Synthesis of cyanophycin (multi-L-arginyl-poly-L-aspartic acid, CGP) in recombinant organisms is an important option to obtain sufficiently large amounts of this polymer with a designed composition for use as putative precursors for biodegradable technically interesting chemicals. Therefore, derivates of CGP, harbouring a wider range of constituents, are of particular interest. As shown previously, cyanophycin synthetases with wide substrate ranges incorporate other amino acids than arginine. Therefore, using an organism, which produces the required supplement by itself, was the next logical step. Former studies showed that Pseudomonas putida strain ATCC 4359 is able to produce large amounts of L-citrulline from L-arginine. By expressing the cyanophycin synthetase of Synechocystis sp. PCC 6308, synthesis of CGP was observed in P. putida ATCC 4359. Using an optimised medium for cultivation, the strain was able to synthesise insoluble CGP amounting up to 14.7 ± 0.7% (w/w) and soluble CGP amounting up to 28.7 ± 0.8% (w/w) of the cell dry matter, resulting in a total CGP content of the cells of 43.4% (w/w). HPLC analysis of the soluble CGP showed that it was composed of 50.4 ± 1.3 mol % aspartic acid, 32.7 ± 2.8 mol % arginine, 8.7 ± 1.6 mol % citrulline and 8.3 ± 0.4 mol % lysine, whereas the insoluble CGP contained less than 1 mol % of citrulline. Using a mineral salt medium with 1.25 or 2% (w/v) sodium succinate, respectively, plus 23.7 mM L-arginine, the cells synthesised insoluble CGP amounting up to 25% to 29% of the CDM with only a very low citrulline content.
The genome sequence of the facultative chemolithoautotrophic bacterium Ralstonia eutropha H16 exhibited two coding sequences with high homologies to cyanophycin synthetases (CphA) as well as one gene coding for a putative cyanophycinase (CphB). To investigate whether or not the genes cphA H16 (H16_A0774), cphA'H16 (H16_A0775) and cphB H16 (H16_B1013) encode active cyanophycin (CGP) metabolism proteins, several functional analyses were performed. Extensive in silico analysis revealed that all characteristic motifs are conserved within CphAH16, whereas CphA'H16 misses a large part of the so-called J-loop present in other active cyanophycin synthetases. Although transcription of both genes was demonstrated by RT-PCR, and heterologously expressed cphA genes led to light-scattering inclusions in recombinant cells of Escherichia coli, no CGP could be isolated from the cells or detected by HPLC analysis. For all enzyme assay experiments carried out, significant enzyme activities were determined for CphA and CphA' in recombinant E. coli cells if crude cell extracts were applied. Homologous expression of cphA genes in cells of R. eutropha H16∆phaC1 did not result in the formation of light-scattering inclusions, and no CGP could be isolated from the cells or detected by HPLC analysis. No transcription of cphB encoding a putative cyanophycinase could be detected by RT-PCR analysis and no overexpression was achieved in several strains of E. coli. Furthermore, no enzyme activity was detected by using CGP overlay agar plates.
enzyme catalyzing the polymerization of two peptidoglycan precursors in bacterial cell wall biogenesis.1. The best investigated mechanism, distributed ubiquitously in living matter, is the template -dependent ribosomal synthesis of proteins. Here the amino acids are activated by adenylation catalyzed by aminoacyl -tRNA synthetases [1] .2. The second mechanism is performed by nonribosomal peptide synthetases ( NRPS ) which are multienzyme complexes consisting of four domains [2, 3] . The adenylation domain required for activation of the substrate at the expense of ATP, via the formation of an enzyme -stabilized aminoacyl adenylate. As the formed adenylate is not stable, the energy is further conserved by transfer of the peptide to: the thiolation domain giving rise to a thioester bond formed between the amino acid and a cysteine of the enzyme complex.The condensation domain is required for formation of peptide bonds between the itemized monomers, and the thioesterase domain catalyzes the release of the fi nal product from the NRPS by cyclization to an amide or ester, or by hydrolysis to the free acid [3] . The transfer of the intermediates is mediated by the cofactor pantetheine. Numerous peptide antibiotics such as penicillin, bacitracin, and actinomycin are synthesized by NRPS (reviewed by [2] ). These compounds are known to contain non -proteinogenic amino acids, d -amino acids, hydroxy acids, methylated or cyclic forms, and other unusual constituents; these modifi cations are catalyzed by the NRPS. As the enzyme complex itself functions as matrix, the resulting peptides have a strictly defi ned length which is in contrast to poly(amino acids).3. The third mechanism is represented by nonmodular one -step peptide synthesis. Enzymes belonging to this group catalyze the biosynthesis of poly(amino acids). Naturally occurring poly(amino acids) comprise cyanophycin [multil -arginyl -poly -( l -aspartic acid); cyanophycin granule polypeptide , ( CGP )], ( poly -( ε -lysine) ( PL ), and poly -( γ -glutamate) ( PGA ). As a consequence of non -ribosomal biosynthesis these peptides reveal a polydisperse mass distribution.Further aspects distinguishing poly(amino acids) from proteins are the following:proteins consist of a mixture of 22 amino acids, whereas poly(amino acids) consist of one amino acid in the polymer backbone;their biosynthesis is not constrained by translational inhibitors such as chloramphenicol [4] ;the amide bonds formed between the monomers are not exclusively linked between the α -carboxylic and α -amino groups, as for proteins, but also between β -or γ -carboxylic groups or ε -amino groups. Polyhydroxyalkanoate Synthases 249
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