Tailoring Bacterial Cellulose through the CRISPR/Cas9-Mediated Gene Editing Tool in <i>Komagataeibacter xylinus</i>

Bacterial cellulose (BC) is a nanocellulose produced by bacteria, formed by glucose units linked through β-1,4 glycosidic bonds. It features a three-dimensional network structure, superior water retention capacity, high porosity, and outstanding biocompatibility, among other notable characteristics. <i>Komagataeibacter xylinus</i> was the predominant strain used for BC production. The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associate-protein 9)-mediated gene editing tool has been applied in various species; however, its application in <i>K. xylinus</i> has not been reported. To facilitate metabolic pathway engineering in <i>K. xylinus</i>, a CRISPR/Cas9-mediated gene editing tool specific to this strain was developed, achieving a gene editing efficiency exceeding 73%. Upon application of the CRISPR/Cas9-mediated gene editing tool in <i>K. xylinus</i>, the strain's ability to synthesize BC was enhanced by 23.6% (5.75 g/L), and the impact of BC synthase-correlated genes (<i>bcsH</i>, <i>bcsX</i>, <i>bcsY</i>, <i>and bcsZ</i>) on BC structure was investigated. The advancement of CRISPR/Cas9-mediated gene editing tools in <i>K. xylinus</i> is expected to accelerate genetic modification of this organism. This advancement has the potential to significantly improve our understanding of the genetic regulatory mechanisms that govern the structure and production of BC, thereby facilitating cost-effective synthesis of BC with tailored structural properties.