Construction of a synthetic biology toolkit for the genetic manipulation in the cellulose-producing strain Kosakonia oryzendophytica

Bacterial cellulose (BC), a robust and highly crystalline nanomaterial composed of glucose polymers, exhibits exceptional properties including superior water retention capacity, biocompatibility, and customizable mechanical strength, positioning it as a promising candidate for advanced material applications. To harness its full potential, we developed a synthetic biology platform for engineering <i>Kosakonia oryzendophytica</i>-a hyperproductive BC synthesis strain. First, we systematically characterized a library of regulatory elements (promoters, ribosome binding sites, and terminators, etc.) in this chassis, demonstrating tunable expression intensities ranging from 1.84 % to 169 % relative to the canonical <i>LacO1</i> promoter. Subsequently, we established a high-efficiency CRISPR/Cas9-mediated scarless genome editing system through coordinated optimization of λ Red recombinase and Cas9 nuclease expression, achieving near-perfect editing efficiency (≈100 %). This system was functionally validated by targeted knockout of key genes (<i>bcsA</i>, <i>fbp</i>, and <i>galU</i>), with scanning electron microscopy analysis confirming the BC synthesis deficiency in Δ<i>bcsA</i> and Δ<i>fbp</i> mutants. The integration of these genetic tools-comprising tunable expression modules and precision genome-editing capabilities-provides a comprehensive toolkit for reprogramming <i>K. oryzendophytica</i> to produce next-generation cellulose-based functional materials with tailored properties.