Construction of a synthetic biology toolkit for the genetic manipulation in the cellulose-producing strain Kosakonia oryzendophytica
Year: 2025
Type: article
Abstract: 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 Kosakonia oryzendophytica —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 LacO1 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 ( bcsA , fbp , and galU ), with scanning electron microscopy analysis confirming the BC synthesis deficiency in Δ bcsA and Δ fbp mutants. The integration of these genetic tools—comprising tunable expression modules and precision genome-editing capabilities—provides a comprehensive toolkit for reprogramming K. oryzendophytica to produce next-generation cellulose-based functional materials with tailored properties.
Authors: Liangtian Miao, Wei Feng, Jiaxun Ren, Keke Sun, Guoqiang Li, Huifeng Jiang
Language: English
Subfield: Biomaterials
Field: Materials Science
Domain: Physical Sciences
Open Access status: gold
Estimated APC paid: $1,500
Award: G4712977176
OpenAlex ID: W4410769559