Abstract

Synthesis of the biopolymer cyanophycin (CGP) in recombinant organisms has been studied for several years due to its unique properties, easy purification and putative use as a precursor for biodegradable chemicals. CGP and CGP dipeptides are also interesting for applications in agriculture, food supplementation, medical and cosmetic purposes. Therefore an efficient production method is key for commercial use of CGP and new variations of CGP in terms of alternative compositions and constituents are always desirable to increase the putative applications. In our recent studies we demonstrated the synthesis of soluble and insoluble CGP in E. coli up to the 30 L-scale. Using an optimized cultivation method and a plasmid-based addiction system we were able to achieve a cell density of 10.2 g/L and a CGP content of 36.2% wt/wt of the CDM (25.1% insoluble and 11.1% soluble CGP). Both forms of the polymer showed high amounts of lysine replacing the arginine residues. The lysine content was identified as a critical factor for the solubility behavior of the polymer and by using a temperature dependent purification method it was possible to fractionate insoluble CGP in dependence of its specific lysine content. Insoluble CGP showing a lysine content of less than 4 mol% was not soluble even at 90°C, while insoluble CGP containing 31 mol% lysine was soluble at 30°C. Higher lysine contents would result in the formation of CGP soluble at even lower temperatures explaining the occurrence of soluble CGP. In our case the soluble CGP isolated from this strain showed a lysine content of over 36 mol%, proving the relation of lysine content and solubility behavior. Using this soluble CGP we established a chemical procedure to modify the polymer. In vitro guanidination of the lysine side chains with o-methylisourea (OMIU) yielded the nonproteinogenic amino acid homoarginine. Analysis of the modified polymer showed that 100% of the lysine side chains had been converted to homoarginine. The conversion also caused a solubility change of the modified CGP, which now resembled the behavior of insoluble CGP showing again the influence of lysine on the solubility of CGP. By establishing a chemical procedure to modify CGP we added another dipeptide to the spectrum of possible applications, and also showed for the first time that modification of CGP after its synthesis is feasible and not only limited to the digestion into dipeptides or a total hydrolysis of the polymer to its basic amino acids. Based on this results we opened a whole new range of possible modifications for further research.