Abstract:Objective Using Sphingomonas paucimobilis as the starting strain, a high-yield gellan gum-producing engineered strain was constructed through metabolic engineering, and fermentation process optimization was performed, providing both theoretical support and technical foundations for the efficient biosynthesis of gellan gum with this bacterium.Methods A CRISPR-Cas9-based gene editing system was developed for S. paucimobilis, and subsequently employed to genomically integrate two key gellan gum biosynthesis genes: the regulatory protein gene (gelA) and the β-1,4-glucuronosyltransferase gene (gelK), both under the control of constitutive promoters. Building upon this foundation, fermentation parameters including carbon source, nitrogen source, pH, and dissolved oxygen were systematically optimized through single-factor experiments.Results The engineered strain FMME-GG08 achieved a gellan gum yield of 10.8 g/L in shake-flask cultivation, representing a 130.2% enhancement over the parental strain. Following fermentation process optimization, the production level reached 20.1 g/L in 15 L scale bioreactors, with a sucrose conversion efficiency of 0.50 g/g.Conclusion This study not only successfully constructed a high-yield gellan gum-producing strain and established an efficient fermentation process, providing a reliable technical solution for industrial production, but also developed a genetic editing strategy that serves as an important reference for metabolic engineering of non-model microorganisms to produce other high-value exopolysaccharides.