代谢工程改造少动鞘氨醇单胞菌发酵生产结冷胶

Metabolic engineering of Sphingomonas paucimobilis for the production of gellan gum

摘要:

目的 以少动鞘氨醇单胞菌为出发菌株，通过代谢工程改造构建高产结冷胶工程菌株，并优化发酵工艺，为利用该菌高效合成结冷胶提供理论支撑与工艺基础。方法 基于CRISPR-Cas9系统开发适用于少动鞘氨醇单胞菌的基因编辑工具，并利用该工具在基因组中整合组成型启动子驱动的结冷胶合成基因簇调控蛋白基因(gelA)和β-1,4-葡萄糖醛酸转移酶基因(gelK)。在此基础上，通过单因素试验优化了碳源、氮源、pH及溶氧等发酵工艺参数。结果 工程菌株FMME-GG08在摇瓶培养中结冷胶产量达到10.8 g/L，较出发菌株提升130.2%。发酵工艺优化后，在15 L规模发酵罐中结冷胶产量达到20.1 g/L，蔗糖转化效率为0.50 g/g。结论 本研究不仅成功构建了结冷胶高产菌株并建立了高效发酵工艺，为工业化生产提供了可靠技术方案，同时所开发的基因编辑策略也为代谢工程改造少动鞘氨醇单胞菌等非模式微生物生产其他高价值胞外多糖提供了重要参考。

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.