bCyanophycin, a polyamide of cyanobacterial or noncyanobacterial origin consisting of aspartate, arginine, and lysine, was synthesized in different recombinant strains of Escherichia coli expressing cphA from Synechocystis sp. strain PCC 6308 or PCC 6803, Anabaena sp. strain PCC 7120, or Acinetobacter calcoaceticus ADP1. The molar aspartate/arginine/lysine ratio of the water-soluble form isolated from a recombinant strain expressing CphA 6308 was 1:0.5:0.5, with a lysine content higher than any ever described before. The water-insoluble form consisted instead of mainly aspartate and arginine residues and had a lower proportion of lysine, amounting to a maximum of only 5 mol%. It could be confirmed that the synthesis of soluble cyanobacterial granule polypeptide (CGP) is independent of the origin of cphA. Soluble CGP isolated from all recombinant strains contained a least 17 mol% lysine. The total CGP portion of cell dry matter synthesized by CphA 6308 from recombinant E. coli was about 30% (wt/ wt), including 23% (wt/wt) soluble CGP, by using terrific broth complex medium for cultivation at 30°C for 72 h. Enhanced production of soluble CGP instead of its insoluble form is interesting for further application and makes recombinant E. coli more attractive as a suitable source for the production of polyaspartic acid or dipeptides. In addition, a new low-cost, time-saving, effective, and common isolation procedure for mainly soluble CGP, suitable for large-scale application, was established in this study.C yanophycin, a highly branched polypeptide that is contained in the cyanobacterial granule, consists of a polyaspartic acid backbone to which arginine residues are linked by isopeptide bonds at free carboxylate groups, and the polymer is therefore called multi-L-arginyl-polyaspartic acid and cyanobacterial granule polypeptide (CGP) (1, 2). The polypeptide is synthesized via ATP-, MgCl 2 -, KCl-, and sulfhydryl reagent-dependent nonribosomal protein biosynthesis by the cphA-encoded cyanophycin synthetase CphA (1, 3). The incorporation of L-arginine is dependent on the presence of L-aspartic acid and results in a branched copolymer with molecular masses ranging from 25 to 100 kDa (1, 4). The accumulation of CGP in cyanobacteria is, for example, triggered by phosphate starvation (5) and serves the cells as a nitrogen and energy storage compound (3). The ability to synthesize CGP is not a unique feature of cyanobacteria but also occurs in other groups of bacteria (6, 7). The properties of CGP concerning solubility are interesting, because it is insoluble at neutral pH but soluble at pHs of Ͻ2 and Ͼ9 (8). This makes it easy to isolate CGP with 0.1 N HCl, because after the treatment of cyanobacterial cells with acid, the only polymer isolated from extracts is a polymer consisting of arginine and aspartic acid at a molar ratio of about 1:1 (3).Heterologously synthesized CGP, e.g., from bacterial hosts like Escherichia coli, Pseudomonas putida, and Ralstonia eutropha or from yeasts, is quite different (9, 10, 11). The water...

Cyanophycin, inclusions in cyanobacteria discovered by the Italian scientist Borzi in 1887, were characterized as a polyamide consisting of aspartic acid and arginine. Its synthesis in cyanobacteria was analyzed regarding growth conditions, responsible gene product, requirements, polymer structure and properties. Heterologous expression of diverse cyanophycin synthetases (CphA) in Escherichia coli enabled further enzyme characterization. Cyanophycin is a polyamide with variable composition and physiochemical properties dependent on host and cultivation conditions in contrast to the extracellular polyamides poly-γ-glutamic acid and poly-ε-l-lysine. Furthermore, recombinant prokaryotes and transgenic eukaryotes, including plants expressing different cphA genes, were characterized as suitable for production of insoluble cyanophycin regarding higher yields and modified composition for other requirements and applications. In addition, cyanophycin was characterized as a source for the synthesis of polyaspartic acid or N-containing bulk chemicals and dipeptides upon chemical treatment or degradation by cyanophycinases, respectively. Moreover, water-soluble cyanophycin derivatives with altered amino acid composition were isolated from transgenic plants, yeasts and recombinant bacteria. Thereby, the range of dipeptides could be extended by biological processes and by chemical modification, thus increasing the range of applications for cyanophycin and its dipeptides, including agriculture, food supplementations, medical and cosmetic purposes, synthesis of the polyacrylate substitute poly(aspartic acid) and other applications.

Soluble cyanobacterial granule polypeptide (CGP), especially that isolated from recombinant Escherichia coli strains, consists of aspartic acid, arginine, and a greater amount of lysine than that in insoluble CGP isolated from cyanobacteria or various other recombinant bacteria. In vitro guanidination of lysine side chains of soluble CGP with o-methylisourea (OMIU) yielded the nonproteinogenic amino acid homoarginine. The modified soluble CGP consisted of 51 mol% aspartate, 14 mol% arginine, and 35 mol% homoarginine. The complete conversion of lysine residues to homoarginine was confirmed by (i) nuclear magnetic resonance spectrometry, (ii) coupled liquid chromatography-mass spectrometry, and (iii) high-performance liquid chromatography. Unlike soluble CGP, this new homoarginine-containing polyamide was soluble only under acidic or alkaline conditions and was insoluble in water or at a neutral pH. Thus, it showed solubility behavior similar to that of the natural insoluble polymer isolated from cyanobacteria, consisting of aspartic acid and arginine only. Polyacrylamide gel electrophoresis revealed similar degrees of polymerization of the native (12- to 40-kDa) and modified (10- to 35-kDa) polymers. This study showed that the chemical structure and properties of a biopolymer could be changed by in vitro introduction of a new functional group after biosynthesis of the native polymer. In addition, the modified CGP could be digested in vitro using the cyanophycinase from Pseudomonas alcaligenes strain DIP1, yielding a new dipeptide consisting of aspartate and homoarginine.