In vitro synthesis of β-aspartyl-basic amino acid dipeptides via a multi-enzyme cascade system with ATP regeneration

Cyanophycin, also known as cyanophycin granule polypeptide (CGP), was a natural polyamide synthesized via a nonribosomal pathway from L-aspartic acid and L-arginine. Its derived dipeptide, β-Asp-Arg, holds potential applications in various fields requiring arginine supplementation in feed or food industries. However, conventional CGP production methods were constrained by strict substrate specificity and high costs. To address these challenges, two-step enzymatic cascade system was constructed integrating CGP biosynthesis with dipeptide production. Central to this system is a novel cyanophycin synthetase (CphA1) from Acidobacteria bacterium, which exhibits remarkable substrate promiscuity. This enzyme enables both primer-independent polymerization of poly-L-Arg/L-Lys-poly(L-Asp) backbones and primer-dependent incorporation of non-canonical substrates (L-ornithine and L-citrulline), thereby expanding the repertoire of cyanophycin derivatives. Furthermore, polyphosphate kinase synergizes with CphA1 to regenerate ATP and promote CGP synthesis, reducing ATP consumption by 90 %. Subsequently, the polymer was efficiently hydrolyzed by CphB from Geminocystis herdmanii to yield the target dipeptides. This method achieved complete conversion of CGP into classical dipeptides β-Asp-Arg and β-Asp-Lys, with the highest titer reaching 80 mM, while also generating non-classical products β-Asp-Orn and β-Asp-Cit. These results highlight the robustness and versatility of this strategy, offering a promising route for the scalable synthesis of β-Asp-basic amino acid dipeptides.