Signal peptides for engineered protein secretion by free and hydrogel-encapsulated cyanobacteria

Cyanobacteria provide a promising platform for light-driven heterologous protein production. In heterologous production systems, cells are typically lysed for product purification or to facilitate interaction with external substrates, unless the product is secreted. Fusion to molecular "shipping labels" known as signal peptides can target chimeric proteins to native membrane translocation pathways and result in secretion. In this work, we identified signal peptides from the model cyanobacterium Synechococcus elongatus PCC 7942 (S. elongatus) and genetically fused to two heterologous enzymes: the reporter nanoluciferase (NLuc) and an oxidative laccase (CotA). Increased extracellular activity was observed for one or more signal-protein variants for both enzymes. Furthermore, cells of S. elongatus producing a signal-laccase chimera were encapsulated within an alginate hydrogel, and the resulting engineered living material released sufficient enzyme to decolorize a dye pollutant in its surroundings. These findings streamline recombinant protein production in cyanobacteria and facilitate environmental interactions with cell products, expanding the potential applications of both free-living and material-encapsulated cyanobacteria.