Increased Lysine Content Is the Main Characteristic of the Soluble Form of the Polyamide Cyanophycin Synthesized by Recombinant Escherichia coli

Abstract

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...

Solubility Behavior of Cyanophycin Depending on Lysine Content

“…Several theories, including differences in the structure of the polymer; variations in the cell milieu influencing the specificity of the cyanophycin synthetase; or the incorporation of alternative constituents, like Lys, citrulline, and ornithine, have been discussed (6,8). We are now confident that we have identified one of the factors leading to the formation of soluble CGP in E. coli.…”

“…Pseudomonas putida, for example, synthesized soluble CGP only when cultivated at an elevated temperature of 37°C, while insoluble CGP was formed only at the usual growth temperature of 30°C (8). Several studies on Escherichia coli showed that synthesis of soluble CGP occurred only at a temperature of 30°C or less, while cells of the same strain grown at 37°C contained much less or no soluble CGP (6,14). Another theory suggested that the increased amounts of alternative constituents influenced the solubility or the structure of CGP, or both (6).…”

“…Several studies on Escherichia coli showed that synthesis of soluble CGP occurred only at a temperature of 30°C or less, while cells of the same strain grown at 37°C contained much less or no soluble CGP (6,14). Another theory suggested that the increased amounts of alternative constituents influenced the solubility or the structure of CGP, or both (6). Also, combinations of these theories were suggested (6).…”

“…Another theory suggested that the increased amounts of alternative constituents influenced the solubility or the structure of CGP, or both (6). Also, combinations of these theories were suggested (6). Having an explanation for the solubility behavior might also be helpful in improving the downstream processing and purification of CGP and the correlated dipeptides.…”

Analysis of the Spore Membrane Proteome in Clostridium perfringens Implicates Cyanophycin in Spore Assembly

“…These ratios suggest that this is a typical cyanophycin polymer with an aspartate backbone and arginine and lysine side chains, since aspartate accounts for 50% of the residues and the other two amino acids account for the remaining 50%. The solubility of cyanophycin is affected by the lysine content (28), and recombinant cphA genes from other bacteria expressed in E. coli produce soluble cyanophycin with increased lysine content (55). This makes it difficult to predict if cyanophycin exists in a soluble or insoluble form in C. perfringens.…”