Extracellular laccase activity in engineered Synechocystis sp. PCC 6803 for mediator-free textile dye decolorization

Industrial dye discharge into aquatic ecosystems poses persistent environmental and health risks, requiring sustainable and scalable remediation strategies. Laccases are versatile multicopper oxidases capable of degrading a wide spectrum of intractable dyes; however, their industrial application is often constrained by production costs and downstream processing requirements. Harnessing phototrophic microorganisms as self-sustaining enzyme production platforms offers a promising alternative. In this study, we engineered the model cyanobacterium Synechocystis sp. PCC 6803 to secrete its native laccase, SLR1573, using two secretion tags: the type IV pilus-associated PilA signal peptide and a novel C-terminal repeat-in-toxin (RTX) fusion tag. Both designs reported extracellular laccase activity and decolorization of the anthraquinone dye Remazol Brilliant Blue R. The RTX-tagged laccase exhibited the highest extracellular activity (370 U/L) reported in cyanobacteria, and remarkable thermal stability, maintaining activity at temperatures up to 80 °C. Under acidic conditions, extracellular SLR1573 achieved 80 % decolorization of Remazol Brilliant Blue R within 1 h without the addition of redox mediators. This is the first report of mediator-free decolorization of an anthraquinone dye by a cyanobacterial laccase acting directly in the culture medium. The combination of phototrophic, low-cost cultivation and continuous extracellular enzyme production without biomass disruption could represent a promising step towards a scalable and economically attractive alternative for textile dye remediation. These findings demonstrate the feasibility of coupling enzyme secretion with dye decolorization in a single microbial chassis. This work advances cyanobacteria as cost-effective and sustainable biocatalytic hosts for industrial wastewater remediation.