Systematic Evaluation of Signal Peptide-Driven Protein Secretion in the Fast-Growing Cyanobacterium Synechococcus sp. PCC 11901

The fast-growing cyanobacterium Synechococcus sp. PCC 11901 is emerging as a promising chassis for photosynthetic biomanufacturing. Here we report recombinant protein production in PCC 11901 via signal peptide-mediated secretion, enabling direct recovery of target proteins from the culture medium without cell disruption. Seven signal peptides spanning both Sec and Tat pathways are screened using eYFP as a reporter, with secretion quantified daily over seven days by fluorescence measurements. FutA, belonging to the Tat pathway from Synechocystis sp. PCC 6803, achieves 92.2% extracellular export by day 7, substantially outperforming all Sec candidates, including the best Sec signal peptide thermitase from Cyanobacterium aponinum PCC 10605 (55.7%). Signal peptide-bearing strains exhibit growth reductions of up to 26% relative to the wild-type, with FutA most affected, indicating a general metabolic cost correlated with secretion efficiency. The best-performing signal peptides from both pathways, FutA and thermitase, are validated with secretion of lichenase. Notably, the rank order of signal peptide performance is reversed for lichenase: thermitase demonstrates 2.6-fold higher extracellular activity than FutA, indicating that optimal signal peptide selection is cargo-dependent. These results establish PCC 11901 as a secretion-competent chassis and provide a rational framework for matching signal peptide pathways to target protein properties.