Modulating bacterial nanocellulose crystallinity through post-transcriptional repression in Komagataeibacter xylinus

Bacterial nanocellulose (BC) is a versatile biomaterial, with its microstructural features fundamentally influencing its properties and functionality across various applications. Conventional BC modifying methods typically require additives or extra steps. We developed synthetic regulatory small RNA (sRNA)-based repression cassettes targeting the bcsD, bcsZ , and ccpA genes to control BC microstructure in situ. Initial tests assessing the specificity of these repression cassettes, which target a genome-integrated fluorescent protein, showed significant growth inhibition (>80%) caused by the overexpressed Hfq RNA chaperone. By designing 12 and 24–25 base sRNA sequences and adjusting inducer concentrations, we achieved differential repression of bcsD (6-34%), bcsZ (8-24%), and ccpA (2-20%) genes. The BC titers in bcsD -repressed cells (2.5–2.7 g L -1 ) were similar to wildtype (2.5–2.8 g L -1 ). However, significant decreases occurred in bcsZ and ccpA repressed cells, especially at higher inducer concentrations and with longer sRNA sequences (1.9 g L -1 and 1.5 g L-1, respectively). These repressions notably changed BC's amorphous content, as verified by X-ray diffraction and wide-angle X-ray scattering analyses. This sRNA-based method addresses the shortcomings of conventional techniques, like the inability to control BC modifications and the need for additional processing, offering a targeted and flexible tool for modifying BC microstructure.