2′-岩藻糖基乳糖(2′-fucosyllactose,2′-FL)是人初乳中含量最高的母乳寡糖组分,在维持肠道生态平衡、抵抗病原菌的黏附、调节免疫系统及促进神经系统发育等方面发挥重要作用。该研究选用大肠杆菌MG1655作为出发菌株,首先在基因组中整合来源于幽门螺旋杆菌NCTC11639的α1,2-岩藻糖基转移酶(α1,2-fucosyltransferase,FutC),实现了以甘油为碳源从头合成2′-FL。进一步的,通过敲除分支途径基因lacZ和wcaJ、过表达内源乳糖渗透酶(lactose permease,LacY)和鸟苷二磷酸-L-岩藻糖合成途径酶等组合代谢工程改造,强化了2′-FL生产过程中糖基供体与受体的合成供应,有效提高了2′-FL的生产强度。最终,替换重组菌株基因组中FutC为高活性突变体FutCV93I,2′-FL产量在摇瓶和5 L发酵罐水平上分别提高至6.57、31.13 g/L,为后续2′-FL的规模化工业生产提供了重要的数据支持。
2′-Fucosyllactose (2′-FL), the most abundant human milk oligosaccharide component in human colostrum, plays crucial roles in maintaining intestinal ecological balance, resisting the adhesion of pathogenic bacteria, regulating the immune system, and promoting the development of the nervous system.In this study, Escherichia coli MG1655 was employed as the host strain.First, the α1,2-fucosyltransferase gene (futC) from Helicobacter pylori NCTC11639 was chromosomally integrated, enabling de novo 2′-FL biosynthesis using glycerol as the sole carbon source.Subsequently, a series of metabolic engineering strategies were implemented, including deletion of competing pathway genes (lacZ and wcaJ), overexpression of endogenous lactose permease (LacY), and enhancement of GDP-L-fucose synthesis pathway enzymes, thereby improving the supply of glycosyl donors and acceptors and significantly boosting 2′-FL productivity.Finally, replacement of wild-type FutC with the high-activity variant FutCV93I elevated 2′-FL titers to 6.57 g/L in shake flasks and 31.13 g/L in a 5 L bioreactor, providing important data support for the subsequent large-scale industrial production of 2′-FL.
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