3-岩藻糖基乳糖(3-FL),母乳中一种重要的岩藻糖基化人乳寡糖,为婴儿提供许多健康益处。以前,我们对大肠杆菌BL21(DE3)进行了代谢工程,用于体内3-FL的生物合成。在这项研究中,我们最初优化了培养条件,使3-FL产量加倍。参与体内鸟苷5'-二磷酸-岩藻糖生物合成的竞争途径基因随后被灭活,以将通量重定向到3-FL生物合成。接下来,使用基于质粒或染色体整合的表达评估了三种有前途的转运蛋白,以最大程度地提高细胞外3-FL的产量。此外,通过分析α1,3-岩藻糖基转移酶(FutM2)结构,我们将Q126残基鉴定为活性位点中的高度可突变残基。位点饱和突变后,表现最好的变种人,FutM2-Q126A,已获得。结构分析和分子动力学模拟表明,小的残留物置换会积极影响螺旋结构的生成。最后,最佳菌株BD3-A在摇瓶和补料分批培养中产生6.91和52.1g/L的3-FL,分别,突出了其大规模工业应用的潜力。
3-Fucosyllactose (3-FL), an important fucosylated human milk oligosaccharide in breast milk, offers numerous health benefits to infants. Previously, we metabolically engineered Escherichia coli BL21(DE3) for the in vivo biosynthesis of 3-FL. In this study, we initially optimized culture conditions to double 3-FL production. Competing pathway genes involved in in vivo guanosine 5\'-diphosphate-fucose biosynthesis were subsequently inactivated to redirect fluxes toward 3-FL biosynthesis. Next, three promising transporters were evaluated using plasmid-based or chromosomally integrated expression to maximize extracellular 3-FL production. Additionally, through analysis of α1,3-fucosyltransferase (FutM2) structure, we identified Q126 residues as a highly mutable residue in the active site. After site-saturation mutation, the best-performing mutant, FutM2-Q126A, was obtained. Structural analysis and molecular dynamics simulations revealed that small residue replacement positively influenced helical structure generation. Finally, the best strain BD3-A produced 6.91 and 52.1 g/L of 3-FL in a shake-flask and fed-batch cultivations, respectively, highlighting its potential for large-scale industrial applications.