Cyanophycin Granules

Cyanophycin is the third known polymer, the others being polyglutamate and polylysine, consisting of a restricted number of amino acids, that is synthesized by a nonribosomal synthetase. Cyanophycin is a comb-like polymer with α-amino-α-carboxy-linked l-aspartate residues representing the poly(aspartate) backbone and with l-arginine residues linked to the β-carboxylic groups of aspartate (Figure 16(a)). It is also referred to as cyanophycin grana protein (CGP) or multi-l-arginyl-poly-l-aspartate in the literature. Lysine and some other amino acids, for example, ornithine or citrulline, may in some bacteria partially replace arginine in the cyanophycin synthesized in the cytoplasm or during in vitro biosynthesis of cyanophycin using the purified enzyme. It is found in the cytoplasm as insoluble membrane-less inclusions (Figure 16(b)). Bacteria accumulate cyanophycin as storage compound for nitrogen, carbon, and energy when growth is limited by another nutrient.

Iminodisuccinic Acid Sodium Salt(IDS-Na)

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Figure 16. (a) Structural formula of cyanophycin. (b) Transmission electron micrograph of cells of the Gram-negative bacterium Acinetobacter baylyi showing cyanophycin granules. Cells were grown in a mineral salts medium containing 75 mmol l⁻¹ arginine and 10 mmol l⁻¹ ammonium sulfate and had accumulated cyanophycin up to about 40% of the cell dry matter. Bar = 1 μm. Micrograph courtesy of Yasser Elbahloul and Rudolf Reichelt, Westfälische Wilhelms-Universität Münstery, with permission from Shively JM, Cannon GC, Heinhorst S, et al. (2006) Bacterial inclusions. In: Encyclopedia of Life Sciences. Chichester: John Wiley & Sons, Ltd. http://www.els.net/[doi: 10.1038/npg.els.0004268].

Cyanophycin is synthesized by many cyanobacteria and also several nonphotosynthetic bacteria like Acinetobacter calcoaceticus independently of ribosomal protein biosynthesis. The key enzyme of cyanophycin synthesis is cyanophycin synthetase encoded by cphA. The enzyme catalyses the consecutive addition of aspartate and arginine residues to a primer or to the growing polymer chain and requires one ATP for addition of each constituent. In addition, the enzyme is dependent on the presence of K⁺, Mg²⁺, a (cyanophycin) primer, and a thiol reagent such as β-mercaptoethanol in the reaction mixture. Cyanophycin is intracellularly degraded by cyanophycinases encoded by cphB if growth resumes yielding β–Asp–Arg dipeptides that are hydrolysed by a peptidase to the monomers. Cyanophycin is extracellularly also degraded by cyanophycinases secreted into the medium by some bacteria. Proteases seem generally not to be capable of hydrolyzing cyanophycin.

So far technical applications are not known for cyanophycin. However, due to the availability of cyanophycin synthetase genes from several cyanobacteria and the occurrence of cyanophycin also in heterotrophic bacteria that grow much faster and to many higher cell densities than cyanobacteria, cyanophycin has become much more readily available. It is foreseen that the material properties and applications will now be intensively investigated. However, cultivation of heterotrophic bacteria for production of large amounts of cyanophycin in the cells seems to be a burden for the organism due to the competition of the ribosomal protein biosynthesis machinery and the cyanophycin synthetases for the amino acids aspartate and arginine. This can be overcome by using amino acid rich complex media or by directly feeding both amino acids. However, this increases the production process costs. A further alternative is to use an addiction system. A recombinant strain of R. eutropha has been recently constructed for the production of cyanophycin with a defective chromosomal KDPG aldolase gene (eda), which is essential for growth of the cells if, for example, fructose is used as carbon source. Instead, the strain contained a plasmid containing not only a cyanophycin synthetase gene (cphA) but also an intact eda gene. Thereby, cphA was coupled to eda, and therefore the cells could not lose the plasmid if they grew on fructose.