代谢工程法改造大肠杆菌合成2’-岩藻糖基乳糖

Metabolic Engineering of Escherichia coli for 2’-fucosyllactose Synthesis

摘要:

母乳寡糖对婴幼儿健康有促进作用，可保护新生儿免受感染和炎症，是婴幼儿最佳的天然食品。2’-岩藻糖基乳糖（2’-fucosyllactose，2’-FL）是母乳寡糖中特有的成分且含量较高，在婴幼儿配方奶粉以及医药产业中有广泛的应用前景。天然产物提取法和化学合成法是合成2’-FL的主要方法，但这两种方法难以大量提取2’-FL，且存在严重污染问题，因此，目前有关2’-FL生产的研究集中在温和环保的全细胞发酵方法上。以大肠杆菌为宿主细胞，在其内异源表达多聚磷酸盐依赖型甘露糖激酶AsPPGMK和α-1，2-岩藻糖基转移酶，同时共表达合成路径关键酶。为了提高2’-FL的产量，采用抑制UDP-葡萄糖脂质载体转移酶基因wcaJ的方法，阻断中间产物的降解过程，并通过优化催化转化条件，在100 mL体系中添加200 mmol/L底物，进一步提高2’-FL产量。结果表明，反应8 h后，转化率为81%，产量为79 g/L。

Abstract:

Human milk oligosaccharides play a crucial role in promoting infant health, safeguarding newborns against infection and inflammation, and serving as the optimal natural source of nutrition for infants. Among these oligosaccharides, 2’-fucosylactose (2’-FL) stands out as a prominent constituent with the highest content. Due to its significant application potential in medicine and infant formula production, 2’-FL has rapidly emerged as a focal point of research interest. Natural product extraction and chemical synthesis are the main methods of traditional synthesis of 2’-FL, but these two methods are difficult to extract 2’-FL in large quantities, and there is serious pollution. Consequently, there is growing attention towards employing whole cell fermentation as an environmentally friendly approach that offers mild reaction conditions for efficient 2’-FL production. By heterologously expressing polyphosphate-dependent mannose kinase AsPPGMK and α-1,2-fucosyltransferase in Escherichia coli while co-expressing key enzymes involved in the synthesis pathway and knocking out the gene encoding UDP-glucose lipid carrier transferase wcaJ to prevent intermediate metabolite degradation, we successfully enhanced the yield of 2’-FL. Ultimately, through optimization of catalytic transformation conditions, the addition of 200 mmol/L substrate to the 100 mL system further enhanced the yield of 2’-FL to 79 g/L in a yield of 81% in 8 h.