**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 *Synechocystis* sp. PCC 6803, *cphA* and *cphB* are adjacent; however, they are expressed independently [60]. A hypothetical protein named slr2003 is located downstream of *cphA* and is transcribed in a polycistronic unit with *cphA* [60]. However, the function of Slr2003 is unknown. In the gene of CphB (slr2001), a small antisense RNA was detected (transcriptional unit 1486) [60].

In *Anabaena* sp. PCC 7120, two clusters containing CphA and CphB were identified [18]. In the *cph1* cluster, *cphB1* and *cphA1* were expressed under ammonia and nitrate supplemented growth, but the expression of both genes was higher in the absence of combined nitrogen in heterocysts and vegetative cells. In the *cph1* operon, *cphB1* and *cphA1* were cotranscribed. In addition, *cphA1* can be expressed from independent promoters, of which one is constitutive and the other regulated by the global nitrogen control transcriptional factor NtcA [18].

In cluster *cph2*, the *cphB2* and *cphA2* genes were found in opposite orientation and both genes were expressed monocistronically. The genes were expressed under conditions of ammonia, nitrate or N2 supplementation, but the expression was higher in the absence of ammonia. Generally, the expression of the *cph2* is lower compared to *cph1* [18].

In addition to these two gene clusters, a third set of ORFs containing putative *cphA* and *cphB* genes was found in *Nostoc punctiforme* PCC 73102 and *Anabaena variabilis* ATCC 29413 [25].

### **5.2. Dependence of CGP metabolism on arginine biosynthesis**

asparaginases, Sll0422 and All3922, have not only a function in asparagine catabolism but also

The mature isoaspartyl dipeptidases of *Synechocystis* sp. PCC 6803 and *Anabaena* sp. PCC 7120 consist of two protein subunits that are generated by autocleavage of the primary translation product between Gly-172 and Thr-173 (numbering according to *Synechocystis* sp. PCC 6803) within the conserved consensus sequence GT(I/V)G [55]. The native molecular weight of

In *Anabaena* sp. PCC 7120, all genes involved in CGP metabolism as well as the isoaspartyl dipeptidases All3922 are expressed in vegetative cells and heterocysts but in different expression levels. Both, CGP synthetases and CGPases are much higher expressed in heterocysts than in vegetative cells [56]. However, asparaginase All3922 is present in significantly lower levels in heterocysts than in vegetative cells [57]. A deletion of All3922 in *Anabaena* sp. PCC 7120 causes an increased accumulation of CGP and β-Asp-Arg dipeptides. Furthermore, a deletion mutant shows an impaired diazotrophic growth similar to the phenotype known from CphB deletion mutants in *Anabaena* sp. PCC 7120 [18, 57]. This observation implies that the first step of CGP catabolism, the cleavage catalyzed by CphB, takes place in the heterocyst. The released β-Asp-Arg dipeptides are transported to the adjacent vegetative cells. Isoaspartyl dipeptidase All3922, present in the vegetative cells, cleaves the β-Asp-Arg dipeptides and releases monomeric aspartate and arginine [57]. When CGP synthesis is not possible, due to a deletion of CphA, arginine and aspartate might be transferred directly from heterocysts. This explains the minor effects on diazotrophic growth in a CphA deletion mutant [15]. These results identified β-Asp-Arg dipeptides as nitrogen vehicle in diazotrophic heterocyst forming cyanobacteria, next to glutamine and arginine alone or with aspartate [57–59]. A benefit of β-Asp-Arg dipeptides as nitrogen transport substance is avoiding the release of free arginine and aspartate in the heterocyst. This indicates that CGP metabolism has evolved in multicellular heterocyst-forming cyanobacteria to increase the efficiency of nitrogen fixation [57].

Usually, genes involved in CGP metabolism are clustered. The organization of these clusters can be different, depending on the respective organism [25]. In *Synechocystis* sp. PCC 6803, *cphA* and *cphB* are adjacent; however, they are expressed independently [60]. A hypothetical protein named slr2003 is located downstream of *cphA* and is transcribed in a polycistronic unit with *cphA* [60]. However, the function of Slr2003 is unknown. In the gene of CphB (slr2001), a

In *Anabaena* sp. PCC 7120, two clusters containing CphA and CphB were identified [18]. In the *cph1* cluster, *cphB1* and *cphA1* were expressed under ammonia and nitrate supplemented growth, but the expression of both genes was higher in the absence of combined nitrogen in

β2

(α derived

approximately 70kD of this enzyme suggests that it has a subunit structure of α<sup>2</sup>

from the N-terminal part and β from the C-terminal part of the precursor) [55].

in the final step of CGP and protein degradation [55].

92 Cyanobacteria

**5. CGP regulation**

**5.1. Genetic organization of CphA and CphB**

small antisense RNA was detected (transcriptional unit 1486) [60].

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 *Calothrix* sp. strain PCC 7601, it was shown that CGP accumulation occurs preferably in the presence of ammonia [62]. The addition of amino acids to the media further increased CGP formation [63]. During process optimization studies for heterotrophic CGP production in the strain *Acinetobacter calcoaceticus* ADP1, it was shown that addition of arginine to the medium as sole carbon source increased CGP accumulation drastically. When, in *A. calcoaceticus* strain ADP1, CGP synthesis is induced by phosphate starvation, it accounts to 3.5% (w/w) of the cell dry matter (CDM) with ammonia as nitrogen source. Additional supply of the medium with arginine increases the CGP amount to 41.4% (w/w) (CDM). Notably, a combined supply of arginine and aspartate has a much lower stimulating effect to CGP accumulation than arginine alone [30].

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 *Nostoc ellipsosporum,* an arginine biosynthesis gene, *argL*, was interrupted by a transposon. This mutation partially impairs arginine biosynthesis but does not strictly result in l-arginine auxotrophy. Without arginine supplementation, heterocysts failed to fix nitrogen, akinetes were unable to germinate and CGP granules did not appear. However, when both nitrate and arginine are present in the media, the impaired arginine biosynthesis is bypassed. Under this condition, the mutant could form CGP and was able to differentiate functional akinetes, which contained CGP granules [64].

In metabolic engineering studies of the CGP production strain *Acinetobacter calcoaceticus* ADP1, several genes related to the arginine biosyntheses or its regulation were modified to yield higher amounts of arginine. As a consequence, significant higher CGP production was observed [65].

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-acetylglutamylphosphate. 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 *Synechocystis* sp. PCC 6803, NAGK remained in a low activity state, which caused impaired CGP accumulation [69].

indicated by a high ATP and low 2-OG level, non-phosphorylated PII forms an activating

Cyanophycin: A Nitrogen-Rich Reserve Polymer http://dx.doi.org/10.5772/intechopen.77049 95

The crystal structure of the PII-NAGK complex from *Synechococcus elongatus* strain PCC 7942 revealed two PII trimers sandwiching a NAGK homohexamer (trimer of dimers) [88]. Each PII subunit contacts one NAGK subunit [88]. Two parts of PII are involved in interaction with NAGK.The first structure, called B-loop, is located on the PII body and interacts with the C-domain of NAGK subunit, involving residue Glu85. The interaction of the B-loop is the first step in complex formation. Second, the T-loop must adopt a bent conformation and insert into the interdomain cleft of NAGK [89]. This enhances the catalytic efficiency of NAGK, with the Vmax increasing fourfold and the Km for N-acetylglutamate decreasing by a factor of 10 [86]. Furthermore, feedback inhibi-

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 *Synechocystis* sp. PCC 6803, it was possible to engineer the first cyanobacterial CGP overproducer strain. Strain BW86, containing the PII(I86N) version, shows an increase of NAGK activity, which causes a more than 10-fold higher arginine content than the wild-type [10]. Under balanced growth conditions with nitrate as nitrogen source, strain BW86 accumulates up to 15.6 ± 5.4% CGP relative to the CDM, i.e., on average almost sixfold more than the wild type. Appropriate starvation conditions can further increase the CGP content of strain BW86 up to 47.4 ± 2.3% per CDM under phosphate starvation and 57.3 ± 11.1% per CDM under potassium starvation, without addition of arginine to the medium [10]. Furthermore, the CGP, which is produced by strain BW86, shows a high polydispersity ranging from 25 to 100 kDa, similar to the polydispersity of cyanobacterial wild-type CGP, which contrasts CGP from recombinant producer strains using heterologous expression systems with heterotrophic bacteria, yeasts or plants [10]. CGP isolated from those strains have a size ranging of 25–45 kDa [27, 31, 32].

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,

tion of NAGK by arginine is strongly decreased in the presence of PII [86].

complex with NAGK.

**6. Industrial applications**

paint, building or oil industry [48, 50].

The nitrogen-regulated response regulator NrrA also has influence on arginine and CGP biosynthesis. An NrrA-deficient mutant in *Synechocystis* sp. PCC 6803 shows reduced intracellular arginine levels and, consequently, reduced CGP amount [70].

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.
