Molecular characterization of the cyanophycin synthetase from Synechocystis sp. strain PCC6308

Abstract.

A 3878-bp genomic region from the cyanobacterium Synechocystis sp. strain PCC6308, amplified by inverse PCR, harbored the structural genes cphA (2625 bp) and cphB (819 bp) encoding cyanophycin synthetase and cyanophycinase, respectively. Both primary structures exhibited a high degree of similarity to the corresponding translational products from other cyanobacteria. Five regions were localized in the cyanophycin synthetase consensus sequence by their resemblance to conserved sites of ATP-dependent carboxylate-amine/thiol ligases and three substrate ligases. The functionality of cphA was proven by heterologous expression of active enzyme and synthesis of cyanophycin in Escherichia coli, which led to a maximum cyanophycin content of 26.6% (w/w) of cell dry mass. Furthermore, a modified radiometric enzyme assay for a more reliable and feasible measurement of cyanophycin synthetase activity was developed and applied to reveal the substrate specificity of the enzyme.