteria. Viability of an Anabaena sp. PCC 7120 mutant strain lacking all1371 was impaired under nitrogen-fixing conditions. GFP promoter studies indicate expression of all1371 under combined nitrogen deprivation. All1371 might play a substantial role in Anabaena sp. PCC 7120 under these conditions.

Degradation of phycobilisomes in Synechocystis sp. PCC6803: evidence for essential formation of an NblA1/NblA2 heterodimer and its codegradation by A Clp protease complex.

When cyanobacteria acclimate to nitrogen deficiency, they degrade their large (3-5-MDa), light-harvesting complexes, the phycobilisomes. This massive, yet specific, intracellular degradation of the pigmented phycobiliproteins causes a color change of cyanobacterial cultures from blue-green to yellow-green, a process referred to as chlorosis or bleaching. Phycobilisome degradation is induced by expression of the nblA gene, which encodes a protein of ~7 kDa. NblA most likely acts as an adaptor protein that guides a Clp protease to the phycobiliproteins, thereby initiating the degradation process. Most cyanobacteria and red algae possess just one nblA-homologous gene. As an exception, the widely used "model organism" Synechocystis sp. PCC6803 expresses two such genes, nblA16803 and nblA26803, both of whose products are required for phycobilisome degradation. Here, we demonstrate that the two NblA proteins heterodimerize in vitro and in vivo using pull-down assays and a Förster energy-transfer approach, respectively. We further show that the NblA proteins form a ternary complex with ClpC (the HSP100 chaperone partner of Clp proteases) and phycobiliproteins in vitro. This complex is susceptible to ATP-dependent degradation by a Clp protease, a finding that supports a proposed mechanism of the degradation process. Expression of the single nblA gene encoded by the genome of the N2-fixing, filamentous cyanobacterium Nostoc sp. PCC7120 in the nblA1/nblA2 mutant of Synechocystis sp. PCC6803 induced phycobilisome degradation, suggesting that the function of the NblA heterodimer of Synechocystis sp. PCC6803 is combined in the homodimeric protein of Nostoc sp. PCC7120.

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.

Impaired glycogen synthesis causes metabolic overflow reactions and affects stress responses in the cyanobacterium Synechocystis sp. PCC 6803.

The biosynthesis of glycogen or starch is one of the main strategies developed by living organisms for the intracellular storage of carbon and energy. In phototrophic organisms, such polyglucans accumulate due to carbon fixation during photosynthesis and are used to provide maintenance energy for cell integrity, function and viability in dark periods. Moreover, it is assumed that glycogen enables cyanobacteria to cope with transient starvation conditions, as observed in most micro-organisms. Here, glycogen accumulates when an appropriate carbon source is available in sufficient amounts but growth is inhibited by lack of other nutrients. In this study, the role of glycogen in energy and carbon metabolism of phototrophic cyanobacteria was first analysed via a comparative physiological and metabolic characterization of knockout mutants defective in glycogen synthesis. We first proved the role of glycogen as a respiratory substrate in periods of darkness, the role of glycogen as a reserve to survive starvation periods such as nitrogen depletion and the role of glycogen synthesis as an ameliorator of carbon excess conditions in the model organism Synechocystis sp. PCC 6803. We provide striking new insights into the complex carbon and nitrogen metabolism of non-diazotrophic cyanobacteria: a phenotype of sensitivity to photomixotrophic conditions and of reduced glucose uptake, a non-bleaching phenotype based on an impaired acclimation response to nitrogen depletion and furthermore a phenotype of energy spilling. This study shows that the analysis of deficiencies in glycogen metabolism is a valuable tool for the identification of metabolic regulatory principles and signals.

Isolation of cyanophycin from tobacco and potato plants with constitutive plastidic cphATe gene expression.

A chimeric cyanophycin synthetase gene composed of the cphATe coding region from the cyanobacterium Thermosynechococcus elongatus BP-1, the constitutive 35S promoter and the plastid targeting sequence of the integral photosystem II protein PsbY was transferred to the tobacco variety Petit Havanna SRI and the commercial potato starch production variety Albatros. The resulting constitutive expression of cyanophycin synthetase leads to polymer contents in potato leaf chloroplasts of up to 35 mg/g dry weight and in tuber amyloplasts of up to 9 mg/g dry weight. Both transgenic tobacco and potato were used for the development of isolation methods applicable for large-scale extraction of the polymer. Two different procedures were developed which yielded polymer samples of 80 and 90% purity, respectively.

The metabolic network of Synechocystis sp. PCC 6803: systemic properties of autotrophic growth.

Unicellular cyanobacteria have attracted growing attention as potential host organisms for the production of valuable organic products and provide an ideal model to understand oxygenic photosynthesis and phototrophic metabolism. To obtain insight into the functional properties of phototrophic growth, we present a detailed reconstruction of the primary metabolic network of the autotrophic prokaryote Synechocystis sp. PCC 6803. The reconstruction is based on multiple data sources and extensive manual curation and significantly extends currently available repositories of cyanobacterial metabolism. A systematic functional analysis, utilizing the framework of flux-balance analysis, allows the prediction of essential metabolic pathways and reactions and allows the identification of inconsistencies in the current annotation. As a counterintuitive result, our computational model indicates that photorespiration is beneficial to achieve optimal growth rates. The reconstruction process highlights several obstacles currently encountered in the context of large-scale reconstructions of metabolic networks.

Tuber-specific cphA expression to enhance cyanophycin production in potatoes.

The production of biodegradable polymers that can be used to substitute petrochemical compounds in commercial products in transgenic plants is an important challenge for plant biotechnology. Nevertheless, it is often accompanied by reduced plant fitness. To decrease the phenotypic abnormalities of the sprout and to increase polymer production, we restricted cyanophycin accumulation to the potato tubers by using the cyanophycin synthetase gene (cphA(Te)) from Thermosynechococcus elongatus BP-1, which is under the control of the tuber-specific class 1 promoter (B33). Tuber-specific cytosolic (pB33-cphA(Te)) as well as tuber-specific plastidic (pB33-PsbY-cphA(Te)) expression resulted in significant polymer accumulation solely in the tubers. In plants transformed with pB33-cphA(Te), both cyanophycin synthetase and cyanophycin were detected in the cytoplasm leading to an increase up to 2.3% cyanophycin of dry weight and resulting in small and deformed tubers. In B33-PsbY-cphA(Te) tubers, cyanophycin synthetase and cyanophycin were exclusively found in amyloplasts leading to a cyanophycin accumulation up to 7.5% of dry weight. These tubers were normal in size, some clones showed reduced tuber yield and sometimes exhibited brown sunken staining starting at tubers navel. During a storage period over of 32 weeks of one selected clone, the cyanophycin content was stable in B33-PsbY-cphA(Te) tubers but the stress symptoms increased. However, all tubers were able to germinate. Nitrogen fertilization in the greenhouse led not to an increased cyanophycin yield, slightly reduced protein content, decreased starch content, and changes in the amounts of bound and free arginine and aspartate, as compared with control tubers were observed.

NblA, a key protein of phycobilisome degradation, interacts with ClpC, a HSP100 chaperone partner of a cyanobacterial Clp protease.

When cyanobacteria are starved for nitrogen, expression of the NblA protein increases and thereby induces proteolytic degradation of phycobilisomes, light-harvesting complexes of pigmented proteins. Phycobilisome degradation leads to a color change of the cells from blue-green to yellow-green, referred to as bleaching or chlorosis. As reported previously, NblA binds via a conserved region at its C terminus to the alpha-subunits of phycobiliproteins, the main components of phycobilisomes. We demonstrate here that a highly conserved stretch of amino acids in the N-terminal helix of NblA is essential for protein function in vivo. Affinity purification of glutathione S-transferase-tagged NblA, expressed in a Nostoc sp. PCC7120 mutant lacking wild-type NblA, resulted in co-precipitation of ClpC, encoded by open reading frame alr2999 of the Nostoc chromosome. ClpC is a HSP100 chaperone partner of the Clp protease. ATP-dependent binding of NblA to ClpC was corroborated by in vitro pull-down assays. Introducing amino acid exchanges, we verified that the conserved N-terminal motif of NblA mediates the interaction with ClpC. Further results indicate that NblA binds phycobiliprotein subunits and ClpC simultaneously, thus bringing the proteins into close proximity. Altogether these results suggest that NblA may act as an adaptor protein that guides a ClpC.ClpP complex to the phycobiliprotein disks in the rods of phycobilisomes, thereby initiating the degradation process.

Plastid targeting strategies for cyanophycin synthetase to achieve high-level polymer accumulation in Nicotiana tabacum.

The production of biodegradable polymers in transgenic plants is an important challenge in plant biotechnology; nevertheless, it is often accompanied by reduced plant fitness. In order to decrease the phenotypic abnormalities caused by cytosolic production of the biodegradable polymer cyanophycin, and to increase polymer accumulation, four translocation pathway signal sequences for import into chloroplasts were individually fused to the coding region of the cyanophycin synthetase gene (cphA(Te)) of Thermosynechococcus elongatus BP-1, resulting in the constructs pRieske-cphA(Te), pCP24-cphA(Te), pFNR-cphA(Te) and pPsbY-cphA(Te). These constructs were expressed in Nicotiana tabacum var. Petit Havana SRI under the control of the constitutive cauliflower mosaic virus (CaMV) 35S promoter. Three of the four constructs led to polymer production. However, only the construct pPsbY-cphA(Te) led to cyanophycin accumulation exclusively in chloroplasts. In plants transformed with the pCP24-cphA(Te) and pFNR-cphA(Te) constructs, water-soluble and water-insoluble forms of cyanophycin were only located in the cytoplasm, which resulted in phenotypic changes similar to those observed in plants transformed with constructs lacking a targeting sequence. The plants transformed with pPsbY-cphA(Te) produced predominantly the water-insoluble form of cyanophycin. The polymer accumulated to up to 1.7% of dry matter in primary (T(0)) transformants. Specific T(2) plants produced 6.8% of dry weight as cyanophycin, which is more than five-fold higher than the previously published value. Although all lines tested were fertile, the progeny of the highest cyanophycin-producing line showed reduced seed production compared with control plants.

PII-regulated arginine synthesis controls accumulation of cyanophycin in Synechocystis sp. strain PCC 6803.

Cyanophycin (multi-L-arginyl-poly-L-aspartic acid) is a nitrogen storage polymer found in most cyanobacteria and some heterotrophic bacteria. The cyanobacterium Synechocystis sp. strain PCC 6803 accumulates cyanophycin following a transition from nitrogen-limited to nitrogen-excess conditions. Here we show that the accumulation of cyanophycin depends on the activation of the key enzyme of arginine biosynthesis, N-acetyl-L-glutamate kinase, by signal transduction protein PII.

Crystal structure of NblA from Anabaena sp. PCC 7120, a small protein playing a key role in phycobilisome degradation.

Cyanobacterial light-harvesting complexes, the phycobilisomes, are proteolytically degraded when the organisms are starved for combined nitrogen, a process referred to as chlorosis or bleaching. Gene nblA, present in all phycobilisome-containing organisms, encodes a protein of about 7 kDa that plays a key role in phycobilisome degradation. The mode of action of NblA in this degradation process is poorly understood. Here we presented the 1.8-A crystal structure of NblA from Anabaena sp. PCC 7120. In the crystal, NblA is present as a four-helix bundle formed by dimers, the basic structural units. By using pull-down assays with immobilized NblA and peptide scanning, we showed that NblA specifically binds to the alpha-subunits of phycocyanin and phycoerythrocyanin, the main building blocks of the phycobilisome rod structure. By site-directed mutagenesis, we identified amino acid residues in NblA that are involved in phycobilisome binding. The results provided evidence that NblA is directly involved in phycobilisome degradation, and the results allowed us to present a model that gives insight into the interaction of this small protein with the phycobilisomes.

Production of cyanophycin, a suitable source for the biodegradable polymer polyaspartate, in transgenic plants.

The production of biodegradable polymers in transgenic plants in order to replace petrochemical compounds is an important challenge for plant biotechnology. Polyaspartate, a biodegradable substitute for polycarboxylates, is the backbone of the cyanobacterial storage material cyanophycin. Cyanophycin, a copolymer of l-aspartic acid and l-arginine, is produced via non-ribosomal polypeptide biosynthesis by the enzyme cyanophycin synthetase. A gene from Thermosynechococcus elongatus BP-1 encoding cyanophycin synthetase has been expressed constitutively in tobacco and potato. The presence of the transgene-encoded messenger RNA (mRNA) correlated with changes in leaf morphology and decelerated growth. Such transgenic plants were found to produce up to 1.1% dry weight of a polymer with cyanophycin-like properties. Aggregated material, able to bind a specific cyanophycin antibody, was detected in the cytoplasm and the nucleus of the transgenic plants.

NblA is essential for phycobilisome degradation in Anabaena sp. strain PCC 7120 but not for development of functional heterocysts.

Phycobilisomes (PBS) are the major light-harvesting complexes of cyanobacteria. These usually blue-coloured multiprotein assemblies are rapidly degraded when the organisms are starved for combined nitrogen. This proteolytic process causes a colour change of the cyanobacterial cells from blue-green to yellow-green ('bleaching'). As is well documented for the unicellular, non-diazotrophic cyanobacteria Synechococcus elongatus PCC 7942 and Synechocystis sp. PCC 6803, a gene termed nblA plays a key role in PBS degradation. Filamentous, diazotrophic cyanobacteria like Anabaena adapt to nitrogen deprivation by differentiation of N(2)-fixing heterocysts. However, during the first hours after nitrogen deprivation all cells degrade their PBS. When heterocysts mature and nitrogenase becomes active, vegetative cells resynthesize their light-harvesting complexes while in heterocysts the phycobiliprotein content remains very low. Expression and function of nblA in Anabaena sp. PCC 7120 was investigated. This strain has two nblA homologous genes, one on the chromosome (nblA) and one on plasmid delta (nblA-p). Northern blot analysis indicated that only the chromosomal nblA gene is up-regulated upon nitrogen starvation. Mutants with interrupted nblA and nblA-p genes, respectively, grew on N(2) and developed functional heterocysts. Mutant DeltanblA-p behaved like the wild-type. However, mutant DeltanblA was unable to degrade its PBS, which was most obvious in non-bleaching heterocysts. The results show that NblA, encoded by the chromosomal nblA gene, is required for PBS degradation in Anabaena but is not essential for heterocyst differentiation.

Structure of the isoaspartyl peptidase with L-asparaginase activity from Escherichia coli.

The crystal structure of the Escherichia coli enzyme (EcAIII) with isoaspartyl dipeptidase and L-asparaginase activity has been solved and refined to a resolution of 1.65 angstroms, with crystallographic R-factor and Rfree values of 0.178 and 0.209, respectively. EcAIII belongs to the family of N-terminal hydrolases. The amino-acid sequence of EcAIII is homologous to those of putative asparaginases from plants. The structure of EcAIII is similar to the structures of glycosylasparaginases. The mature and catalytically active form of EcAIII is a heterotetramer consisting of two alpha-subunits and two beta-subunits. Both of the equivalent active sites present in the EcAIII tetramer is assisted by a metal-binding site. The metal cations, modelled here as Na+, have not previously been observed in glycosylasparaginases. This reported structure helps to explain the inability of EcAIII and other plant-type asparaginases to hydrolyze N4-(beta-N-acetylglucosaminyl)-L-asparagine, the substrate of glycosylasparaginases.

Cyanophycin synthetase-like enzymes of non-cyanobacterial eubacteria: characterization of the polymer produced by a recombinant synthetase of Desulfitobacterium hafniense.

Some bacterial genomes were found to contain genes encoding putative proteins with considerable sequence homology to cyanophycin synthetase CphA of cyanobacteria. Such a gene from the Gram-positive, spore-forming anaerobe Desulfitobacterium hafniense was cloned. Expression in Escherichia coli resulted in the formation of a polydispers copolymer of aspartic acid and arginine, with a minor amount of lysine, of about 30 kDa molecular mass. In contrast to cyanophycin, this polymer was water-soluble. The structure of the polymer formed by the synthetase from Desulfitobacterium hafniense was studied by enzymatic degradation with the cyanophycin-specific hydrolase cyanophycinase, and by chemical and mass-spectroscopic analyses. Despite of the differences in solubility, indicating that both polymers cannot be completely identical, the chemical structure was found to be very similar to that of cyanophycin. The results suggest that the use of cyanophycin-like polymers as a nitrogen-rich reserve material is not restricted to cyanobacteria, and that such polymers may not necessarily be stored in granules.

Isoaspartyl dipeptidase activity of plant-type asparaginases.

Recombinant plant-type asparaginases from the cyanobacteria Synechocystis sp. PCC (Pasteur culture collection) 6803 and Anabaena sp. PCC 7120, from Escherichia coli and from the plant Arabidopsis thaliana were expressed in E. coli with either an N-terminal or a C-terminal His tag, and purified. Although each of the four enzymes is encoded by a single gene, their mature forms consist of two protein subunits that are generated by autoproteolytic cleavage of the primary translation products at the Gly-Thr bond within the sequence GTI/VG. The enzymes not only deamidated asparagine but also hydrolysed a range of isoaspartyl dipeptides. As various isoaspartyl peptides are known to arise from proteolytic degradation of post-translationally altered proteins containing isoaspartyl residues, and from depolymerization of the cyanobacterial reserve polymer multi-L-arginyl-poly-L-aspartic acid (cyanophycin), plant-type asparaginases may not only function in asparagine catabolism but also in the final steps of protein and cyanophycin degradation. The properties of these enzymes are compared with those of the sequence-related glycosylasparaginases.