Cyanophycin Synthetase Research Articles

Structure and function of a hexameric cyanophycin synthetase 2.

Cyanophycin is a natural polymer composed of a poly-aspartate backbone with arginine attached to each of the aspartate sidechains. Produced by a wide range of bacteria, which mainly use it as a store of fixed nitrogen, it has many promising industrial applications. Cyanophycin can be synthesized from the amino acids Asp and Arg by the widespread cyanophycin synthetase 1 (CphA1), or from the dipeptide β-Asp-Arg by the cyanobacterial enzyme cyanophycin synthetase 2 (CphA2). CphA2 enzymes display a range of oligomeric states, from dimers to dodecamers. Recently, the crystal structure of a CphA2 dimer was solved but could not be obtained in complex with substrate. Here, we report cryo-EM structures of the hexameric CphA2 from Stanieria sp. at ~2.8 Å resolution, both with and without ATP analog and cyanophycin. The structures show a two-fold symmetrical, trimer-of-dimers hexameric architecture, and substrate-binding interactions that are similar to those of CphA1. Mutagenesis experiments demonstrate the importance of several conserved substrate-binding residues. We also find that a Q416A/R528G double mutation prevents hexamer formation and use this double mutant to show that hexamerization augments the rate of cyanophycin synthesis. Together, these results increase our mechanistic understanding of how an interesting green polymer is biosynthesized.

Recombinant Multi-l-Arginyl-Poly-l-Aspartate (Cyanophycin) as an Emerging Biomaterial.

Multi-l-arginyl-poly-l-aspartate (MAPA) is a non-ribosomal polypeptide which synthesis is directed by cyanophycin synthetase, and its production can be achieved using recombinant microorganisms carrying the cphA gene. Along its poly-aspartate backbone, arginine or lysine links to each aspartate via an isopeptide bond. MAPA is a zwitterionic polyelectrolyte full of charged carboxylic, amine, and guanidino groups. In aqueous solution, MAPA exhibits dual thermal and pH responses similar to those stimuli-responsive polymers. Being biocompatible, the films containing MAPA can support cell proliferation and elicits minimal immune response in macrophages. Dipeptides from MAPA after enzymatic treatments can provide nutritional benefits. In light of the increasing interest in MAPA, this article focuses on the recent discovery of the function of cyanophycin synthetase and the potentials of MAPA as a biomaterial.

Bioinformatics of cyanophycin metabolism genes and characterization of promiscuous isoaspartyl dipeptidases that catalyze the final step of cyanophycin degradation

Cyanophycin is a bacterial biopolymer used for storage of fixed nitrogen. It is composed of a backbone of L-aspartate residues with L-arginines attached to each of their side chains. Cyanophycin is produced by cyanophycin synthetase 1 (CphA1) using Arg, Asp and ATP, and is degraded in two steps. First, cyanophycinase breaks down the backbone peptide bonds, releasing β-Asp-Arg dipeptides. Then, these dipeptides are broken down into free Asp and Arg by enzymes with isoaspartyl dipeptidase activity. Two bacterial enzymes are known to possess promiscuous isoaspartyl dipeptidase activity: isoaspartyl dipeptidase (IadA) and isoaspartyl aminopeptidase (IaaA). We performed a bioinformatic analysis to investigate whether genes for cyanophycin metabolism enzymes cluster together or are spread around the microbial genomes. Many genomes showed incomplete contingents of known cyanophycin metabolizing genes, with different patterns in various bacterial clades. Cyanophycin synthetase and cyanophycinase are usually clustered together when recognizable genes for each are found within a genome. Cyanophycinase and isoaspartyl dipeptidase genes typically cluster within genomes lacking cphA1. About one-third of genomes with genes for CphA1, cyanophycinase and IaaA show these genes clustered together, while the proportion is around one-sixth for CphA1, cyanophycinase and IadA. We used X-ray crystallography and biochemical studies to characterize an IadA and an IaaA from two such clusters, in Leucothrix mucor and Roseivivax halodurans, respectively. The enzymes retained their promiscuous nature, showing that being associated with cyanophycin-related genes did not make them specific for β-Asp-Arg dipeptides derived from cyanophycin degradation.

Soluble and stable cyanophycin synthetase expression enhances heterologous cyanophycin production in Escherichia coli

Microbial production of biopolymers represents a promising, sustainable alternative to current approaches for plastic production. Cyanophycin synthetase 1 (CphA1) produces cyanophycin - an attractive biopolymer consisting of a poly-L-aspartic acid backbone decorated with L-arginine side groups. In this work, a series of CphA1 enzymes from different bacteria that were each previously identified to express as soluble and stable enzymes in Escherichia coli were screened for heterologous cyanophycin production in engineered E. coli. In each case, expression of stable CphA1 enzymes resulted in greater insoluble cyanophycin production, and it was found that CphA1 from Tatumella morbirosei (TmCphA1) was especially productive. Under these conditions, TmCphA1 was capable of supporting up to ∼2-fold greater yields of insoluble cyanophycin than any other tested CphA1 enzymes, including 10.8-times more than CphA1 from Synechocystis sp. PCC6308. Finally, using a bench-scale bioreactor, cyanophycin production by TmCphA1-expressing E. coli reached up to 1.9 g per liter of culture by 48 h.

Cyanophycin and its biosynthesis: not hot but very cool.

Covering: 1878 to early 2023Cyanophycin is a biopolymer consisting of a poly-aspartate backbone with arginines linked to each Asp sidechain through isopeptide bonds. Cyanophycin is made by cyanophycin synthetase 1 or 2 through ATP-dependent polymerization of Asp and Arg, or β-Asp-Arg, respectively. It is degraded into dipeptides by exo-cyanophycinases, and these dipeptides are hydrolyzed into free amino acids by general or dedicated isodipeptidase enzymes. When synthesized, chains of cyanophycin coalesce into large, inert, membrane-less granules. Although discovered in cyanobacteria, cyanophycin is made by species throughout the bacterial kingdom, and cyanophycin metabolism provides advantages for toxic bloom forming algae and some human pathogens. Some bacteria have developed dedicated schemes for cyanophycin accumulation and use, which include fine temporal and spatial regulation. Cyanophycin has also been heterologously produced in a variety of host organisms to a remarkable level, over 50% of the host's dry mass, and has potential for a variety of green industrial applications. In this review, we summarize the progression of cyanophycin research, with an emphasis on recent structural studies of enzymes in the cyanophycin biosynthetic pathway. These include several unexpected revelations that show cyanophycin synthetase to be a very cool, multi-functional macromolecular machine.

Structural bases for aspartate recognition and polymerization efficiency of cyanobacterial cyanophycin synthetase

Cyanophycin is a natural biopolymer consisting of equimolar amounts of aspartate and arginine as the backbone and branched sidechain, respectively. It is produced by a single enzyme, cyanophycin synthetase (CphA1), and accumulates as a nitrogen reservoir during N2 fixation by most cyanobacteria. A recent structural study showed that three constituent domains of CphA1 function as two distinct catalytic sites and an oligomerization interface in cyanophycin synthesis. However, it remains unclear how the ATP-dependent addition of aspartate to cyanophycin is initiated at the catalytic site of the glutathione synthetase-like domain. Here, we report the cryogenic electron microscopy structures of CphA1, including a complex with aspartate, cyanophycin primer peptide, and ATP analog. These structures reveal the aspartate binding mode and phosphate-binding loop movement to the active site required for the reaction. Furthermore, structural and mutational data show a potential role of protein dynamics in the catalytic efficiency of the arginine condensation reaction.

A cryptic third active site in cyanophycin synthetase creates primers for polymerization

Cyanophycin is a nitrogen reserve biopolymer in many bacteria that has promising industrial applications. Made by cyanophycin synthetase 1 (CphA1), it has a poly-L-Asp backbone with L-Arg residues attached to each aspartate sidechain. CphA1s are thought to typically require existing segments of cyanophycin to act as primers for cyanophycin polymerization. In this study, we show that most CphA1s will not require exogenous primers and discover the surprising cause of primer independence: CphA1 can make minute quantities of cyanophycin without primer, and an unexpected, cryptic metallopeptidase-like active site in the N-terminal domain of many CphA1s digests these into primers, solving the problem of primer availability. We present co-complex cryo-EM structures, make mutations that transition CphA1s between primer dependence and independence, and demonstrate that primer dependence can be a limiting factor for cyanophycin production in heterologous hosts. In CphA1, domains with opposite catalytic activities combine into a remarkable, self-sufficient, biosynthetic nanomachine.

Structure and Function of the β-Asp-Arg Polymerase Cyanophycin Synthetase 2.

Cyanophycin is a biopolymer composed of long chains of β-Asp-Arg. It is widespread in nature, being synthesized by many clades of bacteria, which use it as a cellular reservoir of nitrogen, carbon, and energy. Two enzymes are known to produce cyanophycin: cyanophycin synthetase 1 (CphA1), which builds cyanophycin from the amino acids Asp and Arg by alternating between two separate reactions for backbone extension and side chain modification, and cyanophycin synthetase 2 (CphA2), which polymerizes β-Asp-Arg dipeptides. CphA2 is evolutionarily related to CphA1, but questions about CphA2's altered structure and function remain unresolved. Cyanophycin and related molecules have drawn interest as green biopolymers. Because it only has a single active site, CphA2 could be more useful than CphA1 for biotechnological applications seeking to produce modified cyanophycin. In this study, we report biochemical assays on nine cyanobacterial CphA2 enzymes and report the crystal structure of CphA2 from Gloeothece citriformis at 3.0 Å resolution. The structure reveals a homodimeric, three-domain architecture. One domain harbors the polymerization active site and the two other domains have structural roles. The structure and biochemical assays explain how CphA2 binds and polymerizes β-Asp-Arg and highlights differences in in vitro oligomerization and activity between CphA2 enzymes. Using the structure and distinct activity profile as a guide, we introduced a single point mutation that converted Gloeothece citriformis CphA2 from a primer-dependent enzyme into a primer-independent enzyme.

Cyanophycin Modifications-Widening the Application Potential.

A circular bioeconomy approach is essential to slowing down the fearsome ongoing climate change. Replacing polymers derived from fossil fuels with biodegradable biobased polymers is one crucial part of this strategy. Cyanophycin is a polymer consisting of amino acids produced by cyanobacteria with many potential applications. It consists mainly of aspartic acid and arginine, however, its composition may be changed at the production stage depending on the conditions of the polymerization reaction, as well as the characteristics of the enzyme cyanophycin synthetase, which is the key enzyme of catalysis. Cyanophycin synthetases from many sources were expressed heterologously in bacteria, yeast and plants aiming at high yields of the polymer or at introducing different amino acids into the structure. Furthermore, cyanophycin can be modified at the post-production level by chemical and enzymatic methods. In addition, cyanophycin can be combined with other compounds to yield hybrid materials. Although cyanophycin is an attractive polymer for industry, its usage as a sole material remains so far limited. Finding new variants of cyanophycin may bring this polymer closer to real-world applications. This short review summarizes all modifications of cyanophycin and its variants that have been reported within the literature until now, additionally addressing their potential applications.

Structures and function of the amino acid polymerase cyanophycin synthetase.

Cyanophycin is a natural biopolymer produced by a wide range of bacteria, consisting of a chain of poly-L-Asp residues with L-Arg residues attached to the β-carboxylate sidechains by isopeptide bonds. Cyanophycin is synthesized from ATP, aspartic acid and arginine by a homooligomeric enzyme called cyanophycin synthetase (CphA1). CphA1 has domains that are homologous to glutathione synthetases and muramyl ligases, but no other structural information has been available. Here, we present cryo-electron microscopy and X-ray crystallography structures of cyanophycin synthetases from three different bacteria, including cocomplex structures of CphA1 with ATP and cyanophycin polymer analogs at 2.6 Å resolution. These structures reveal two distinct tetrameric architectures, show the configuration of active sites and polymer-binding regions, indicate dynamic conformational changes and afford insight into catalytic mechanism. Accompanying biochemical interrogation of substrate binding sites, catalytic centers and oligomerization interfaces combine with the structures to provide a holistic understanding of cyanophycin biosynthesis.

Incorporation of alternative amino acids into cyanophycin by different cyanophycin synthetases heterologously expressed in Corynebacterium glutamicum

Cyanophycin (multi-l-arginyl-poly-l-aspartic acid; also known as cyanophycin grana peptide [CGP]) is a biopolymer that could be used in various fields, for example, as a potential precursor for the synthesis of polyaspartic acid or for the production of CGP-derived dipeptides. To extend the applications of this polymer, it is therefore of interest to synthesize CGP with different compositions. A recent re-evaluation of the CGP synthesis in C. glutamicum has shown that C. glutamicum is a potentially interesting microorganism for CGP synthesis with a high content of alternative amino acids. This study shows that the amount of alternative amino acids can be increased by using mutants of C. glutamicum with altered amino acid biosynthesis. With the DM1729 mutant, the lysine content in the polymer could be increased up to 33.5 mol%. Furthermore, an ornithine content of up to 12.6 mol% was achieved with ORN2(Pgdh4). How much water-soluble or insoluble CGP is synthesized is strongly related to the used cyanophycin synthetase. CphADh synthesizes soluble CGP exclusively. However, soluble CGP could also be isolated from cells expressing CphA6308Δ1 or CphA6308Δ1_C595S in addition to insoluble CGP in all examined strains. The point mutation in CphA6308Δ1_C595S partially resulted in a higher lysine content. In addition, the CGP content could be increased to 36% of the cell dry weight under optimizing growth conditions in C. glutamicum ATCC13032. All known alternative major amino acids for CGP synthesis (lysine, ornithine, citrulline, and glutamic acid) could be incorporated into CGP in C. glutamicum.

Overexpression of cphA gene from Nostoc flagelliforme improves the drought tolerance of Arabidopsis thaliana

Nostoc flagelliforme is a terrestrial cyanobacterium distributed in arid or semiarid steppes in western and northwestern China. Cyanophycin is synthesized by cyanophycin synthetase (CphA), which serves as a storage polymer for nitrogen, carbon, and energy in N. flagelliforme. In the present study, cphA (GenBank Accession No.: KX469292), an open reading frame (ORF) of 2706 bp encoding a protein of 901 amino acid residues, was cloned. We found that cyanophycin synthesis was promoted by the expression of cphA in E. coli. The function of the cphA gene in Arabidopsis thaliana under drought stress was investigated, and stable integration and expression of transgenes in transgenic lines were confirmed by PCR, Western blot, Southern blot, and real-time PCR analyses. The transcript abundance of cphA was markedly increased in transgenic A. thaliana compared with that in the untransformed plants. A subcellular localization assay confirmed that cphA protein was targeted to the cytoplasm. The survival rate was also significantly higher in cphA overexpression lines than that in untransformed plants under drought stress, which verified by drought tolerance assay of the transgenic plants. These findings highlight the potential importance of cphA in the drought tolerance of N. flagelliforme.

Re-evaluation of cyanophycin synthesis in Corynebacterium glutamicum and incorporation of glutamic acid and lysine into the polymer.

Corynebacterium glutamicum was only examined in the early 2000s as a possible microorganism for the production of the polyamide cyanophycin (multi-L-arginyl-poly-[L-aspartic acid], CGP). CGP is a potential precursor for the synthesis of polyaspartic acid and CGP-derived dipeptides which may be of use in peptide-based clinical diets, as dietary supplements, or in livestock feeds. In the past, C. glutamicum was disregarded for CGP production due to low CGP contents and difficulties in isolating the polymer. However, considering recent advances in CGP research, the capabilities of this organism were revisited. In this study, several cyanophycin synthetases (CphA) as well as expression vectors and cultivation conditions were evaluated. The ability of C. glutamicum to incorporate additional amino acids such as lysine and glutamic acid was also examined. The strains C. glutamicum pVWEx1::cphAΔ1 and C. glutamicum pVWEx1::cphABP1 accumulated up to 14% of their dry weight CGP, including soluble CGP containing more than 40mol% of the alternative side-chain amino acid lysine. The soluble, lysine-rich form of the polymer was not detected in C. glutamicum in previous studies. Additionally, an incorporation of up to 6mol% of glutamic acid into the backbone of CGP synthesized by C. glutamicum pVWEx1::cphADh was detected. The strain accumulated up to 17% of its dry weight in soluble CGP. Although glutamic acid had previously been found to replace arginine in the side chain, this is the first time that glutamic acid was found to substitute aspartic acid in the backbone.

Optimization of conditions and partial characterization of cyanophycin synthetase from a thermophilic cyanobacterium Chlorogloeopsis fritschii

Abstract Cyanophycin granule polypeptide (CGP), a polymer of aspartic acid and arginine, synthesized by cyanophycin synthetase can be converted to polyaspartate which has many industrial applications. Cyanophycin and cyanophycin synthetase from a thermophilic cyanobacterium Chlorogloeopsis fritschii have been studied expecting enzyme from this organism to be thermostable. The organism exhibited best growth in Chu-10 medium at 45 °C. Maximum amount of cyanophycin was observed on 21 d when organism entered into stationary phase. Optimum conditions for cyanophycin synthetase were 30–40 °C and pH 8.0–9.5. The enzyme showed high specificity to aspartic acid and arginine but synthesized polyaspartate when arginine was omitted from the assay mixture. The enzyme activity doubled when Zn2+ were used as cofactor in place of Mg2+. The enzyme exhibited good thermal stability as it showed 66% activity when treated with 45 °C for one hour. Since cyanophycin synthetase formed an industrially valuable molecule polyaspartic acid and showed thermal stability, it has potential applications which needs to be further investigated.

Ammonium recycling supports toxic Planktothrix blooms in Sandusky Bay, Lake Erie: Evidence from stable isotope and metatranscriptome data

Sandusky Bay, Lake Erie, receives high nutrient loadings (nitrogen and phosphorus) from the Sandusky River, which drains an agricultural watershed. Eutrophication and cyanobacterial harmful algal blooms (cyanoHABs) persist throughout summer. Planktothrix agardhii is the dominant bloom-forming species and the main producer of microcystins in Sandusky Bay. Non-N2 fixing cyanobacteria, such as Planktothrix and Microcystis, thrive on chemically reduced forms of nitrogen, such as ammonium (NH4+) and urea. Ammonium regeneration and potential uptake rates and total microbial community demand for NH4+ were quantified in Sandusky Bay. Potential NH4+ uptake rates in the light increased from June to August at all stations. Dark uptake rates also increased seasonally and, by the end of August, were on par with light uptake rates. Regeneration rates followed a similar pattern and were significantly higher in August than June. Ammonium uptake kinetics during a Planktothrix-dominated bloom in Sandusky Bay and a Microcystis-dominated bloom in Maumee Bay were also compared. The highest half saturation constant (Km) in Sandusky Bay was measured in June and decreased throughout the season. In contrast, Km values in Maumee Bay were lowest at the beginning of summer and increased in October. A significant increase in Vmax in Sandusky Bay was observed between July and the end of August, reflective of intense competition for depleted NH4+. Metatranscriptome results from Sandusky Bay show a shift from cyanophycin synthetase (luxury NH4+ uptake; cphA1) expression in early summer to cyanophycinase (intracellular N mobilization; cphB/cphA2) expression in August, supporting the interpretation that the microbial community is nitrogen-starved in late summer. Combined, our results show that, in late summer, when nitrogen concentrations are low, cyanoHABs in Sandusky Bay rely on regenerated NH4+ to support growth and toxin production. Increased dark NH4+ uptake late in summer suggests an important heterotrophic contribution to NH4+ depletion in the phycosphere. Kinetic experiments in the two bays suggest a competitive advantage for Planktothrix over Microcystis in Sandusky Bay due to its higher affinity for NH4+ at low concentrations.

Reprint of “Fast and sensitive in vivo studies under controlled environmental conditions to substitute long-term field trials with genetically modified plants”

We introduce an easy, fast and effective method to analyze the influence of genetically modified (GM) plants on soil and model organisms in the laboratory to substitute laborious and time consuming field trials. For the studies described here we focused on two GM plants of the so-called 3rd generation: GM plants producing pharmaceuticals (PMP) and plant made industrials (PMI). Cyanophycin synthetase (cphA) was chosen as model for PMI and Choleratoxin B (CTB) as model for PMP. The model genes are expressed in transgenic roots of composite Vicia hirsuta plants grown in petri dishes for semi-sterile growth or small containers filled with non-sterile soil. No significant influence of the model gene expression on root induction, growth, biomass, interaction with symbionts such as rhizobia (number, size and functionality of nodules, selection of nodulating strains) or arbuscular mycorrhizal fungi could be detected.In vitro, but not in situ under field conditions, structural diversity of the bulk soil microbial community between transgenic and non-transgenic cultivars was determined by PLFA pattern-derived ratios of bacteria: fungi and of gram+: gram− bacteria. Significant differences in PLFA ratios were associated with dissimilarities in the quantity and molecular composition of rhizodeposits as revealed by Py-FIMS analyses.Contrary to field trials, where small effects based on the transgene expression might be hidden by the immense influence of various environmental factors, our in vitro system can detect even minor effects and correlates them to transgene expression with less space, time and labour.

Fast and sensitive in vivo studies under controlled environmental conditions to substitute long-term field trials with genetically modified plants

We introduce an easy, fast and effective method to analyze the influence of genetically modified (GM) plants on soil and model organisms in the laboratory to substitute laborious and time consuming field trials. For the studies described here we focused on two GM plants of the so-called 3rd generation: GM plants producing pharmaceuticals (PMP) and plant made industrials (PMI). Cyanophycin synthetase (cphA) was chosen as model for PMI and Choleratoxin B (CTB) as model for PMP. The model genes are expressed in transgenic roots of composite Vicia hirsuta plants grown in petri dishes for semi-sterile growth or small containers filled with non-sterile soil. No significant influence of the model gene expression on root induction, growth, biomass, interaction with symbionts such as rhizobia (number, size and functionality of nodules, selection of nodulating strains) or arbuscular mycorrhizal fungi could be detected.In vitro, but not in situ under field conditions, structural diversity of the bulk soil microbial community between transgenic and non-transgenic cultivars was determined by PLFA pattern-derived ratios of bacteria: fungi and of gram+: gram− bacteria. Significant differences in PLFA ratios were associated with dissimilarities in the quantity and molecular composition of rhizodeposits as revealed by Py-FIMS analyses.Contrary to field trials, where small effects based on the transgene expression might be hidden by the immense influence of various environmental factors, our in vitro system can detect even minor effects and correlates them to transgene expression with less space, time and labour.

Analysis of the Spore Membrane Proteome in Clostridium perfringens Implicates Cyanophycin in Spore Assembly.

Heat-resistant endospore formation plays an important role in Clostridium perfringens-associated foodborne illnesses. The spores allow the bacterium to survive heating during normal cooking processes, followed by germination and outgrowth of the bacterium in contaminated foods. To identify proteins associated with germination and other spore functions, a comparative spore membrane proteome analysis of dormant and germinated spores of C. perfringens strain SM101 was performed by using gel-based protein separation and liquid chromatography coupled with matrix-assisted laser desorption ionization-tandem time of flight (MALDI-TOF/TOF) mass spectrometry. A total of 494 proteins were identified, and 117 of them were predicted to be integral membrane or membrane-associated proteins. Among these membrane proteins, 16 and 26 were detected only in dormant and germinated spores, respectively. One protein that was detected only in germinated spore membranes was the enzyme cyanophycinase, a protease that cleaves the polymer cyanophycin, which is composed of l-arginine-poly(l-aspartic acid), to β-Asp-Arg. Genes encoding cyanophycinase and cyanophycin synthetase have been observed in many species of Clostridium, but their role has not been defined. To determine the function of cyanophycin in C. perfringens, a mutation was introduced into the cphA gene, encoding cyanophycin synthetase. In comparison to parent strain SM101, the spores of the mutant strain retained wild-type levels of heat resistance, but fewer spores were made, and they were smaller, suggesting that cyanophycin synthesis plays a role in spore assembly. Although cyanophycin could not be extracted from sporulating C. perfringens cells, an Escherichia coli strain expressing the cphA gene made copious amounts of cyanophycin, confirming that cphA encodes a cyanophycin synthetase. Clostridium perfringens is a common cause of food poisoning, and germination of spores after cooking is thought to play a significant role in the disease. How C. perfringens controls the germination process is still not completely understood. We characterized the proteome of the membranes from dormant and germinated spores and discovered that large-scale changes occur after germination is initiated. One of the proteins that was detected after germination was the enzyme cyanophycinase, which degrades the storage compound cyanophycin, which is found in cyanobacteria and other prokaryotes. A cyanophycin synthetase mutant was constructed and found to make spores with altered morphology but normal heat resistance, suggesting that cyanophycin plays a different role in C. perfringens than it does in cyanobacteria.

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 β-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.

Effect of Cyanophycin Metabolism in Recombinant Sinorhizobium (Ensifer) meliloti 1021 on the Symbiosis with Alfalfa (Medicago sativa)

The aim of this study was to investigate the effect of the synthesis and degradation of the nitrogen-rich polymer cyanophycin in rhizobia on symbiotic nitrogen fixation and crop yield in legumes. For this, cyanophycin synthetase from Anabaena sp. PCC7120 was expressed in the bacteroids of the symbiont S. meliloti 1021 either alone or together with an intracellular cyanophycinase from the same bacterium, either in the wild type or in a polyhydroxybutyratenegative (PHB-) mutant, and the effect on the growth of alfalfa host plants was studied. All strains induced the formation of nitrogen-fixing nodules in the host, but clear differences in various parameters were noticeable. Alfalfa plants infected with the wild type expressing only cyanophycin synthetase showed significantly lower shoot nitrogen contents and higher nitrogen fixation rates than plants inoculated with wild type, but the wild type phenotype was exceeded in S. meliloti 1021 expressing cyanophycinase in addition to cyanophycin synthetase. Growth of plants infected with the PHB- mutant expressing only cyanophycin synthetase was severely impaired in comparison to growth of plants infected with the PHB- mutant expressing both or neither of the two proteins. Transmission electron micrographs of sections of nodules induced by wild type S. meliloti 1021 producing cyanophycin synthetase and cyanophycinase showed that the rough endoplasmic reticulum and Golgi membranes were extended further in comparison to the nodules induced by the wild type, the PHB- mutant, or all other recombinant S. meliloti strains, indicating higher metabolic activities in these nodules.