Utilization of phosphate-responsive promoters for auto-inducible management of biotechnological processes based on cyanobacteria

Controllable gene expression is essential in microbial biotechnology, yet most systems rely on costly external inducers that limit large-scale applicability. Here, we employed phosphate-responsive promoters controlled by the SphS–SphR phosphate-sensing two-component system as auto-inducible expression systems in cyanobacteria. Specifically, we characterized the promoters of phoA , sphX , and pstS2, which are activated under low phosphate availability and achieved induction folds of up to 13, as well as P urtA and a synthetic promoter P P i -neg , which are repressed under these conditions. Replacement of the ribosome binding site in the native promoter systems further expanded the accessible range of expression levels, with context-dependent increases or decreases depending on the promoter. By systematically adjusting the phosphate concentration and cell density, the timing of gene expression could be precisely controlled. Notably, intracellular phosphate storage during early growth enables transient buffering of external depletion, allowing promoter activation prior to growth limitation. This, in turn, enables their use in continuously growing cultures. As a proof of concept, we established a sucrose production process that autonomously transitioned from a growth phase with basal production to a production phase with a 5.5-fold higher sucrose titre, triggered by phosphate depletion during cultivation. Overall, this auto-inducible expression system expands the cyanobacterial genetic toolbox and enhances the applicability of cyanobacteria in scalable production processes.