**6. Industrial applications**

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].

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 intracel-

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.

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– 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 part-

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

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,

lular arginine levels and, consequently, reduced CGP amount [70].

**5.3. PII regulation of arginine metabolism**

94 Cyanobacteria

ners and thereby regulate the cellular C/N balance [83].

biosynthesis via regulation of NAGK [68, 69, 86].

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]).

strongly affected by the expression system. A stabilized multi-copy *cphA* expression system, using the KDPG-aldolase gene (*eda*)-dependent addiction system, allows cultivation without antibiotic selection. The multi-copy *cphA* expression results in a CGP yield between 26.9% and 40.0% (w/w) of CDM. The maximum amount of 40.0% (w/w) of CDM was observed in a 30- and 500-l pilot plant. In the absence of the amino acids arginine and aspartic acid in the

Cyanophycin: A Nitrogen-Rich Reserve Polymer http://dx.doi.org/10.5772/intechopen.77049 97

The industrially established host *Saccharomyces cerevisiae* has also been used for CGP production, by expression of *cphA* from *Synechocystis* sp. PCC 6803. *S. cerevisiae* harboring *cphA* accumulated up to 6.9% (w/w) of CDM. Two CGP species were observed in this strain: water-soluble and the typical water-insoluble CGP. Furthermore, the isolated polymer from this transgenic yeast contained 2 mol% lysine, which can be increased up to 10 mol% when cultivation occurs with lysine in the medium [31]. During metabolic engineering studies, several arginine biosynthesis mutants have been analyzed concerning their CGP accumulation abilities. Surprisingly, strains with defects in arginine degradation accumulated only 4% CGP (w/w) of CDM; however, arginine auxotrophic strains were able to accumulate up to 15.3%. Depending on the cultivation conditions, between 30 and 90% of the extracted CGP was soluble at neutral pH. In addition to arginine, aspartate and lysine, further amino acids, such as citrulline and ornithine, have been detected in isolated CGP from different arginine biosynthesis mutants [98]. Furthermore, it was also possible to produce CGP and CGP derivates in *Pseudomonas putida* and the yeast *Pichia pastoris* [99, 100].

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 *Pseudomonas alcaligenes*. This allows the large-scale production of customized

Production of CGP has also been attempted in several transgenic plants. Here, ectopic expression of the primer-independent CphA from *Thermosynechococcus elongatus* BP-1 leads to an accumulation of CGP up to 6.8% (w/w) in tobacco leafs and to 7.5% (w/w) of CDM in potato tubers [102, 103]. CGP production and extraction in plants can be coupled with the production of other plant products like starch [103]. The peculiarities and challenges of plant-produced

Compared to bacteria that are used so far in biotechnological industry, cyanobacteria are

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. *Synechocystis* sp. PCC 6803 strain BW86 is the first reported bulk chemical producing cyanobacterial strain in the literature. CGP production in *Synechocystis* BW86 does not require organic carbon or CGP precursor substances. Growth limiting conditions like phosphate and potassium starvation can further increase the CGP production up to 47.4 ± 2.3% and 57.3 ± 11.1% per CDM, respectively. The studies of Trautmann et al. showed that strain BW86 can be cultivated in flat plate photobioreactors (Midiplate reactor system [105]). During this optimization study, the optimal light intensity as well as the phosphate concentration was determined to maximize CGP synthesis. Under optimal production conditions, highest amount of CGP was around 40% of CDM with a total yield of 340 mg CGP per liter in 9 days [106].

as energy and carbon source. Cyanobacteria have been

β-dipeptides, depending on the composition of the CGP derivates [92, 101].

CGP have been reviewed by Nausch et al. [32].

unique as they use sunlight and CO2

medium, the CGP amount was still between 26.9% and 27.7% (w/w) of CDM [97].

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 *E. coli*, giving new insights in the nature of this polymer as bulk chemical [91]. They describe CGP as an amorphous, glassy polyzwitterion with high thermostability. The dry material is stiff and brittle. According to these properties, CGP could be used to synthesize zwitterionomeric copolymers or as reinforcing fillers [91].
