A novel recombinant PHB production platform in filamentous cyanobacteria avoiding nitrogen starvation while preserving cell viability.

During the past decades, the importance of developing sustainable, carbon dioxide (CO₂)-neutral and biodegradable alternatives to conventional plastic has become evident in the context of global pollution issues. Therefore, heterotrophic bacteria such as Cupriavidus sp. have been intensively explored for the synthesis of the biodegradable polymer polyhydroxybutyrate (PHB). PHB is also naturally produced by a variety of phototrophic cyanobacteria, which only need sunlight and CO_2, thereby allowing a CO₂ negative, eco-friendly synthesis of this polymer. However, a major drawback of the use of cyanobacteria is the need of a two-stage production process, since relevant amount of PHB synthesis only occurs after transferring the cultures to conditions of nitrogen starvation, which hinders continuous, large-scale production.This study aimed at generating, by means of genetic engineering, a cyanobacterium that continuously produces PHB in large amounts. We choose a genetically amenable filamentous cyanobacterium of the genus Nostoc sp., which is a diazotrophic cyanobacterium, capable of atmospheric nitrogen (N₂) fixation but naturally does not produce PHB. We transformed this Nostoc strain with various constructs containing the constitutive promotor P_psbA and the PHB synthesis operon phaC1AB from Cupriavidus necator H16. In fact, while the transformants initially produced PHB, the PHB-producing strains rapidly lost cell viability. Therefore, we next attempted further optimization of the biosynthetic gene cluster. The PHB operon was expanded with phasin gene phaP1 from Cupriavidus necator H16 in combination with the native intergenic region of apcBA from Nostoc sp. 7120. Finally, we succeeded in stabilized PHB production, whilst simultaneously avoiding decreasing cell viability. In conclusion, the recombinant Nostoc strain constructed in the present work constitutes the first example of a continuous and stable PHB production platform in cyanobacteria, which has been decoupled from nitrogen starvation and, hence, harbours great potential for sustainable, industrial PHB production.