Abstract
Cyanophycin is a nitrogen/carbon reserve polymer present in most cyanobacteria as well as in a few heterotrophic bacteria. It is a non-ribosomally synthesized polyamide consisting of aspartate and arginine (multi-l-arginyl-poly-l-aspartic acid). The following chapter provides an overview of the characteristics and occurrence of cyanophycin in cyanobacteria. Information about the enzymes involved in cyanophycin metabolism and the regulation of cyanophycin accumulation is also summarized. Herein, we focus on the main regulator, the PII signal transduction protein and its regulation of arginine biosynthesis. Since cyanophycin could be used in various medical or industrial applications, it is of high biotechnological interest. In the last few years, many studies were published aiming at the large-scale production of cyanophycin in different heterotrophic bacteria, yeasts and plants. Recently, a cyanobacterial production strain has been reported, which shows the highest so ever reported cyanophycin yield. The potential and possibilities of biotechnological cyanophycin production will be reviewed in this chapter.
Keywords
- cyanophycin
- cyanophycin synthetase
- cyanophycinase
- nitrogen reserve
- polyamide
- l-arginine
- l-aspartate
- PII protein
1. Introduction
Cyanophycin, abbreviated CGP (cyanophycin granule peptide), is next to poly-γ-glutamic acid and poly-ε-lysine, the third polyamino acid known to occur in nature [1]. It serves as a nitrogen/carbon reserve polymer in many cyanobacterial strains as well as in a few heterotrophic bacteria. CGP consists of the two amino acids, aspartate and arginine, forming a poly-l-aspartic acid backbone with arginine side chains. The arginine residues are linked to the β-carboxyl group of every aspartyl moiety via isopeptide bond [2].
CGP was discovered in 1887 by the botanist Antonio Borzi during microscopic studies of filamentous cyanobacteria [3]. He observed opaque and light scattering inclusions by using light microscopy and created the name
With a C/N ratio of 2:1, CGP is extremely rich in nitrogen and consequently an excellent nitrogen storage compound. During the degradation of CGP and subsequent degradation of arginine, a function as energy source was also proposed [8].
2. CGP occurrence
Most cyanobacteria, including unicellular and filamentous, as well as diazotrophic and non-diazotrophic groups are able to accumulate CGP (Figure 1).
In non-diazotrophic cyanobacteria, the amount of CGP is usually less than 1% of the cell dry mass during exponential growth. CGP accumulates conspicuously under unbalanced growth conditions including stationary phase, light stress or nutrient limitation (sulfate, phosphate or potassium starvation) that do not involve nitrogen starvation [9, 10]. Under such unbalanced conditions, the amount of CGP may increase up to 18% of the cell dry mass [10]. During the recovery from nitrogen starvation by the addition of a usable nitrogen source, CGP is transiently accumulated [11, 12].
In the unicellular diazotrophic cyanobacterium
Furthermore, in heterocysts of diazotrophic cyanobacteria of the order
Akinetes are resting spore-like cells of a subgroup of heterocyst-forming cyanobacteria for surviving long periods of unfavorable conditions. During akinete development, the cells transiently accumulate storage compounds, namely glycogen, lipid droplets and CGP [19, 20] (Figure 1). CGP granules also appear during germination of dormant akinetes [21].
CGP was formally thought to be unique in cyanobacteria. In 2002, Krehenbrink et al. and Ziegler et al. discovered through evaluation of obligate heterotrophic bacteria genomes that many heterotrophic bacteria possess CGP synthetase genes [23, 24]. Genes of CGP metabolism occur in a wide range of different phylogenetic taxa and not closely related to cyanobacteria [25].
3. CGP characteristics
In 1971, Robert Simon isolated CGP granules for the first time by using differential centrifugation. Along with this study, CGP has shown its special and unique solubility behavior [26]. CGP is insoluble at physiological ionic strength and at neutral pH, but soluble in solutions which are acidic, basic or highly ionic. In non-ionic detergents such as Triton X-100, CGP is insoluble; however, in ionic detergents like SDS, it is soluble [6]. Present-day CGP extraction methods are based on its solubility at low pH and insolubility at neutral pH [27].
The chemical structure of CGP was proposed in 1976 by Simon and Weathers [2]. According to this model, CGP has a polymer backbone consisting of α-linked aspartic acid residues. The α-amino group of arginine is linked via isopeptide bonds to the β-carboxylic group of every aspartyl moiety. Because every aspartate residue is linked to an arginine residue, CGP contains equimolar amounts of aspartate and arginine [2]. This structure has been confirmed via enzymatic degradation studies. CGP-degrading enzymes (see below) release β-Asp-Arg dipeptides [28]. CD spectroscopy data suggest that the acid-soluble and neutral insoluble forms of CGP have similar conformations. Both forms contain substantial fractions of β-pleated sheet structure [29].
Cyanobacterial CGP has a molecular weight and polydispersity ranging from 25 to 100 kDa [26]. In contrast, the native CGP producer
Native CGP is exclusively composed of aspartate and arginine. By contrast, in CGP isolated from recombinant
4. CGP metabolism
4.1. Cyanophycin synthetase
CGP is non-ribosomally synthesized from aspartate and arginine by cyanophycin synthetase (CphA1) (Figure 2). In 1976, CphA1 was enriched and characterized for the first time by Simion [35]. The enzyme incorporates aspartate and arginine in an elongation reaction, which requires ATP, KCl, MgCl2 and a sulfhydryl reagent (β-mercaptoethanol or DTT). For its activity, CphA1 needs a so far unknown CGP primer, as a starting point of the elongation reaction [35]. By using synthetically primers, Berg et al. could show that a single building block of CGP (β-Asp-Arg) does not serve as an efficient primer for CphA1 elongation reaction in vitro. The primers need to consist of at least three Asp-Arg building blocks (β-Asp-Arg)3 to detect CphA1 activity [36]. Other peptides, like cell wall or other cellular components, have been suggested to serve as an alternative priming substance for the CphA1 reaction [37]. This could be an explanation for the functionality of CGP synthesis in recombinant bacteria, without the ability to produce native CGP primers [38]. Interestingly, the CphA1 of
Today, CphA1 enzymes from several bacteria, including cyanobacteria and heterotrophic bacteria, have been purified and characterized [33, 39, 40, 41, 42]. The molecular mass of the characterized CphA1 enzymes ranges from 90 to 130 kDa. The active form of CphA1s from
The mechanism of CGP synthesis by CphA1 has been suggested by Berg et al. in 2000, by measuring the step-wise incorporation of amino acids to the C-terminus of the CGP primer. The putative CGP elongation cycle starts at the C-terminal end of the poly-aspartate backbone. First, the carboxylic acid group of the poly-aspartate backbone is activated by transfer of the γ-phosphoryl group of ATP. In the second step, one aspartate is bound at the C-terminus of the growing polymer by its amino group, forming a peptide bound. Subsequently, the intermediate (β-Asp-Arg)n-Asp is transferred to the second active site of CphA1 and phosphorylated at the β-carboxyl group of the aspartate. Finally, the α-group of arginine is linked to the β-carboxyl group of aspartate, forming an isopeptide bound [36].
Various CphA1 enzymes have been characterized with respect to their substrate affinity and specificity. For CphA1 of
CphA homologs are widely distributed in eubacteria. In silico analysis proposes 10 different groups of cyanophycin synthetases [25]. In cyanobacteria, cyanophycin synthetases of group I–III (CphA, CphA2 and CphA2’) can be found.
Recently, the function of a cyanophycin synthetase of group II (CphA2) has been characterized. Most non-diazotrophic cyanobacteria use a single type of cyanophycin synthetase (CphA1). However, in many nitrogen-fixing cyanobacteria, an additional version of CphA is present, termed CphA2. In 2016, Klemke et al. resolved the function of CphA2 [44]. Compared to CphA1, CphA2 has a reduced size and just one ATP-binding site. CphA2 uses the product of CGP hydrolysis, β-aspartyl-arginine dipeptide as substrate to resynthesize cyanophycin, consuming one molecule of ATP per elongation. A mutant lacking CphA2 shows only a minor decrease in the overall CGP content. However, a CphA2-deficient mutant displays similar defects under diazotrophic and high light conditions than a CphA1 mutant [15, 44]. This observation suggests that the apparent “futile cycle” of CGP hydrolysis and immediate repolymerization is probably of physiological significance in the context of nitrogen fixation [17].
4.2. Cyanophycinase
Since 1976, it is known that CGP is resistant against hydrolytic cleavage by several proteases or arginase [2, 45]. This resistance is probably due to the branched structure of CGP [38]. Therefore, the presence of a highly specified peptidase for CGP hydrolysis was suggested.
In 1999, Richter et al. reported a CGP hydrolyzing enzyme from the unicellular cyanobacterium
In addition to CphB, which catalyzes the intracellular cleavage of CGP, other versions of cyanophycinase exist, catalyzing the extracellular hydrolysis of CGP. In 2002, Obst et al. isolated several Gram-negative bacteria from different habitats, which were able to utilize CGP as a source of carbon and energy [47, 48]. One isolate was affiliated as
In 2007, in silico analysis showed that CphB homologs are widely distributed in eubacteria, proposing eight different groups including intracellular and extracellular CGPases. CGPases from cyanobacteria belong to group I, II and partially group III (CphB1–3). Groups IV–VIII, including CphE, are present in a large variety of non-photosynthetic bacteria [25].
4.3. Aspartyl-arginine dipeptidase
The last step in catabolism of CGP is the cleavage of β-Asp-Arg dipeptides to monomeric amino acids, arginine and aspartate (Figure 2). In 1999, Richter et al. found β-Asp-Arg dipeptides hydrolyzing activity in extracts of
The mature isoaspartyl dipeptidases of
In
5. CGP regulation
5.1. Genetic organization of CphA and CphB
Usually, genes involved in CGP metabolism are clustered. The organization of these clusters can be different, depending on the respective organism [25]. In
In
In cluster
In addition to these two gene clusters, a third set of ORFs containing putative
5.2. Dependence of CGP metabolism on arginine biosynthesis
Generally, CGP accumulation is triggered by cell growth arresting stress conditions, such as entry into stationary phase, light or temperature stress, limitation of macronutrients (with the exception of nitrogen starvation) or inhibition of translation by adding antibiotics like chloramphenicol [9, 10, 61]. All of these CGP triggering conditions result in a reduced or arrested growth. In exponential growth phase the amino acids arginine and aspartate are mostly used for protein biosynthesis with the consequence of a low intracellular level of free amino acids. Under growth-limiting conditions, protein biosynthesis is slowed down, which yields an excess of monomeric amino acids in the cytoplasm, triggering the CGP biosynthesis [10].
CGP accumulation also requires an excess of nitrogen. For the filamentous cyanobacterium
A potential link between regulation of arginine biosynthesis and GCP metabolism was suggested in many previous studies. In a transposon mutagenesis study in the filamentous cyanobacterium
In metabolic engineering studies of the CGP production strain
Bacteria produce arginine from glutamate in eight steps. The first five steps involving N-acetylated intermediates lead to ornithine. The conversion of ornithine to arginine requires three additional steps [66]. The second enzyme of ornithine biosynthesis is the N-acetylglutamate kinase (NAGK), which catalyzes the phosphorylation of N-acetyl glutamate to N-acetylglutamyl-phosphate. NAGK catalyzes the controlling step in arginine biosynthesis [67]. NAGK activity is subjected to allosteric feedback inhibition by arginine and is, moreover, positively controlled by the PII signal transduction protein (see below) [67, 68]. Maheswaran et al. showed that arginine production and the following CGP accumulation depend on the catalytic activation of NAGK by the signal transduction protein PII [69]. In a PII-deficient mutant of
The nitrogen-regulated response regulator NrrA also has influence on arginine and CGP biosynthesis. An NrrA-deficient mutant in
All these results and observations point towards arginine as main bottleneck of CGP biosynthesis, while aspartate plays a minor role. CGP accumulation occurs as a result of arginine enrichment in the cytoplasm. Reasons for increased arginine content in the cell are lowered protein biosynthesis as a result of various growth limiting conditions. Furthermore, an excess of nitrogen and energy sensed by PII leads to NAGK activation and thereby increased arginine biosynthesis.
5.3. PII regulation of arginine metabolism
The PII signal transduction proteins are widely distributed in prokaryotes and chloroplasts, where they play a coordinating role in the regulation of nitrogen assimilatory processes [71, 72, 73]. For this purpose, PII senses the energy status of the cell by binding ATP or ADP in a competitive way [74]. Binding of ATP and synergistic binding of 2-oxoglutarate (2-OG) allows PII to sense the current carbon/nitrogen status of the cell [75]. 2-OG is the carbon skeleton for the GS/GOGAT reactions and thereby links the carbon and nitrogen metabolism in all domains of life [76, 77]. The pool size of 2-OG reacts quickly to changes in nitrogen availability, wherefore 2-OG is an indicator of the carbon/nitrogen balance [78, 79]. Depending on the nitrogen supply, PII may be phosphorylated at the apex of the T-loop at position Ser49 [80, 81]. Binding of the effector molecules ATP, ADP and 2-OG as well as phosphorylation leads to conformational rearrangements of the large surface-exposed T-loop, PII’s major protein-interaction structure [82]. These conformational states direct the interaction of PII with its various interaction partners and thereby regulate the cellular C/N balance [83].
In cyanobacteria, PII regulates the global nitrogen control transcriptional factor NtcA, through binding to the NtcA co-activator PipX [84]. In common with other bacteria, cyanobacterial PII proteins can interact with the biotin carboxyl carrier protein (BCCP) of acetyl-CoA carboxylase (ACC) and thereby control the acetyl-CoA levels [85]. Furthermore, PII controls arginine biosynthesis via regulation of NAGK [68, 69, 86].
PII proteins form a cylindrical-shaped homotrimer with 12–13 kDa per subunits. The T-loop, a large and surface-exposed loop, protrudes from each subunit. The effector binding sites are positioned in the three inter-subunit clefts [87, 88]. If sufficient energy and nitrogen are available, indicated by a high ATP and low 2-OG level, non-phosphorylated PII forms an activating complex with NAGK.
The crystal structure of the PII-NAGK complex from
During PII mutagenesis, a PII variant was identified that binds constitutively NAGK in vitro. This PII variant exhibits a single amino acid replacement, Ile86 to Asn86, hereafter referred as PII(I86N) [89]. The crystal structure of PII(I86N) has been solved, showing an almost identical backbone than wild-type PII. However, the T-loop adopts a compact conformation, which is a structural mimic of PII in the NAGK complex [89, 90]. Addition of 2-OG in the presence of ATP normally leads to a dissociation of the PII-NAGK complex, however PII(I86N) no longer responds to 2-OG [90].
The PII(I86N) variant enables a novel approach of metabolic pathway engineering by using custom-tailored PII signaling proteins. By replacing the wild-type PII with a PII carrying the mutation for I86N in
6. Industrial applications
Industrial applications for CGP have previously mainly focused on chemical derivatives. CGP can be converted via hydrolytic β-cleavage to poly(α-l-aspartic acid) (PAA) and free arginine. PAA is biodegradable and has a high number of negatively charged carboxylic groups, making PAA to a possible substituent for polyacrylates [48, 50, 91]. PAA can be employed as antiscalant 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 [48, 50].
CGP can also serve as a source for 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 industry, with various commercial products already available on the market (reviewed by Sallam and Steinbuchel [92]).
Potential applications of non-modified CGP have been discussed but remain so far largely unexplored. This can partially be explained by the lack of research being conducted on the material properties of CGP. Recently in 2017, the first study regarding CGP material properties has been published. In this study, Khlystov et al. focused on the structural, thermal, mechanical and solution properties of CGP produced by recombinant
7. Biotechnological production
Previous ventures to produce CGP in high amounts were mainly focused on heterotrophic bacteria, yeasts and plants as production host. These recombinant production hosts heterologously express CGP synthetase genes, mostly from cyanobacteria. In this way, heterotrophic bacteria, which are established in biotechnological industry including
Strain
The industrially established host
CGP and CGP derivates are important sources for β-dipeptides for several applications. A large-scale method was developed to convert CGP into its constituting β-dipeptides by using CphE from
Production of CGP has also been attempted in several transgenic plants. Here, ectopic expression of the primer-independent CphA from
Compared to bacteria that are used so far in biotechnological industry, cyanobacteria are unique as they use sunlight and CO2 as energy and carbon source. Cyanobacteria have been identified as rich source of various biologically active compounds, biofertilizers, bioplastics, energy, food and feed [104]. Obviously, the importance of environmentally friendly production processes increases more and more. Hence, Cyanobacteria are expected to play a major role in future industry.
The main bottleneck of CGP production in Cyanobacteria is the relatively slow growth rate, which is much lower than in biotechnologically established bacteria. Conventional cultivation methods of cyanobacteria reach a biomass of roughly 1 g dry mass per liter [107]. To overcome this limitation, a new cultivation method was developed, using a two-tier vessel with membrane-mediated CO2 supply. By using this cultivation setup, it was possible to enable rapid growth of
In comparison, the recombinant
8. Conclusions
CGP is well researched and its occurrence in cyanobacteria is known for more than 100 years. However, many questions are still open. Most obviously, the cell biology of the CGP granules remains largely unknown. In the last decades, research on CGP mainly focused on biotechnological purposes, like strain or process optimization. Most work has been carried out with short-chain CGP from recombinant producer strains; however the biophysical properties of the long-chain native CGP remain largely unexplored. So far, heterotrophic bacteria were mainly used to produce industrial biocompounds including CGP. In this chapter, we discussed the possibility of a cyanobacterial CGP production strain. The main disadvantages of cyanobacteria, their slower growth and the low abundance of product can be compensated using genetic engineering together with appropriate production processes. Future industry has to cope with the manifold challenges to counteract environmental pollution and climate change. The use of cyanobacteria in CGP production and, more generally, in biotechnological applications for bioproduct synthesis provides an environmentally friendly alternative to conventional biotechnological approaches.
Acknowledgments
This work was supported by grants from the DFG (Fo195/9), the research training group GRK 1708 and the Baden-Württemberg foundation grant 7533-10-5-92B. We thank Iris Maldener for provision of the electron micrographs of
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