Abstract
Cyanophycin (multi-L-arginyl-poly-L-aspartic acid) is a biopolymer of industrial interest, which naturally occurs in most cyanobacterial species and in a few heterotrophic bacteria. It is a non-ribosomally synthesized polyamide consisting of L-Arginine and L-Aspartate and serves as a biological nitrogen/carbon storage compound.
This chapter provides an overview about the natural occurrence of cyanophycin in cyanobacteria. Herein, we focus on the physiological function of cyanophycin in filamentous and unicellular cyanobacteria. Information about the enzymes involved in cyanophycin metabolism and their regulations are also provided. Due to its unique properties, cyanophycin could be used in various industrial applications. This chapter also summarizes the current state of biotechnological cyanophycin production.
Access provided by Autonomous University of Puebla. Download chapter PDF
Similar content being viewed by others
1 Introduction
Cyanobacteria are Gram-negative prokaryotes that perform oxygenic photosynthesis and fix carbon dioxide through the Calvin-Benson Cycle to produce reduced carbohydrates (Rippka et al. 1979). For the synthesis of cellular building blocks, required for cell growth, Cyanobacteria assimilate various inorganic and organic nitrogen sources, such as nitrate, ammonium, urea or a few other organic nitrogen compounds (e.g., amino acids, cyanate). The preferred nitrogen source is ammonium that is converted to glutamate via the coupled reactions of glutamine synthetase and glutamine-oxoglutarate aminotransferase (GS/GOGAT pathway) (Flores and Herrero pp.; Luque and Forchhammer 2008). According to their morphology, cyanobacteria are subdivided into five divisions (Rippka et al. 1979). Unicellular forms belong to the divisions I (order Chroococcales) and II (order Pleurocapsales). Cyanobacteria of division III (order Oscillatoriales) and IV (order Nostocales) form filaments of vegetative cells, with the latter being able to differentiate specialized cells. Division V cyanobacteria (order Stigenomaticales) are similar to division IV, but the filaments are branched. The capability to fix dinitrogen (N2), called diazotrophy, is found in both unicellular and filamentous forms of cyanobacteria (Rippka et al. 1979; Fredriksson and Bergman 1997; Bandyopadhyay et al. 2013). In cyanobacteria of the order Nostocales and Stigenomaticales, nitrogen fixation takes place in specialized cells termed heterocysts (Herrero et al. 2016).
Cyanobacteria are widespread and well adapted to changing environmental conditions in terms of light conditions and nutrient supply. They colonize nearly all light-exposed habitats ranging from aquartical, including fresh- and seawater to terrestrial habitats (Whitton 2012). Some species can even tolerate extreme environments like hot springs, deserts or the arctic tundra (Miller and Castenholz 2000; Whitton 2012). Cyanobacteria are very interesting targets for biotechnological research because of their ability to synthesize reduced carbon compounds and various biopolymers, only by using water, carbon dioxide and light as an energy source. This chapter will focus on the biopolymer cyanophycin, a major nitrogen storage compound produced by many cyanobacteria.
2 Cyanophycin: A Nitrogen-Rich Storage Compound
Cyanophycin is a branched non-ribosomally synthesized polypeptide first found in cyanobacteria (Borzi 1887). Due to its composition of aspartate and arginine, cyanophycin has a high nitrogen to carbon ratio of 2:1, making it an excellent nitrogen storage substance (Simon and Weathers 1976). It serves as a dynamic nitrogen reservoir in most diazotrophic and non-diazotrophic cyanobacteria as well as in some heterotrophic bacteria (Ziegler et al. 2002; Füser and Steinbüchel 2007). Cyanophycin accumulates in the form of opaque and light-scattering cytoplasmic granules and was therefore mentioned cyanophycin granule peptide (CGP). Electron microscopic studies described CGP granules as electron dense, highly structured and membrane-less inclusions (Lang et al. 1972; Allen and Weathers 1980).
The polymer is synthesized by a single enzyme termed cyanophycin synthetase (CphA) (see below for details). Genes homologous to cyanobacterial cphA are found in a wide range of prokaryotes (Krehenbrink et al. 2002), indicating that cyanophycin has a broader biologicals significance, which is, however, mostly unexplored to date. In general, accumulation of the polymer increased under unbalanced growth conditions, e.g., in stationary growth phase or stress condition (high light, high CO2 or nutrient stress) but only when nitrogen is available (Allen et al. 1980; Allen and Weathers 1980; Carr 1988; Herrero and Burnat 2014; Watzer and Forchhammer 2018b). These findings stand for a putative role of the polymer as a temporary nitrogen reserve molecule (Li et al. 2001).
CGP has been noticed for potential biotechnological use. Biodegradable polyamino acids like CGP could substitute synthetical polymers with similar properties in the fields of medical, food, feed and pharmaceutical industry (Frommeyer et al. 2016). Further, CGP can serve as a natural source of amino acids such as arginine and aspartate, and the poly-aspartate backbone of this polymer could also be used as a biodegradable substitute for polyacrylates (Obst and Steinbüchel 2004; Sallam et al. 2009a, b).
3 Structure of Cyanophycin
The nitrogen-rich polymer CGP consists of two amino acids, aspartate and arginine in equimolar amounts. The aspartic acid residues were linked by peptide bounds forming a poly-L-aspartic acid backbone. Arginine is linked by iso-peptide bond to the backbone, forming the branched polymer also called [multi-(L-arginyl-poly-Laspartic acid)] (Simon and Weathers 1976). One building unit of the polymer consists of the dipeptide β-Asp-Arg (Richter et al. 1999). Circular dichroism (CD) spectroscopy data suggest that a substantial fraction of CGP has β-pleated sheet structure (Simon et al. 1980). The individual CGP chains have no fixed length but show polydisperse molecular masses ranging from 25 to 100 kDa in cyanobacteria. This is in contrast to the native CGP producing heterotrophic bacteria Acinetobacter sp. ADP1, which synthesizes CGP with a lower molecular weight ranging from 21 to 28 kDa (Elbahloul et al. 2005). Cyanophycins, produced by genetically modified microorganisms (GMOs) harboring cyanobacterial CGP synthesizing enzymes, have molecular masses in the range of 25–45 kDa (Frey et al. 2002; Steinle et al. 2009). Possible explanations for these differences could be that different ratios of CGP synthase to precursor molecules exist in GMOs or that CGP synthesis in a cyanobacterial background involves additional factors contributing the polymer length. These additional factors should also be absent in Acinetobacter sp. ADP1 (Watzer and Forchhammer 2018b). Cyanobacterial CGP is exclusively composed of aspartate and arginine. By contrast, CGP of recombinant microorganisms, further amino acids like lysin, citrulline and ornithine, have been detected (Steinle et al. 2009; Ziegler et al. 1998) (see below).
CGP granules were isolated for the first time in 1971 from Anabaena cylindrical by differential centrifugation (Simon 1971). Along with this study, CGP has shown its unique solubility behavior. CGP is insoluble under physiological conditions but soluble in either acidic, basic or highly ionic solutions. CGP can be solubilized by SDS but not by non-ionic detergents such as Triton X-100 (Wiefel and Steinbüchel 2014; Lang et al. 1972). Routinely, CGP is extracted by acidic treatment and subsequent precipitation at neutral pH (Frey et al. 2002).
4 Physiological Function of Cyanophycin in the Context of Nitrogen Fixation
CGP is produced by many different cyanobacteria, both diazotrophic and non-diazotrophic strains of unicellular or filamentous nature (Fig. 1). In diazotrophic strains, the formation of CGP is directly linked to the nitrogen-fixation process, whereas in non-diazotrophic strains, the biological significance remained elusive until recently (see below).
In principle, nature has evolved two solutions to the problem that oxygenic photosynthesis and nitrogen fixation are incompatible processes due to the extremely high oxygen sensitivity of the nitrogenase enzyme. Therefore, the two processes cannot take place at the same time at the same place. To solve this issue, the two processes may be separated on a temporal basis or may be confined to different compartments. Simple filamentous (Sect. 3) and unicellular nitrogen-fixing cyanobacteria temporarily separate nitrogen fixation and photosynthesis on a diurnal basis, as shown in the cyanobacterium Cyanothece sp. ATCC 51142; the cells perform photosynthesis during the day where they accumulate fixed carbon in the form of glycogen. This storage material is catabolized during the night to support nitrogen fixation with energy and carbon skeletons (Sherman et al. 1998). The nitrogen fixation products are immediately converted to CGP, which accumulates during the night. In the subsequent day period, when photosynthesis is active again, CGP is degraded to mobilize the fixed nitrogen for anabolic reactions to support cell growth (Sherman et al. 1998). A similar diurnal CPG accumulation during dark periods was also reported in the filamentous cyanobacterium Trichodesmium sp., which is the dominant diazotrophic cyanobacterium in tropical and subtropical seas and therefore of global importance in N and C cycling (Finzi-Hart et al. 2009).
Multicellular cyanobacteria of the order Nostocales are characterized by a spatial separation of photosynthesis and nitrogen fixation through a cell differentiation process which results in the formation of specialized cells for nitrogen fixation, the heterocysts.
4.1 Cyanophycin in Heterocyst-Forming Cyanobacteria
Heterocystous cyanobacteria comprise the orders Nostocales and Stigenomaticales. They are filamentous and true multicellular organisms in which two differently specialized cell types have a division of labor. Both processes, photosynthesis and nitrogen fixation, take place in different cells. Nitrogen fixation occurs only in the heterocysts, which are terminally differentiated cells specialized for nitrogen fixation (Kumar et al. 2010; Maldener and Muro-Pastor 2010). On the other hand, oxygenic photosynthesis takes place only in the vegetative cells. When no combined nitrogen sources are available, the multicellular organisms differentiate between 5 and 10% of cells into heterocysts.
The heterocysts provide a microoxic compartment for the activity of nitrogenase (Adams and Carr 1981). To achieve this functional state, numerous morphological and metabolic changes take place during the transition of vegetative cells into heterocysts, which are based on cell-specific gene expression occurring in the differentiated heterocysts (Wolk et al. 1994; Golden and Yoon 1998; Maldener et al. 2014; Flores et al. 2019). Concerning the photosynthetic apparatus, heterocysts show remarkably differences to the vegetative cells (Magnuson and Cardona 2016): The oxygen-producing photosystem II is absent and phycobilisomes are largely degraded. Thylakoid membranes are re-organized and support cyclic electron transport via the photosystem I, to provide ATP (Wolk et al. 1994). In addition, respiration takes place in the thylakoid-derived honeycomb-membranes, where the molecular oxygen is reduced to water (Wolk 1996). In the microoxic environment of heterocysts, instead of CO2 fixation by RubisCO, atmospheric N2 is fixed by the key enzyme for nitrogen fixation, the nitrogenase. The nitrogenase encoded by nifHDK genes is cell specific and exclusively active in mature heterocysts (Golden et al. 1985; Fay 1992). The enzyme is very oxygen sensitive (Gallon 1981) and is irreversibly inactivated by oxygen (Hill et al. 1981; Golden et al. 1985; Fay 1992). It reduces molecular nitrogen to ammonium using reduction equivalents and ATP (Wolk et al. 1994; Kumar et al. 2010). The high energy demand for nitrogen fixation is partially provided from products of oxygenic photosynthesis performed in the neighboring vegetative cells of the filament. The division of labor between heterocysts and vegetative cells requires an efficient exchange of metabolites and signals along the filament (Wolk 1968; Wolk et al. 1974). Molecular diffusion of small molecules from cytoplasm to cytoplasm takes place through special cell-cell connections, so-called “septal junctions” (Maldener and Muro-Pastor 2010; Flores et al. 2016). The structure of the septal junctions has been resolved recently in more detail in Anabaena sp. PCC 7120 (Weiss et al. 2019). Each septal junction complex comprises a channel that spans the gap through the periplasmic space in the septal region and traverses the septal peptidoglycan though approx. 20 nm nanopores to connect two adjacent cells. On the cytoplasmic site of each entrance point, the channel is covered by a cap and plug structure that enables closure of the septal junctions (Weiss et al. 2019).
Mature heterocysts are characterized by a specialized cell wall composed of polysaccharide and glycolipid layers, which minimizes the diffusion of molecular oxygen into the specialized cell (Walsby 2007; Nicolaisen et al. 2009). A distinct morphological feature of heterocysts is so-called “polar plaques” or “polar nodes,” which are specific structures containing CGP (Sherman et al. 2000) closely located to the neck and the adjacent vegetative cell. The polar nodes are easily recognized by light microscopy, and as such, they are characteristic morphological signatures of heterocysts (Ziegler et al. 2001) (Fig. 1). It was assumed that CGP located at these heterocystous structures is a key intermediate in the storage of fixed nitrogen products in heterocysts and acts as a dynamic nitrogen reservoir (Carr 1988; Sherman et al. 2000). CGP in the polar nodes could serve as a sink for fixed nitrogen in the heterocyst to avoid feedback inhibition of nitrogen assimilatory reactions from soluble products of nitrogen fixation (Burnat et al. 2014; Forchhammer and Watzer 2016).
Vegetative cells support the metabolism of heterocysts by supplying them with glutamate and reduced carbohydrates, e.g., alanine and the chemically inert disaccharide sucrose (Jüttner 1983; Curatti et al. 2002; Vargas et al. 2011) as a source for energy, reductant power, and the required carbon skeletons for assimilation of fixed nitrogen. Invertases degrade the disaccharides into glucose and fructose in heterocysts (Lopez-Igual et al. 2010; Vargas et al. 2011), which can be further catabolized via the oxidative pentose phosphate cycle to generate reducing power for nitrogen fixation and respiration. Ammonium produced by nitrogenase activity is immediately assimilated by glutamine synthetase, whereas the GOGAT reaction takes place in adjacent vegetative cells (Martin-Figueroa et al. 2000), which requires an efficient shuffling of glutamate and glutamine between heterocysts and vegetative cells.
Vegetative cells are supplied by adjacent heterocysts with reduced nitrogen such as glutamine and the dipeptide β-aspartyl-arginine (Thomas et al. 1977; Burnat et al. 2014). Therefore, CGP in the outer region of the polar nodes is cleaved by the action of cyanophycinase into β-aspartyl-arginine dipeptides. CGP catabolic enzymes are present at significantly higher levels in vegetative cells than in heterocysts, indicating a sophisticated spatial organization of nitrogen metabolism (Burnat and Flores 2014; Burnat et al. 2014) (Fig. 2).
Surprisingly, under standard laboratory conditions, this mode of metabolic organization appears to be dispensable, since mutants of Anabaena sp. PCC 7120 or Anabaena variabilis, lacking the CGP synthetic genes, were little affected in diazotrophic growth under usual cultivation conditions (Ziegler et al. 2001). In this case, arginine and aspartate might be transferred directly to the vegetative cells, without an intermediate storage in CGP. However, mutants lacking the CGP synthetic genes were affected in diazotrophic growth under stressful high light conditions (Ziegler et al. 2001; Klemke et al. 2016), indicating that cyanopyhcin plays a role in coping with metabolic stress.
Moreover, diazotrophic growth is significantly decreased in strains, which are unable to degrade CGP (Burnat and Flores 2014; Burnat et al. 2014). These results identified β-aspartyl-arginine dipeptides as a nitrogen vehicle in heterocyst-forming cyanobacteria, next to glutamine, arginine, or aspartate alone. A benefit of β-aspartyl-arginine dipeptides as a nitrogen transport substance is avoiding the release of free arginine and aspartate back in the heterocyst. This indicates that CGP metabolism has evolved in heterocyst-forming cyanobacteria to increase the efficiency of nitrogen fixation (Watzer and Forchhammer 2018b; Flores et al. 2019a; Burnat et al. 2014, 2019).
Several of the heterocyst-forming cyanobacteria can differentiate another type of specialized cells, the akinetes. These are resting cells able to survive long periods of unfavorable conditions. During akinete development, the cells transiently accumulate CGP in addition to other storage compounds such as glycogen or lipid droplets (Perez et al. 2016) (Fig. 1). In mature akinetes, CGP disappears gradually, and when the akinetes germinate, CGP reappears transiently (Perez et al. 2018). Apparently, synthesis of CGP seems inessential, since Anabaena variabilis akinetes lacking CGP granules are not impaired in germination. This behavior agrees with early observations suggesting that CGP is not the direct nitrogen source for protein biosynthesis in germinating akinetes (Sutherland et al. 1985).
4.2 Cyanophycin in Non-diazotrophic Cyanobacteria
In non-diazotrophic cyanobacteria, CGP can only be detected under unbalanced growth conditions. In exponentially growing cells, its amount is usually less than 1% of the cell dry mass. However, when growth is retarded or arrested due to unfavorable conditions under nitrogen supply, (antibiotic treatment; sulfate, phosphate or potassium starvation), the cells may start to build up CGP, which can amount up to 18% of the cell dry mass (Allen et al. 1980; Watzer et al. 2015; Simon 1973). When Synechocystis cells recover from prolonged nitrogen starvation, by adding nitrate to the starved cells, they transiently accumulate CGP (Klotz et al. 2016; Allen and Hutchison 1980; Watzer and Forchhammer 2018a). This raised the question regarding the biological significance of transient CGP accumulation. This issue was recently solved by Watzer and Forchhammer (Watzer and Forchhammer 2018a). The transient accumulation of CGP becomes beneficial to the cells under conditions of fluctuating or limiting nitrogen supply. Whereas wild-type Synechocystis cells are able to fully recover from prolonged nitrogen chlorosis when they are provided with pulses of nitrate, recovery is severely delayed in a mutant deficient in CGP synthesis (cphA mutant, see below). This phenotype becomes even more prominent when cells are cultivated in 12 h day/night cycles. From these findings, it can be concluded that under natural conditions, characterized by fluctuating nutrient and light supply, cyanophycin synthesis allows intracellular sequestration of nitrogen assimilation products, so that under periods of nitrogen shortage, CGP degradation provides anabolic precursors to continue growth. Even under constant illumination and a constant but limited nitrogen supply, the wild type has a growth advantage over the cphA mutant. This suggests that CGP synthesis optimizes the nitrogen assimilation process.
The recent discovery of the ornithine–ammonia cycle (OAC) in cyanobacteria emphasizes the role of CGP as dynamic nitrogen storage (Zhang et al. 2018). The pathway starts with carbamoyl phosphate synthesis and ends with the conversion of arginine to ornithine, CO2, and ammonia by a novel arginine dihydrolase (ArgZ). Disruption of the OAC-pathway by mutating argZ leads to impaired cell growth under fluctuating nitrogen supply. This demonstrates that the OAC-pathway allows rapid remobilization of nitrogen reserves under temporal starvation conditions. Since the cphA-deficient mutant showed similar growth defects under fluctuating nitrogen supply, it can be concluded that nitrogen, which is stored in CGP, can be remobilized by the OAC-pathway (Zhang et al. 2018).
5 Enzymes of Cyanophycin Metabolism
Enzymes of the CGP metabolism occur in a wide of range of phototrophic and chemolithoautotrophic bacteria (Füser and Steinbüchel 2007). In general, CGP is synthesized from aspartate and arginine by the cyanophycin synthetase (CphA1) in an ATP-depending elongation reaction. Intracellular CGP degradation is catalyzed by the cyanophycinase (CphB). The β-Asp-Arg dipeptides resulting from the cleavage of CGP are further hydrolyzed by isoaspartyl dipeptidase, releasing aspartate and arginine (Watzer and Forchhammer 2018b) (Fig. 3). The following paragraphs provide detailed information about the enzymes involved in CGP metabolism.
5.1 Cyanophycin Synthetase (CphA)
The non-ribosomal biosynthesis of cyanophycin takes place, as long as the cell has sufficient nitrogen and energy available by a single enzyme called cyanophycin synthetase (CphA) (Simon 1976). In the synthesis the amino acids aspartate and arginine condense in two steps with one ATP consumption per step (Simon 1976; Berg et al. 2000). Additionally, the reaction requires potassium ions, magnesium ions, cyanophycin primers (low-molecular-mass CGP), and a thiol reagent (such as β-mercaptoetanol or dithiothreitol) (Simon 1976). The requirement of low-molecular mass CGP in the reaction of CphA leads to the conclusion that CphA catalyzes the elongation of a CGP-filament but not its de novo synthesis.
By using synthetic primers, Berg et al. could show that a single building unit (β Asp-Arg) does not serve as an efficient primer for the CphA1 elongation reaction. CphA1 needs at least an Asp-Arg trimer (β Asp-Arg)3 as a primer for activity (Berg et al. 2000). Other peptides, like the peptidoglycan-pentapeptide or other cellular components, have been suggested to serve as an alternative priming substance, which allows CphA1 activity in recombinant bacteria without ability to produce native CGP primers (Hai et al. 2002; Obst and Steinbüchel 2006). These alternative priming substances are also present in yeasts like Saccharomyces cerevisiae or Pichia pastoris but absent in higher plants (Steinle et al. 2008, 2010; Nausch et al. 2016). The primer-independent CphA of Thermosynechococcus elongatus BP-1 allows CGP production in higher plants (Arai and Kino 2008). Challenges and peculiarities of CGP production in plants have been reviewed by Nausch et al. (2016).
Up to date, several CphA1 enzymes from different bacteria, including cyanobacteria and heterotrophic bacteria, have been characterized (Table 1) (Ziegler et al. 1998; Arai and Kino 2008; Hai et al. 2002; Füser and Steinbüchel 2007; Aboulmagd et al. 2001a; Elbahloul et al. 2005; Du et al. 2019). The molecular masses of the characterized CphA1 enzymes range between 90 and 130 kDa. CphA1 from Synechocystis sp. PCC6803 and Anabaena variabilis PCC 7937 forms most likely a homodimer, while the primer-independent CphA1 from Thermosynechoccus elongatus forms a homotetramer (Ziegler et al. 1998; Aboulmagd et al. 2001a; Arai and Kino 2008). The primary structure of cyanobacterial CphA1 can be divided in two regions; each region contains an active site and an ATP binding site (Ziegler et al. 1998; Berg et al. 2000). Experiments revealed that arginine is probably bound at the C-terminal and aspartate at the N-terminal active site (Berg 2003).
The putative mechanism of CphA1 has been suggested by Berg et al. by measuring the step-wise incorporation of amino acids to the C-terminus of a CGP primer. First, the carboxylic acid group at the C-terminus of the polyaspartate backbone becomes activated by phosphorylation. Subsequently, one aspartate is bound at the C-terminus by its amino group, forming a peptide bound. The intermediate (β-Asp-Arg)n—Asp is transferred to the second active site of the N-terminal region of CphA1. The β-carboxyl group of aspartate becomes phosphorylated and is then linked to the α-aminogroup of arginine, forming an isopeptide-bound (Berg et al. 2000).
For CphA1 of Synechocystis sp. PCC 6308, the apparent KM value in vitro for aspartate is 450 μM, for aginine 49 μM, for ATP 200 μM and for CGP as priming substance 35 μg/ml, indicating a higher affinity toward arginine compared to aspartate. During the in vitro reaction, CphA1 converts 1.3 ± 0.1 mol ATP per mol incorporated amino acid (Aboulmagd et al. 2001a).
Previous microscopical studies revealed different localizations of CphA depending on its activity in the non-diazortphic cyanobacterium Synechocystis sp. PCC 6803 (see below for more details) (Watzer and Forchhammer 2018a). Under balanced growth conditions, in which no CGP synthesis occurs, CphA is equally distributed in the cytoplasm. Immediately after the induction of CGP synthesis, CphA aggregates in foci that were randomly localized in the cell. These foci increase in size due to the cyanophycin synthesizing reaction of CphA and become visible as CGP granules. Doing this, CphA is exclusively attached on the granule surface. As CGP accumulates, the number of granules per cell continuously decreases (Watzer and Forchhammer 2018a). Electron micrographs could show that large CGP granule fuse when they collide, forming larger amorphous aggregates (Watzer et al. 2015). During CGP degradation, the localization of CphA changes from the granule surface back to the cytoplasm. This suggests that as CGP is degraded, CphA dissociates from the surface and converts to the inactive form, where it remains silent until CGP accumulation is triggered again.
In addition to the main enzyme for CGP synthesis CphA1, there is a second enzyme, CphA2 (Picossi et al. 2004). The gene for the enzyme CphA2 has been identified predominantly in diazotrophic cyanobacterial species that also possess the gene for CphA1 (Klemke et al. 2016). Therefore, in heterocystous cyanobacteria of Sect. 4, 71% and 82% of Sect. 5 contain a cphA2 gene. CphA2 is a truncated enzyme version of CphA1, showing only a high homology in amino acid sequence to the N-terminal part of CphA1, but not to the C terminal part. The function of CphA2 is the addition of β-Asp-Arg dipeptide units to the growing CGP chain using one molecule of ATP (Klemke et al. 2016). CphA2 was found directly attached to the polymer like CphA1 in vegetative cells and heterocysts. By enzymatic activities of CphA2 and the degradation enzyme cyanophycinase (see below), a continuous pool of the dipeptide β-Asp-Arg in the filament is provided. If needed, CGP can be polymerized directly from β-Asp-Arg. A cphA2 deletion mutant displays only a minor decrease in the overall CGP content. However, such a mutant showed defects under diazotrophic growth at high light conditions like a CphA1-deficient mutant. This observation implies that CGP hydrolysis and re-polymerization by CphA1 and CphA2 could run as a “futile cycle” in case of high photosynthetic activity (Fig. 3). Furthermore, CphA2 may be involved in regulating the cellular β-Asp-Arg pool and thereby restrict the degradation of the dipeptide by the isoaspartyl dipeptidase into the amino acids arginine and aspartate (Forchhammer and Watzer 2016).
5.2 Cyanophycinase (CphB)
CGP is resistant against hydrolytic cleavage by several arginases and proteases, probably due to its branched structure (Simon and Weathers 1976; Obst and Steinbüchel 2006). In 1999, Richter et al. reported a CGP hydrolyzing from the unicellular cyanobacterium Synechocystis sp. PCC 6803, called CphB (Richter et al. 1999). CphB is a 29.4 kDa, dimeric C-terminal exopeptidase, catalyzing the enzymatic cleavage of CGP to β-Asp-Arg dipeptides (Richter et al. 1999; Law et al. 2009). Based on sequence analysis, its sensitivity to serine protease inhibitors and site-directed mutagenesis, CphB can be classified as serine-type exopeptidase with the catalytic Ser at position 132 (Richter et al. 1999; Law et al. 2009). Structural modeling indicated that the substrate specificity of CphB for CGP is based on an extended conformation of the active site. The unique conformation of the active site requires β-linked aspartyl peptides for binding and catalysis, preventing CphB from non-specific cleavage of other polypeptides (Law et al. 2009; Watzer and Forchhammer 2018b).
In addition to the intracellular CphB, other versions of CGP hydrolyzing enzymes exist, catalyzing the extracellular cleavage of CGP. In 2002, Obst et al. isolated several Gram-negative, heterotrophic bacteria from different habitats, which were able to utilize CGP as a carbon, nitrogen, and energy source (Obst et al. 2002). One stain was identified as Pseudomonas anguillisptica strain BI. A novel CGPase was identified and called CphE in culture supernatant of this strain (Obst et al. 2002). CphE exhibits an amino acid sequence identity of 27–28% to cyanobacterial CphB. The primary degradation product of CGP digested with CphE are β-Asp-Arg dipeptides. Sensitivity to serine protease inhibitors has been reported, indicating that the catalytic mechanism of CphE is related to serein-type proteases (Obst et al. 2002). Today, extracellular CGPases have been reported in several bacteria ranging from Gram-positive to Gram-negative, from areobic to an anaerobic. This indicates that utilization of CGP as a carbon, nitrogen, and energy source seems to be a common principle in nature (Obst et al. 2002, 2004, 2005; Obst and Steinbüchel 2004; Sallam and Steinbüchel 2008; Watzer and Forchhammer 2018b).
5.3 Aspartyl-Arginine Dipeptidase
The last step of CGP catabolism is the cleavage of β-Asp-Arg dipeptides to monomeric amino acids. The ORF sll0422 from Synechocysis sp. PCC 6803 and all3922 from Anabaena sp. PCC 7120 are annotated on the basis of sequence similarity as “plant-type asparaginase” (PTA). In 2002, Hejazi et al. characterized several PTAs for substrate specificity, showing that sll0422 and all3922 are able to hydrolyze a wide range of isoaspartyl dipeptides including β-Asp-Arg.
The mature PTA of Synechocysis sp. PCC 6803 and Anabaena sp. PCC 7120 consists of two subunits, which are generated by autocleavage of the primary translation product (Hejazi et al. 2002). The native enzyme has a molecular mass of approximately 70 kDa, suggesting a subunit structure of α 2 β 2 (α derived from N-terminal part and β derived from the C-terminal part of the immature translation product).
A deletion mutant of gene all3922 (coding for PTA in Anabaena sp. strain 7120) accumulated CGP and β-Asp-Arg and was strongly impaired in diazotrophic growth (Burnat et al. 2014). Further results showed that PTA was present in vegetative cells more than in heterocysts, although β-Asp-Arg was found in substantial amounts in heterocysts. It can be assumed that the dipeptide produced by cyanophycin degradation in heterocysts should be transferred to vegetative cells.
Furthermore, a compartmentalization was also found for arginine catabolizing enzymes. The arginine decarboxylase pathway that produces sym-homospermidine is also more present in vegetative cells than in nitrogen-fixing heterocysts (Burnat and Flores 2014). Distortion of the arginine decarboxylase pathway in Anabaena sp. PCC 7120 through the deletion of agmatinase (speB) led to the accumulation of high amounts of CGP and to impairment of diazotrophic growth (Burnat and Flores 2014). The arginine catabolic pathway of Anabaena sp. PCC 7120 has recently been elucidated by Burnat et al. 2019. This pathway consists of two genes, the arginine-guanidine removing enzyme agrE (alr4995) and a proline oxidase putA (alr0540). The AgrE-PutA pathway catabolizes arginine to produce proline and glutamate, with concomitant release of ammonium. Both genes, agrE and putA, were found to be expressed at higher levels in vegetative cells than in heterocysts, implying that arginine is catabolized mainly in the vegetative cells (Burnat et al. 2019; Flores et al. 2019a).
All these results suggest that CGP is degraded in the heterocysts to the dipeptide β-Asp-Arg and the dipeptide could serve as a mobile nitrogen reserve for the vegetative cells in addition to other molecules such as glutamine (Burnat et al. 2014, 2019; Flores et al. 2019a).
6 Genetic Organization and Expression of cphA and cphB
Genes involved in CGP metabolism are usually organized in clusters (Füser and Steinbüchel 2007). In the genome of Synechocystis sp. PCC 6803 cphA and cphB are localized side by side but are expressed independently (Mitschke et al. 2011). A hypothetical protein with unknown function (orf slr2003) is located downstream of cphA and both are transcribed in a polysistronic unit (Mitschke et al. 2011).
Anabaena sp. PCC 7120 contains two clusters, cph1 and cph2, both containing cyanophycin biosynthetic genes with cphA and cphB (Picossi et al. 2004). The cph1 cluster contains cphB1 and cphA1, which are cotranscribed and form an operon. In addition, cphA1 can be expressed from two independent promotors, of which one is constitutive and the other regulated by the global transcription factor NtcA (Picossi et al. 2004). The chp1 operon was expressed under nitrate- and ammonia-supplemented growth, but the expression increased at diazotrophic growth conditions in heterocysts and vegetative cells. Cell-specific expression under diazotrophic conditions of the cph1 gene cluster showed a global expression of cphA1 and cphB1 in vegetative cells and heterocysts, but an increased expression level was observed in heterocysts (Picossi et al. 2004). A higher enzyme activity of CphA1 and CphB1 was also observed in heterocysts than in vegetative cells (Carr 1988), which suggests a role of cyanophycin as a dynamic reservoir for fixed nitrogen (Li et al. 2001).
In the cph2 cluster, cphA2 and cphB2 were found in opposite orientations and both were expressed monoistronically. Both genes were expressed under ammonia-, nitrate-, and N2-supplemented growth, but the expression was higher in the absence of ammonia. Generally, the expression of cph1 was higher compared to cph2 (Picossi et al. 2004).
In addition to cph1 and cph2 clusters, a third cluster containing putative cphA and cphB genes was found in Nostoc punctiforme PCC 73102 and Anabaena variabilis ATCC 29413 (Füser and Steinbüchel 2007).
7 Biotechnological Production of Cyanophycin
The natural compound cyanophycin has up to now no industrial application whereas chemical derivatives of CGP are of potential biotechnological interest. CGP can be converted via hydrolytic β-cleavage to poly(α-L-aspartic acid) (PAA) and free arginine. Arginine is a valuable product and PAA is biodegradable and has a high number of negatively charged carboxylic groups, making PAA a possible substituent for polyacrylates (Obst and Steinbüchel 2004; Sallam et al. 2009a, b; Khlystov et al. 2017). PAA can be employed as an anti-sealant or dispersing ingredient in many fields of applications, including washing detergents or suntan lotions. Furthermore, PAA has potential application areas as an additive in paper, paint, building, or oil industry (Sallam et al. 2009a, b; Obst and Steinbüchel 2004; Watzer and Forchhammer 2018b).
In addition to its use as polymer, CGP can also serve as a source for constituent dipeptides and amino acids in food, feed, and pharmaceutical industry. The amino acids arginine (semi-essential), aspartate (non-essential) and lysine (essential) derived from CGP have a broad spectrum of nutritional or therapeutic applications. Large-scale production of these amino acids, as mixtures or dipeptides, is established in the industry, with various commercial products already available on the market (Sallam and Steinbüchel 2010; Watzer and Forchhammer 2018b).
7.1 Cyanophycin Production Using Recombinant Production Hosts
Previous approaches to produce CGP mainly focused on heterotrophic bacteria, yeasts, or plants as production hosts. These recombinant production hosts were generated by transforming CGP synthetase genes, mostly from cyanobacterial origin. Therefore, biotechnologically established production strains like E. coli, Cupriavidus necator (formally known as Ralstonia eutropha), Pseudomonas putida, Corynebacterium glutamicum, or Saccharomyces cerevisiae were used for heterologous production of CGP (Aboulmagd et al. 2001b; Steinle et al. 2008; Frey et al. 2002; Watzer and Forchhammer 2018b; Du et al. 2019). In addition to production in microorganisms, production of CGP has also been attempted in several crop plants (reviewed by (Nausch et al. 2016).
In 1998, Ziegler et al. produced the first recombinant CGP by heterologous expression of cphA from Synechocystis sp. PCC 6803 in E. coli. CGP isolated from this recombinant producer strain showed some differences to those from cyanobacteria. In particular, a reduced polydispersity ranging from 20 to 40 kDa and an altered amino acid composition, containing low amounts of lysin next to arginine and aspartate, haven been reported (Ziegler et al. 1998). Based on this finding, several CGP producer strains have been generated in the following years to establish CGP for biotechnological industry.
E.coli DH1 harboring cphA from Synechocystis sp. PCC 6803 was used in the first large-scale CPG production study in a culture volume up to 500 liter, allowing CGP quantities in kilogram scale. During process optimization, the highest observed CGP amount was 24% (w/w) per cell dry mass (CDM). However, this optimal production rate required Terrific-Broth-Medium. In mineral salts medium, CGP production only occurred in the presence of additional casamino acids (Frey et al. 2002). An engineered CphA from Nostoc ellipsosprum could further increase the maximal CGP production up to 34.5% (w/w) of CDM, but also required complex growth media to yield such high amounts (Hai et al. 2006).
Cupriavidus necator and Pseudomonas putida are industry-established producer stains for several products and have therefore been considered as candidates for biotechnological CGP production (Aboulmagd et al. 2001b; Diniz et al. 2006). CPG synthase genes from Synechocystis sp. PCC 6308 or Anabaena sp. PCC 7120 have been used to enable CGP production in these species (Voss et al. 2004; Aboulmagd et al. 2001b). Metabolic engineering and process optimization studies showed that accumulation of CGP mainly depends on the origin of cphA gene, the accumulation of other storage compounds like polyhydroxyalkanoates (PHA), the availability of intermediates derived from the tricarboxylic acid cycle (TCA-cycle), and the addition of CGP precursor components like arginine to the medium (Voss et al. 2004; Diniz et al. 2006). Using this knowledge, it was possible to obtain CGP amounts up to 17.9% (w/w) per CDM at 30 liter scale using mineral salt medium (Diniz et al. 2006). CGP accumulation of Cupriavidus necator is also strongly affected by the cphA expression system. A stabilized multi-copy cphA expression system, using the 2-keto-3-deoxy-6-phospho-gluconate (KDPG) aldolase gene-dependent addiction system, allows cultivation without antibiotic selection. The multi-copy expression of cphA results in an increased CGP production between 26.9% and 40% (w/w) per CDM. The high amount of 40% (w/w) of CDM could be also observed in a 30- and 500-liter scale. Without the addition of amino acids to the medium, the amount of CGP was still between 26.9 and 27.7% (w/w) of CDM (Voss and Steinbüchel 2006).
The industry-established host Saccharomyces cerevisiae has also been considered for biotechnological CGP production (Steinle et al. 2008, 2009). By expressing cphA from Synechocystis sp. PCC 6803, it was possible to obtain CGP in amounts up to 6.9% (w/w) of CDM. In this case, two species of CGP could be observed: water-soluble and the typical water-insoluble CGP. Depending on the cultivation conditions, the ratio between water-soluble and insoluble CGP-species could be varied. Furthermore, the isolated polymer contained 2 mol% lysine, which can be increased up to 10 mol% by adding lysin to the medium (Steinle et al. 2008). During metabolic engineering studies, several arginine biosynthesis mutants of Saccharomyces cerevisiae have been characterized concerning their abilities to accumulate CGP. Next to arginine, aspartate, and lysine, further non-proteinogenic amino acids like citrulline and ornithine could be detected in isolated CGP derivates (Steinle et al. 2009). Depending on these results, customized CGP derivates with altered amino acid composition could also be produced using Pseudomonas putida ant the yeast Pichia pastoris as a production host (Wiefel et al. 2011; Frommeyer et al. 2016; Steinle et al. 2010). Customized CGP derivates are important sources for β-dipeptides, which can be used in several applications. The extracellular CGPase, CphE from Pseudomonas alcaligens, enables large-scale production of these customized β-dipeptides from CGP derivates (Sallam et al. 2009a, b; Sallam and Steinbüchel 2010).
The biotechnologically well-established bacterium Corynebacterium glutamicum has so far not been considered for CGP production due to low CGP quantities and difficulties in polymer extraction. Very recently, Wiefel et al. re-evaluated the capability of Corynebacterium glutamicum as a CGP producer strain, by using different expression vectors, cphA-genes, and cultivation conditions (Wiefel et al. 2019). It was possible to create strains which produce CGP up to 14% of the CDM, including water-soluble CGP containing more than 40 mol % of lysin as a side-chain. Additionally, a strain was generated which incorporated up to 6 mol % glutamic acid into the backbone as a substitution of aspartate. Interestingly, a previous study already reported a CGP-species from nitrogen-limited Synechocystis sp. PCC6308 which contained high amounts of glutamic acid (Merritt et al. 1994); however, the position in the polymer could not be resolved. The re-discovery of glutamic acid in the backbone of CGP in recombinant Corynebacterium glutamicum raises again the question of the biological significance of this peculiar CGP species in Cyanobacteria (Merritt et al. 1994; Wiefel et al. 2019).
7.2 Dependence of CGP Production from Arginine Biosynthesis
Generally, CGP accumulation is triggered by unfavorable conditions, such as entry into stationary phase of growth, limitation of macronutrients (with the exception of nitrogen starvation) or inhibition of translation by adding antibiotics like chloramphenicol (Allen et al. 1980; Simon 1973; Watzer and Forchhammer 2018b). All these conditions result in a reduced or arrested growth. In the exponential growth phase, amino acids like arginine and aspartate are mostly used for de novo protein biosynthesis. Under growth-limiting conditions, the protein biosynthesis is slowed down, resulting in an excess of free amino acids. This enrichment of monomeric amino acids allows CGP accumulation.
Acinetobacter calcoaceticus ADP1 accumulates only 3.5% (w/w) CGP of the cellular dry matter (CDM) when grown with ammonia as a nitrogen source and under phosphate starvation (Elbahloul et al. 2005). During process optimization it was shown that the CGP production of A. calcoaceticus ADP1 during growth with arginine as a sole carbon and nitrogen source increased drastically to 41.4% (w/w) of CDM. Surprisingly, a combined supply of arginine and aspartate had a much lower stimulating effect as arginine alone (Elbahloul et al. 2005). In the following metabolic engineering studies of A. calcoaceticus ADP1, several genes related to the arginine biosynthesis were modified to yield higher amounts of arginine. In consequence, significant higher amounts of CGP could be detected (Elbahloul and Steinbüchel 2006).
Another link between the arginine biosynthesis and CGP metabolism was observed during transposon mutagenesis studies in the filamentous cyanobacterium Nostoc ellipsosorum. Here, an arginine biosynthesis gene, argL, was interrupted by a transposon, leading to a partially impaired arginine biosynthesis. Without arginine supplementation, heterocyst failed to fix nitrogen, akinetes were unable to germinate and CGP granules did not appear. However, when arginine is provided, mutant could form CGP granules and was able to differentiate functional akinetes (Leganes et al. 1998).
These results point towards arginine as main bottleneck of CGP biosynthesis, while aspartate plays a minor role. High concentrations of arginine surpass the Km value of CphA, triggering CGP production. In agreement, the Km value for arginine is much lower compared to aspartate, indicating a higher sensitivity of CphA towards arginine in the unicellular cyanobacterium Synechocystis sp. PCC 6803 (Aboulmagd et al. 2001a). Whether CphA is subjected to additional activity control remains to be elucidated (Watzer and Forchhammer 2018a, b).
7.3 Overproduction of Cyanophycin by Genetic Engineering the PII Signaling System
Since arginine synthesis is a limiting factor for CGP accumulation, overproduction of CGP requires enhanced arginine synthesis. Arginine synthesis is strictly controlled by the nutritional status of the cyanobacteria, mainly through tight regulation of the committed step of ornithine synthesis, the N-acetylglutamate kinase (NAGK) reaction. The NAGK enzyme is feedback controlled by arginine levels, and in addition, NAGK activity and arginine-feedback inhibition are modulated by the PII signal transduction protein. The PII signaling protein responds to the effector molecules 2-oxoglutarate (which reports the C/N metabolic balance) and to the adenylnucleotides ATP or ADP, which compete for the same binding site. Thereby, the PII signaling protein integrates the energy status of the cells (ATP/ADP ratio) with the C/N metabolic balance, where high 2-OG levels indicates carbon-sufficient but nitrogen-limiting conditions (for review see (Forchhammer and Lüddecke 2016; Forcada-Nadal et al. 2018). PII in the ATP-complexed state binds to NAGK, thereby enhancing its catalytic activity and tuning down arginine-feedback inhibition. This activation by PII results in an increased metabolic flux into the arginine pathway (Maheswaran et al. 2004; Maheswaran et al. 2006; Watzer et al. 2015). A single amino acid replacement in the cyanobacterial PII signaling protein (Ile86 to Asp86) generated a PII variant (PII-I86N) that constitutively binds to NAGK in vitro and enhances its activity (Fokina et al. 2010). Replacing the wild-type allele for PII in Synechocystis by this variant resulted in a strain, which has constitutively high NAGK activity and ten-fold increased levels of arginine (BW86) (Watzer et al. 2015). Already under balanced growth conditions with nitrate as a nitrogen source, strain BW86 accumulated up to 15.6 ± 5.4% CGP relative to the cell dry mass (CDM), while CGP in the wild type was barely detectable. Phosphate or potassium starvation further increased the CGP content of strain BW86 up to 47.4 ± 2.3% or 57.3 ± 11.1% per CDM, respectively (Watzer et al. 2015). The cyanophycin, which is produced by strain BW86, showed a high polydispersity ranging from 25 to 100 kDa, similar to the polydispersity of wild-type cyanobacterial CGP (Simon 1971). This contrasted CGP from recombinant producer strains using heterologous expression systems such as heterotrophic bacteria, yeasts, or plants, whose recombinantly produced CGP shows polydispersity ranging from 25 to only 45 kDa (Frey et al. 2002; Steinle et al. 2008). The novel cyanobacterial producer stain for cyanophycin may provide a valuable resource for the synthesis of large quantities of natural long-chain cyanophycin with so-far unexplored properties.
References
Aboulmagd E, Sanio FBO, Steinbüchel A (2001a) Purification of Synechocystis sp strain PCC6308 cyanophycin synthetase and its characterization with respect to substrate and primer specificity. Appl Environ Microb 67(5):2176–2182. https://doi.org/10.1128/AEM.67.5.2176-2182.2001
Aboulmagd E, Voss I, Oppermann-Sanio FB, Steinbüchel A (2001b) Heterologous expression of cyanophycin synthetase and cyanophycin synthesis in the industrial relevant bacteria Corynebacterium glutamicum and Ralstonia eutropha and in Pseudomonas putida. Biomacromolecules 2(4):1338–1342. https://doi.org/10.1021/bm010075a
Adams DG, Carr NG (1981) The developmental biology of heterocyst and akinete formation in cyanobacteria. Crc Rev Microbiol 9(1):45–100. https://doi.org/10.3109/10408418109104486
Allen MM, Hutchison F (1980) Nitrogen limitation and recovery in the cyanobacterium Aphanocapsa sp. PCC 6308. Arch Microbiol 128(1):1–7. https://doi.org/10.1007/Bf00422297
Allen MM, Weathers PJ (1980) Structure and composition of cyanophycin granules in the cyanobacterium Aphanocapsa 6308. J Bacteriol 141(2):959–962
Allen MM, Hutchison F, Weathers PJ (1980) Cyanophycin granule polypeptide formation and degradation in the cyanobacterium Aphanocapsa 6308. J Bacteriol 141(2):687–693
Arai T, Kino K (2008) A cyanophycin synthetase from Thermosynechococcus elongatus BP-1 catalyzes primer-independent cyanophycin synthesis. Appl Microbiol Biotechnol 81(1):69–78. https://doi.org/10.1007/s00253-008-1623-y
Bandyopadhyay A, Elvitigala T, Liberton M, Pakrasi HB (2013) Variations in the rhythms of respiration and nitrogen fixation in members of the unicellular diazotrophic cyanobacterial genus Cyanothece. Plant Physiol 161(3):1334–1346. https://doi.org/10.1104/pp.112.208231
Berg H (2003) Untersuchungen zu Funktion und Struktur der Cyanophycin-Synthetase von Anabaena variabilis ATCC 29413. Dissertation Humboldt–Universität zu Berlin, Germany
Berg H, Ziegler K, Piotukh K, Baier K, Lockau W, Volkmer-Engert R (2000) Biosynthesis of the cyanobacterial reserve polymer multi-L-arginyl-poly-L-aspartic acid (cyanophycin) – Mechanism of the cyanophycin synthetase reaction studied with synthetic primers. Eur J Biochem 267(17):5561–5570. https://doi.org/10.1046/j.1432-1327.2000.01622.x
Borzi A (1887) Le communicazioni intracellulari delle Nostochinee. Malpighia 1:28–74
Burnat M, Flores E (2014) Inactivation of agmatinase expressed in vegetative cells alters arginine catabolism and prevents diazotrophic growth in the heterocyst-forming cyanobacterium Anabaena. Microbiology 3(5):777–792. https://doi.org/10.1002/mbo3.207
Burnat M, Herrero A, Flores E (2014) Compartmentalized cyanophycin metabolism in the diazotrophic filaments of a heterocystforming cyanobacterium. Proc Natl Acad Sci U S A 111(10):3823–3828. https://doi.org/10.1073/pnas.1318564111
Burnat M, Picossi S, Valladares A, Herrero A, Flores E (2019) Catabolic pathway of arginine in Anabaena involves a novel bifunctional enzyme that produces proline from arginine. Mol Microbiol 111(4):883–897. https://doi.org/10.1111/mmi.14203
Carr N (1988) Nitrogen reserves and dynamic reservoirs in cyanobacteria. In: Rogers LJ, Gallon JR (eds) Biochemistry of the algae and cyanobacteria. Annual proceedings of the phyto-chemical society of Europe. Clarendon Press, Oxford, UK, pp 13–21
Curatti L, Flores E, Salerno G (2002) Sucrose is involved in the diazotrophic metabolism of the heterocyst-forming cyanobacterium Anabaena sp. FEBS Lett 513(2–3):175–178. https://doi.org/10.1016/S0014-5793(02)02283-4
Diniz SC, Voss I, Steinbüchel A (2006) Optimization of cyanophycin production in recombinant strains of Pseudomonas putida and Ralstonia eutropha employing elementary mode analysis and statistical experimental design. Biotechnol Bioeng 93(4):698–717. https://doi.org/10.1002/bit.20760
Du J, Li L, Zhou S (2019) Microbial production of cyanophycin: from enzymes to biopolymers. Biotechnol Adv 37(7):107400. https://doi.org/10.1016/j.biotechadv.2019.05.006
Elbahloul Y, Steinbüchel A (2006) Engineering the genotype of Acinetobacter sp strain ADP1 to enhance biosynthesis of cyanophycin. Appl Environ Microbiol 72(2):1410–1419. https://doi.org/10.1128/Aem.72.2.1410-1419.2006
Elbahloul Y, Krehenbrink M, Reichelt R, Steinbüchel A (2005) Physiological conditions conducive to high cyanophycin content in biomass of Acinetobacter calcoaceticus strain ADP1. Appl Environ Microbiol 71(2):858–866. https://doi.org/10.1128/Aem.71.2.858-866.2005
Fay P (1992) Oxygen relations of nitrogen-fixation in cyanobacteria. Microbiol Rev 56(2):340–373
Finzi-Hart JA, Pett-Ridge J, Weber PK, Popa R, Fallon SJ, Gunderson T, Hutcheon ID, Nealson KH, Capone DG (2009) Fixation and fate of C and N in the cyanobacterium Trichodesmium using nanometer-scale secondary ion mass spectrometry. Proc Natl Acad Sci U S A 106(24):9931–9931. https://doi.org/10.1073/pnas.0904281106
Flores E, Herrero A, Forchhammer K, Maldener I (2016) Septal junctions in filamentous heterocyst-forming cyanobacteria. Trends Microbiol 24(2):79–82. https://doi.org/10.1016/j.tim.2015.11.011
Flores E, Picossi S, Valladares A, Herrero A (2019) Transcriptional regulation of development in heterocyst-forming cyanobacteria. Biochim Biophys Acta Gene Regul Mech 1862(7):673–684. https://doi.org/10.1016/j.bbagrm.2018.04.006
Flores E, Arevalo S, Burnat M (2019a) Cyanophycin and arginine metabolism in cyanobacteria. Algal Res 42:101577. https://doi.org/10.1016/j.algal.2019.101577
Fokina O, Chellamuthu VR, Zeth K, Forchhammer K (2010) A novel signal transduction protein PII variant from Synechococcus elongatus PCC 7942 indicates a two-step process for NAGK-PII complex formation. J Mol Biol 399(3):410–421. https://doi.org/10.1016/j.jmb.2010.04.018
Forcada-Nadal A, Llacer JL, Contreras A, Marco-Marin C, Rubio V (2018) The PII-NAGK-PipX-NtcA regulatory axis of cyanobacteria: a tale of changing partners, allosteric effectors and non-covalent interactions. Front Mol Biosci 5:91. https://doi.org/10.3389/fmolb.2018.00091
Forchhammer K, Lüddecke J (2016) Sensory properties of the PII signalling protein family. FEBS J 283(3):425–437. https://doi.org/10.1111/febs.13584
Forchhammer K, Watzer B (2016) Closing a gap in cyanophycin metabolism. Microbiology 162:727–729. https://doi.org/10.1099/mic.0.000260
Fredriksson C, Bergman B (1997) Ultrastructural characterisation of cells specialised for nitrogen fixation in a non-heterocystous cyanobacterium, Trichodesmium sp. Protoplasma 197(1–2):76–85. https://doi.org/10.1007/Bf01279886
Frey KM, Oppermann-Sanio FB, Schmidt H, Steinbüchel A (2002) Technical-scale production of cyanophycin with recombinant strains of Escherichia coli. Appl Environ Microbiol 68(7):3377–3384. https://doi.org/10.1128/AEM.68.7.3377-3384.2002
Frommeyer M, Wiefel L, Steinbüchel A (2016) Features of the biotechnologically relevant polyamide family “cyanophycins”’ and their biosynthesis in prokaryotes and eukaryotes. Crit Rev Biotechnol 36(1):153–164. https://doi.org/10.3109/07388551.2014.946467
Füser G, Steinbüchel A (2007) Analysis of genome sequences for genes of cyanophycin metabolism: identifying putative cyanophycin metabolizing prokaryotes. Macromol Biosci 7(3):278–296. https://doi.org/10.1002/mabi.200600207
Gallon J (1981) The oxygen sensitivity of nitrogenase: a problem for biochemists and micro-organisms. Trends Biochem Sci 6:19–23. https://doi.org/10.1016/0968-0004(81)90008-6
Golden JW, Yoon HS (1998) Heterocyst formation in Anabaena. Curr Opin Microbiol 1(6):623–629. https://doi.org/10.1016/S1369-5274(98)80106-9
Golden JW, Robinson SJ, Haselkorn R (1985) Rearrangement of nitrogen-fixation genes during heterocyst differentiation in the cyanobacterium Anabaena. Nature 314(6010):419–423. https://doi.org/10.1038/314419a0
Hai T, Oppermann-Sanio FB, Steinbüchel A (1999) Purification and characterization of cyanophycin and cyanophycin synthetase from the thermophilic Synechococcus sp. MA19. FEMS Microbiol. Lett 181(2):229–236. https://doi.org/10.1111/j.1574-6968.1999.tb08849.x
Hai T, Oppermann-Sanio FB, Steinbüchel A (2002) Molecular characterization of a thermostable cyanophycin synthetase from the thermophilic cyanobacterium Synechococcus sp strain MA19 and in vitro synthesis of cyanophycin and related polyamides. Appl Environ Microbiol 68(1):93–101. https://doi.org/10.1128/Aem.68.1.93-101.2002
Hai T, Frey KM, Steinbüchel A (2006) Engineered cyanophycin synthetase (CphA) from Nostoc ellipsosporum confers enhanced CphA activity and cyanophycin accumulation to Escherichia coli. Appl Environ Microbiol 72(12):7652–7660. https://doi.org/10.1128/Aem.01132-06
Hejazi M, Piotukh K, Mattow J, Deutzmann R, Volkmer-Engert R, Lockau W (2002) Isoaspartyl dipeptidase activity of plant-type asparaginases. Biochem J 364:129–136. https://doi.org/10.1042/bj3640129
Herrero A, Burnat M (2014) Cyanophycin: a cellular nitrogen reserve material. In: Flores E, Herrero A (eds) The cell biology of cyanobacteria. Caister Academic Press, Norfolk, pp 211–220
Herrero A, Stavans J, Flores E (2016) The multicellular nature of filamentous heterocyst-forming cyanobacteria. FEMS Microbiol Rev 40(6):831–854. https://doi.org/10.1093/femsre/fuw029
Hill S, Kennedy C, Kavanagh E, Goldberg RB, Hanau R (1981) Nitrogen-fixation gene (Nifl) involved in oxygen regulation of nitrogenase synthesis in K Pneumoniae. Nature 290(5805):424–426. https://doi.org/10.1038/290424a0
Jüttner F (1983) C-14-labeled metabolites in heterocysts and vegetative cells of Anabaena cylindrica filaments and their presumptive function as transport vehicles of organic-carbon and nitrogen. J Bacteriol 155(2):628–633
Khlystov NA, Chan WY, Kunjapur AM, Shi WC, Prather KU, Olsen BD (2017) Material properties of the cyanobacterial reserve polymer multi-L- arginyl-poly-L-aspartate (cyanophycin). Polymer 109:238–245. https://doi.org/10.1016/j.polymer.2016.11.058
Klemke F, Nürnberg DJ, Ziegler K, Beyer G, Kahmann U, Lockau W, Volkmer T (2016) CphA2 is a novel type of cyanophycin synthetase in N-2-fixing cyanobacteria. Microbiology 162:526–536. https://doi.org/10.1099/mic.0.000241
Klotz A, Georg J, Budinska L, Watanabe S, Reimann V, Januszewski W, Sobotka R, Jendrossek D, Hess WR, Forchhammer K (2016) Awakening of a dormant cyanobacterium from nitrogen chlorosis reveals a genetically determined program. Curr Biol 26(21):2862–2872. https://doi.org/10.1016/j.cub.2016.08.054
Krehenbrink M, Oppermann-Sanio FB, Steinbüchel A (2002) Evaluation of non-cyanobacterial genome sequences for occurrence of genes encoding proteins homologous to cyanophycin synthetase and cloning of an active cyanophycin synthetase from Acinetobacter sp strain DSM 587. Arch Microbiol 177(5):371–380. https://doi.org/10.1007/s00203-001-0396-9
Kumar K, Mella-Herrera RA, Golden JW (2010) Cyanobacterial heterocysts. Csh Perspect Biol 2(4):a000315. https://doi.org/10.1101/cshperspect.a000315
Lang NJ, Simon RD, Wolk CP (1972) Correspondence of cyanophycin granules with structured granules in Anabaena cylindrica. Arch Mikrobiol 83(4):313–320. https://doi.org/10.1007/Bf00425243
Law AM, Lai SW, Tavares J, Kimber MS (2009) The structural basis of beta-peptide-specific cleavage by the serine protease cyanophycinase. J Mol Biol 392(2):393–404. https://doi.org/10.1016/j.jmb.2009.07.001
Leganes F, Fernandez-Pinas F, Wolk CP (1998) A transposition-induced mutant of Nostoc ellipsosporum implicates an arginine-biosynthetic gene in the formation of cyanophycin granules and of functional heterocysts and akinetes. Microbiology 144:1799–1805. https://doi.org/10.1099/00221287-144-7-1799
Li H, Sherman DM, Bao SL, Sherman LA (2001) Pattern of cyanophycin accumulation in nitrogen-fixing and non-nitrogen-fixing cyanobacteria. Arch Microbiol 176(1–2):9–18. https://doi.org/10.1007/s002030100281
Lopez-Igual R, Flores E, Herrero A (2010) Inactivation of a heterocyst-specific invertase indicates a principal role of sucrose catabolism in heterocysts of Anabaena sp. J Bacteriol 192(20):5526–5533. https://doi.org/10.1128/Jb.00776-10
Luque I, Forchhammer K (2008) Nitrogen assimilation and C/N balance sensing. In: Herrero A, Flores E (eds) The cyanobacteria: molecular biology, genomics and evolution. Caister Academic Press, Norfolk, pp 335–382
Magnuson A, Cardona T (2016) Thylakoid membrane function in heterocysts. BBA-Bioenergetics 1857(3):309–319. https://doi.org/10.1016/j.bbabio.2015.10.016
Maheswaran M, Urbanke C, Forchhammer K (2004) Complex formation and catalytic activation by the PII signaling protein of N-acetyl-L-glutamate kinase from Synechococcus elongatus strain PCC 7942. J Biol Chem 279(53):55202–55210. https://doi.org/10.1074/jbc.M410971200
Maheswaran M, Ziegler K, Lockau W, Hagemann M, Forchhammer K (2006) PII-regulated arginine synthesis controls accumulation of cyanophycin in Synechocystis sp strain PCC 6803. J Bacteriol 188(7):2730–2734. https://doi.org/10.1128/Jb.188.7.2730-2734.2006
Maldener I, Muro-Pastor AM (2010) Cyanobacterial heterocysts. In: eLS. Wiley, Chichester. https://doi.org/10.1002/9780470015902.a0000306.pub2
Maldener I, Summers ML, Sukenik A (2014) Cellular differentiation in filamentous cyanobacteria. In: Flores E, Herrero A (eds) The cell biology of cyanobacteria. Caister Academic Press, Norfolk, pp 263–291
Martin-Figueroa E, Navarro F, Florencio FJ (2000) The GS-GOGAT pathway is not operative in the heterocysts. Cloning and expression of glsF gene from the cyanobacterium Anabaena sp PCC 7120. FEBS Lett 476(3):282–286. https://doi.org/10.1016/S0014-5793(00)01722-1
Merritt MV, Sid SS, Mesh L, Allen MM (1994) Variations in the amino acid composition of cyanophycin in the cyanobacterium Synechocystis sp. PCC 6308 as a function of growth conditions. Arch Microbiol 162(3):158–166
Miller SR, Castenholz RW (2000) Evolution of thermotolerance in hot spring cyanobacteria of the genus Synechococcus. Appl Environ Microbiol 66(10):4222–4229. https://doi.org/10.1128/aem.66.10.4222-4229.2000
Mitschke J, Georg J, Scholz I, Sharma CM, Dienst D, Bantscheff J, Voss B, Steglich C, Wilde A, Vogel J, Hess WR (2011) An experimentally anchored map of transcriptional start sites in the model cyanobacterium Synechocystis sp PCC 6803. Proc Natl Acad Sci U S A 108(5):2124–2129. https://doi.org/10.1073/pnas.1015154108
Nausch H, Huckauf J, Broer I (2016) Peculiarities and impacts of expression of bacterial cyanophycin synthetases in plants. Appl Microbiol Biotechnol 100(4):1559–1565. https://doi.org/10.1007/s00253-015-7212-y
Nicolaisen K, Mariscal V, Bredemeier R, Pernil R, Moslavac S, Lopez-Igual R, Maldener I, Herrero A, Schleiff E, Flores E (2009) The outer membrane of a heterocyst-forming cyanobacterium is a permeability barrier for uptake of metabolites that are exchanged between cells. Mol Microbiol 74(1):58–70. https://doi.org/10.1111/j.1365-2958.2009.06850.x
Obst M, Steinbüchel A (2004) Microbial degradation of poly(amino acid)s. Biomacromolecules 5(4):1166–1176. https://doi.org/10.1021/bm049949u
Obst M, Steinbüchel A (2006) Cyanophycin—an ideal bacterial nitrogen storage material with unique chemical properties. In: Shively JM (ed) Inclusions in prokaryotes. Springer, Berlin, Heidelberg, pp 167–193. https://doi.org/10.1007/3-540-33774-1_7
Obst M, Oppermann-Sanio FB, Luftmann H, Steinbüchel A (2002) Isolation of cyanophycin-degrading bacteria, cloning and characterization of an extracellular cyanophycinase gene (cphE) from Pseudomonas anguilliseptica strain BI. The cphE gene from P anguilliseptica BI encodes a cyanophycin hydrolyzing enzyme. J Biol Chem 277(28):25096–25105. https://doi.org/10.1074/jbc.M112267200
Obst M, Sallam A, Luftmann H, Steinbüchel A (2004) Isolation and characterization of gram-positive cyanophycin-degrading bacteria – kinetic studies on cyanophycin depolymerase activity in aerobic bacteria. Biomacromolecules 5(1):153–161. https://doi.org/10.1021/bm034281p
Obst M, Krug A, Luftmann H, Steinbüchel A (2005) Degradation of cyanophycin by Sedimentibacter hongkongensis strain KI and Citrobacter amalonaticus strain G isolated from an anaerobic bacterial consortium. Appl Environ Microbiol 71(7):3642–3652. https://doi.org/10.1128/Aem.71.7.3642-3652.2005
Perez R, Forchhammer K, Salerno G, Maldener I (2016) Clear differences in metabolic and morphological adaptations of akinetes of two Nostocales living in different habitats. Microbiology 162:214–223. https://doi.org/10.1099/mic.0.000230
Perez R, Wörmer L, Sass P, Maldener I (2018) A highly asynchronous developmental program triggered during germination of dormant akinetes of filamentous diazotrophic cyanobacteria. FEMS Microbiol Ecol 94(1):fix131. https://doi.org/10.1093/femsec/fix131
Picossi S, Valladares A, Flores E, Herrero A (2004) Nitrogen-regulated genes for the metabolism of cyanophycin, a bacterial nitrogen reserve polymer – Expression and mutational analysis of two cyanophycin synthetase and cyanophycinase gene clusters in the heterocyst-forming cyanobacterium Anabaena sp PCC 7120. J Biol Chem 279(12):11582–11592. https://doi.org/10.1074/jbc.M311518200
Richter R, Hejazi M, Kraft R, Ziegler K, Lockau W (1999) Cyanophycinase, a peptidase degrading the cyanobacterial reserve material multi-L-arginyl-poly-L-aspartic acid (cyanophycin) – Molecular cloning of the gene of Synechocystis sp PCC 6803, expression in Escherichia coli, and biochemical characterization of the purified enzyme. Eur J Biochem 263(1):163–169. https://doi.org/10.1046/j.1432-1327.1999.00479.x
Rippka R, Deruelles J, Waterbury JB, Herdman M, Stanier RY (1979) Generic assignments, strain histories and properties of pure cultures of cyanobacteria. J Gen Microbiol 111(Mar):1–61. https://doi.org/10.1099/00221287-111-1-1
Sallam A, Steinbüchel A (2008) Anaerobic and aerobic degradation of cyanophycin by the denitrifying bacterium Pseudomonas alcaligenes strain DIP1 and role of three other coisolates in a mixed bacterial consortium. Appl Environ Microbiol 74(11):3434–3443. https://doi.org/10.1128/Aem.02575-07
Sallam A, Steinbüchel A (2010) Dipeptides in nutrition and therapy: cyanophycin-derived dipeptides as natural alternatives and their biotechnological production. Appl Microbiol Biotechnol 87(3):815–828. https://doi.org/10.1007/s00253-010-2641-0
Sallam A, Kast A, Przybilla S, Meiswinkel T, Steinbüchel A (2009a) Biotechnological process for production of beta-dipeptides from cyanophycin on a technical scale and its optimization. Appl Environ Microbiol 75(1):29–38. https://doi.org/10.1128/AEM.01344-08
Sallam A, Steinle A, Steinbüchel A (2009b) Cyanophycin: biosynthesis and applications. In: Rehm BHA (ed) Microbial production of biopolymers and polymer precursors. Caister Academic Press, Norfolk, pp 79–99
Sherman LA, Meunier P, Colon-Lopez MS (1998) Diurnal rhythms in metabolism: a day in the life of a unicellular, diazotrophic cyanobacterium. Photosynth Res 58(1):25–42. https://doi.org/10.1023/A:1006137605802
Sherman DM, Tucker D, Sherman LA (2000) Heterocyst development and localization of cyanophycin in N2-fixing cultures of Anabaena sp PCC 7120 (cyanobacteria). J Phycol 36(5):932–941. https://doi.org/10.1046/j.1529-8817.2000.99132.x
Simon RD (1971) Cyanophycin granules from the blue-green alga Anabaena cylindrica: a reserve material consisting of copolymers of aspartic acid and arginine. Proc Natl Acad Sci U S A 68(2):265–267. https://doi.org/10.1073/pnas.68.2.265
Simon RD (1973) The effect of chloramphenicol on the production of cyanophycin granule polypeptide in the blue green alga Anabaena cylindrica. Arch Mikrobiol 92(2):115–122. https://doi.org/10.1007/bf00425009
Simon RD (1976) The biosynthesis of multi-L-arginyl-poly(L-aspartic acid) in the filamentous cyanobacterium Anabaena cylindrica. Biochim Biophys Acta 422(2):407–418. https://doi.org/10.1016/0005-2744(76)90151-0
Simon RD, Weathers P (1976) Determination of the structure of the novel polypeptide containing aspartic acid and arginine which is found in cyanobacteria. Biochim Biophys Acta Protein Struct 420(1):165–176. https://doi.org/10.1016/0005-2795(76)90355-x
Simon RD, Lawry NH, McLendon GL (1980) Structural characterization of the cyanophycin granule polypeptide of Anabaena cylindrica by circular dichroism and Raman spectroscopy. Biochim Biophys Acta 626(2):277–281. https://doi.org/10.1016/0005-2795(80)90121-x
Steinle A, Oppermann-Sanio FB, Reichelt R, Steinbüchel A (2008) Synthesis and accumulation of cyanophycin in transgenic strains of Saccharomyces cerevisiae. Appl Environ Microbiol 74(11):3410–3418. https://doi.org/10.1128/AEM.00366-08
Steinle A, Bergander K, Steinbüchel A (2009) Metabolic engineering of Saccharomyces cerevisiae for production of novel cyanophycins with an extended range of constituent amino acids. Appl Environ Microbiol 75(11):3437–3446. https://doi.org/10.1128/Aem.00383-09
Steinle A, Witthoff S, Krause JP, Steinbüchel A (2010) Establishment of cyanophycin biosynthesis in Pichia pastoris and optimization by use of engineered cyanophycin synthetases. Appl Environ Microbiol 76(4):1062–1070. https://doi.org/10.1128/AEM.01659-09
Sutherland JM, Reaston J, Stewart WDP, Herdman M (1985) Akinetes of the cyanobacterium Nostoc PCC 7524 – Macromolecular and biochemical-changes during syndronous germination. J Gen Microbiol 131:2855–2863. https://doi.org/10.1099/00221287-131-11-2855
Thomas J, Meeks J, Wolk CP, Shaffer P, Austin S (1977) Formation of glutamine from [13n] ammonia, [13n] dinitrogen, and [14C] glutamate by heterocysts isolated from Anabaena cylindrica. J Bacteriol 129(3):1545–1555
Vargas WA, Nishi CN, Giarrocco LE, Salerno GL (2011) Differential roles of alkaline/neutral invertases in Nostoc sp. PCC 7120: Inv-B isoform is essential for diazotrophic growth. Planta 233(1):153–162. https://doi.org/10.1007/s00425-010-1288-5
Voss I, Steinbüchel A (2006) Application of a KDPG-aldolase gene-dependent addiction system for enhanced production of cyanophycin in Ralstonia eutropha strain H16. Metab Eng 8(1):66–78. https://doi.org/10.1016/j.ymben.2005.09.003
Voss I, Diniz SC, Aboulmagd E, Steinbüchel A (2004) Identification of the Anabaena sp. strain PCC 7120 cyanophycin synthetase as suitable enzyme for production of cyanophycin in gram-negative bacteria like Pseudomonas putida and Ralstonia eutropha. Biomacromolecules 5(4):1588–1595. https://doi.org/10.1021/bm049861g
Walsby AE (2007) Cyanobacterial heterocysts: terminal pores proposed as sites of gas exchange. Trends Microbiol 15(8):340–349. https://doi.org/10.1016/j.tim.2007.06.007
Watzer B, Forchhammer K (2018a) Cyanophycin synthesis optimizes nitrogen utilization in the unicellular cyanobacterium Synechocystis sp strain PCC 6803. Appl Environ Microbiol 84(20):e01298-18. https://doi.org/10.1128/AEM.01298-18
Watzer B, Forchhammer K (2018b) Cyanophycin: a nitrogen-rich reserve polymer. In: Tiwari A (ed) Cyanobacteria. IntechOpen, London, 85–107, https://doi.org/10.5772/intechopen.77049
Watzer B, Engelbrecht A, Hauf W, Stahl M, Maldener I, Forchhammer K (2015) Metabolic pathway engineering using the central signal processor PII. Microb Cell Factories 14:192. https://doi.org/10.1186/s12934-015-0384-4
Weiss GL, Kieninger AK, Maldener I, Forchhammer K, Pilhofer M (2019) Structure and function of a bacterial gap junction analog. Cell 178(2):374–384. https://doi.org/10.1016/j.cell.2019.05.055
Whitton BA (2012) Ecology of cyanobacteria II: their diversity in space and time. Springer, New York
Wiefel L, Steinbüchel A (2014) Solubility behavior of cyanophycin depending on lysine content. Appl Environ Microbiol 80(3):1091–1096. https://doi.org/10.1128/Aem.03159-13
Wiefel L, Bröker A, Steinbüchel A (2011) Synthesis of a citrulline-rich cyanophycin by use of Pseudomonas putida ATCC 4359. Appl Microbiol Biotechnol 90(5):1755–1762. https://doi.org/10.1007/s00253-011-3224-4
Wiefel L, Wohlers K, Steinbüchel A (2019) Re-evaluation of cyanophycin synthesis in Corynebacterium glutamicum and incorporation of glutamic acid and lysine into the polymer. Appl Microbiol Biotechnol 103(10):4033–4043. https://doi.org/10.1007/s00253-019-09780-5
Wolk CP (1968) Movement of carbon from vegetative cells to heterocysts in Anabaena cylindrica. J Bacteriol 96(6):2138–2143
Wolk CP (1996) Heterocyst formation. Annu Rev Genet 30(1):59–78. https://doi.org/10.1146/annurev.genet.30.1.59
Wolk CP, Austin SM, Bortins J, Galonsky A (1974) Autoradiographic localization of N-13 after fixation of N-13-labeled nitrogen gas by a heterocyst-forming blue-green-alga. J Cell Biol 61(2):440–453. https://doi.org/10.1083/jcb.61.2.440
Wolk CP, Ernst A, Elhai J (1994) Heterocyst metabolism and development. In: Bryant DA (ed) The molecular biology of cyanobacteria. Advances in photosynthesis, vol 1. Springer, Dordrecht, pp 769–823
Zhang H, Liu Y, Nie X, Liu L, Hua Q, Zhao GP, Yang C (2018) The cyanobacterial ornithine-ammonia cycle involves an arginine dihydrolase. Nat Chem Biol 14(6):575–581. https://doi.org/10.1038/s41589-018-0038-z
Ziegler K, Diener A, Herpin C, Richter R, Deutzmann R, Lockau W (1998) Molecular characterization of cyanophycin synthetase, the enzyme catalyzing the biosynthesis of the cyanobacterial reserve material multi-L-arginyl-poly-L-aspartate (cyanophycin). Eur J Biochem 254(1):154–159. https://doi.org/10.1046/j.1432-1327.1998.2540154.x
Ziegler K, Stephan DP, Pistorius EK, Ruppel HG, Lockau W (2001) A mutant of the cyanobacterium Anabaena variabilis ATCC 29413 lacking cyanophycin synthetase: growth properties and ultrastructural aspects. FEMS Microbiol Lett 196(1):13–18. https://doi.org/10.1016/S0378-1097(01)00027-1
Ziegler K, Deutzmann R, Lockau W (2002) Cyanophycin synthetase-like enzymes of non-cyanobacterial eubacteria: characterization of the polymer produced by a recombinant synthetase of Desulfitobacterium hafniense. Zeitschrift Für Naturforschung Section C-a J Biosci 57(5–6):522–529. https://doi.org/10.1515/znc-2002-5-621
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2020 Springer Nature Switzerland AG
About this chapter
Cite this chapter
Watzer, B., Klemke, F., Forchhammer, K. (2020). The Cyanophycin Granule Peptide from Cyanobacteria. In: Jendrossek, D. (eds) Bacterial Organelles and Organelle-like Inclusions. Microbiology Monographs, vol 34. Springer, Cham. https://doi.org/10.1007/978-3-030-60173-7_7
Download citation
DOI: https://doi.org/10.1007/978-3-030-60173-7_7
Published:
Publisher Name: Springer, Cham
Print ISBN: 978-3-030-60172-0
Online ISBN: 978-3-030-60173-7
eBook Packages: Biomedical and Life SciencesBiomedical and Life Sciences (R0)