Primer-Independent Synechococcus Cyanophycin Synthetase Enables Cell-Free Synthesis of Cyanophycin and Its Homoarginine-Substituted Analogue via Adenosine Triphosphate Regeneration

Abstract Cyanophycin granule polypeptide (CGP) is a poly(aspartate−arginine) biopolymer with potential for biomedical and sustainable material applications. However, its cell-free synthesis is constrained by the strict primer dependence of typical synthetases and the stoichiometric adenosine triphosphate (ATP) consumption. Here, cyanophycin synthetase (SyCphA1) from Synechococcus sp. PCC 7002 was characterized as a cyanophycin synthetase capable of primer-independent CGP synthesis. Mutagenesis of the conserved N-domain residue Glu82 severely impaired polymerization, indicating that Glu82 is required for efficient catalysis, rather than merely dictating primer independence. Exploiting the substrate promiscuity of SyCphA1 enabled the incorporation of homoarginine to synthesize hCGP, an analogue exhibiting higher crystallinity and enhanced thermal stability relative to native CGP. To overcome energetic limitations, SyCphA1 was coupled with a polyphosphate kinase to establish a one-pot ATP regeneration cascade driven by polyphosphate. This dual-enzyme system achieved 24-h substrate conversions of 80% and 68% for CGP and hCGP, respectively. Importantly, spontaneous polymer precipitation streamlined downstream product recovery and facilitated the proof-of-concept recycling of the soluble biocatalyst. Collectively, this work expands the synthetic utility of the CphA family and establishes a preparative cell-free platform combining noncanonical cyanophycin synthesis with ATP recycling and precipitation-assisted product recovery.