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Engineering signal peptide-mediated recombinant protein secretion in Komagataeibacter rhaeticus for biological Engineered Living Material applications

Vannas, J.; Singh, A.; Hannikainen, B.; de Assis, M. A.; Ledesma-Amaro, R.; Ellis, T.; Mangayil, R.

2025-12-17 bioengineering
10.64898/2025.12.15.694344 bioRxiv
Show abstract

Bacterial nanocellulose (BC) from Komagataeibacter spp. is an ideal scaffold for biological Engineered Living Materials (bioELMs). Engineering Komagataeibacter to simultaneously synthesize and functionalize BC could transform current workflows, but protein secretion in this organism remains poorly understood. Here, we demonstrate recombinant protein secretion in K. rhaeticus iGEM by leveraging its genome-encoded Sec-translocase. Using secretome analysis to identify native Sec signal peptides (SPs), secretion efficiencies of mScarlet and {beta}-lactamase variant libraries were benchmarked under pellicle-forming and non-pellicle conditions. Inducible expression eliminated metabolic burden and enabled secretion without cell lysis, as confirmed by fluorescence and scanning electron microscopy. Comparative analyses revealed strong condition-dependent variations in secretion performance, with native SPs exhibiting higher efficiency under pellicle-forming conditions. Finally, BC functionalization using secreted {beta}-lactamase was demonstrated, with no detrimental effects on the BC material. This work establishes the first comparative framework for SP-mediated secretion in Komagataeibacter and provides a foundation for next-generation BC-based bioELMs.

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