Cyanophycin Optimizes Growth and Nitrogen Fixation in the Unicellular Diazotroph Cyanothece sp. ATCC 51142

Many cyanobacteria synthesize cyanophycin, a nitrogen-rich amino acid polymer with metabolic engineering and biomanufacturing potential. In non-diazotrophic cyanobacteria, cyanophycin serves as a source of nitrogen under nitrogen stress conditions. However, the role of these storage granules in diazotrophic cyanobacteria, which fix nitrogen on demand, is yet to be understood. The enzyme cyanophycin synthetase, encoded by cphA, synthesizes cyanophycin from the amino acids aspartate and arginine. We probed the consequences of the inability to synthesize cyanophycin on the physiology of a nitrogen-fixing unicellular cyanobacterium, Cyanothece sp. ATCC 51142, by generating a markerless cphA deletion strain (∆cphA) using CRISPR/Cpf1. Under continuous high light and N2-fixing conditions, the ∆cphA strain exhibited a growth defect and phycobilisome degradation, implying nitrogen starvation. Interestingly, under low light conditions, the nitrogen starvation phenotype was not observed. This suggests a critical role for the nitrogen storage bodies in maintaining an optimal cellular carbon/nitrogen balance, especially when the cellular nitrogen fixing machinery cannot match high levels of carbon fixation. Thus, when photosynthetic efficiency is high, the cyanophycin storage granules act as a readily available nitrogen source that ensures optimal metabolism and growth. This study illustrates the essential role of cyanophycin when engineering unicellular nitrogen-fixing cyanobacteria for use as production chassis.