State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, 214122, Jiangsu, China.
International Joint Laboratory on Food Safety, Jiangnan University, Wuxi, 214122, Jiangsu, China.
Mol Biotechnol. 2023 Sep;65(9):1485-1497. doi: 10.1007/s12033-023-00657-7. Epub 2023 Jan 18.
2'-Fucosyllactose (2'-FL), one of the most abundant oligosaccharides in human milk, has gained increased attention owing to its nutraceutical and pharmaceutical potential. However, limited availability and high-cost of preparation have limited its widespread application and in-depth investigation of its potential functions. Here, a modular pathway engineering was implemented to construct an Escherichia coli strain to improve the biosynthesis titer of 2'-FL. Before overexpression of manB, manC, gmd, wcaG, and heterologous expression of futC, genes wcaJ and lacZ encoding UDP-glucose lipid carrier transferase and β-galactosidase, respectively, were inactivated from E. coli BL21 (DE3) with the CRISPR-Cas9 system, which inhibited the production of 2'-FL. The results showed that final shake flask culture yielded a 3.8-fold increase in 2'-FL (0.98 g/L) from the engineered strain ELC07. Fed-batch fermentation conditions were optimized in a 3-L bioreactor. The highest titer of 2'-FL (18.22 g/L) was obtained, corresponding to a yield of 0.25 g/g glycerol and a substrate conversion of 0.88 g/g lactose.
2'-岩藻糖基乳糖（2'-FL）是母乳中最丰富的寡糖之一，由于其具有营养和药用潜力，引起了越来越多的关注。然而，其有限的可用性和高制备成本限制了其广泛应用和对其潜在功能的深入研究。在这里，实施了模块化途径工程，以构建能够提高 2'-FL 生物合成产率的大肠杆菌菌株。在过表达 manB、manC、gmd、wcaG 和异源表达 futC 之前，使用 CRISPR-Cas9 系统敲除了编码 UDP-葡萄糖脂质载体转移酶和β-半乳糖苷酶的基因 wcaJ 和 lacZ，从而抑制了 2'-FL 的产生。结果表明，最终摇瓶培养使工程菌株 ELC07 中的 2'-FL（0.98 g/L）产量增加了 3.8 倍。在 3-L 生物反应器中优化了分批补料发酵条件。获得了最高的 2'-FL 浓度（18.22 g/L），对应的 2'-FL 产率为 0.25 g/g 甘油，底物转化率为 0.88 g/g 乳糖。
