Abstract:

Human milk oligosaccharides (HMOs) are essentially functional and nutritional components in human milk. They can be mainly classified into fucosylated, neutral core, and sialylated HMOs. Lacto-N-triose II (LNT II), lacto-N-neotetraose (LNnT), and lacto-N-tetraose (LNT) are common neutral core human milk oligosaccharides (ncHMOs), which can be extended to form longer-chain HMOs and play important roles in intestine health. In recent years, the biosynthesis of ncHMOs has developed rapidly, and industrial-scale production is from theoretical possibility to practical reality. The synthesis approaches for ncHMOs include chemical synthesis, enzymatic synthesis, and microbial cell synthesis. As the rapid development of biotechnology, enzymatic and microbial cell synthesis have emerged as prominent methods in ncHMOs biosynthesis. Enzymatic synthesis is highly efficient, regioselective, and stereoselective. Currently, glycosyltransferases and glycoside hydrolases represent the two major types of enzymes used for biosynthesizing ncHMOs. Glycosidase-based enzymatic synthesis has demonstrated high conversion rates for LNT II and LNnT production. However, the enzymatic synthesis of LNT remains less efficient and requires further improvement. Notably, the production of LNnT and LNT typically depends on LNT II as a key precursor, requiring a multi-step synthetic strategy. Microbial cell synthesis employs metabolic engineering to construct continuously synthetic pathways in microbial cells such as Escherichia coli and Bacillus subtilis. Knocking out genes in competitive pathway, optimizing genes expression, regenerating cofactors have significantly enhanced the yields of ncHMOs. The biosynthesis of ncHMOs faces several critical challenges, including the low activity and poor substrate specificity of key glycosyltransferases, such as β-1,3-N-acetylglucosaminyltransferase and β-1,3-galactosyltransferase. Additionally, the transporters of LNT II and LNnT are not clear in microbial cell. Furthermore, the yields of LNT II should be substantially improved for industrial-scale production. Thus, it is important to overcome the interconnected limitations in enzyme engineering (particularly glycosyltransferase specificity and activity), microbial cell modification (focusing on metabolic compatibility and pathway design), and bioprocess optimization (through rational pathway redesign) via an integrated synthetic biology and fermentation engineering approach in the future. These strategies are essential to achieve efficient, cost-effective biosynthesis of ncHMOs at industrial scale.

Key words: human milk oligosaccharides, lacto-N-triose II, lacto-N-neotetraose, lacto-N-tetraose, biosynthesis

摘要：

母乳寡糖（human milk oligosaccharides，HMOs）是母乳中重要的功能和营养成分。其中，中性核心母乳寡糖（neutral core human milk oligosaccharides，ncHMOs）主要包括乳糖-N-三糖（LNT II）、乳糖-N-新四糖（LNnT）和乳糖-N-四糖（LNT），在婴幼儿健康发育过程中发挥着不可替代的生理作用。近年来，中性核心母乳寡糖的生物合成技术快速发展，其工业化生产从可能走向现实。本文综述了中性核心母乳寡糖的酶法和微生物细胞法合成研究现状，介绍了糖基转移酶和糖苷酶在酶法合成中性核心母乳寡糖的应用，讨论了糖基转移酶的催化特性、底盘细胞的种类及改造等关键因素对微生物细胞合成中性核心母乳寡糖产量的影响，进一步对比了两种方法生物合成中性核心母乳寡糖的优缺点。目前，中性核心母乳寡糖的生物合成存在酶催化效率低、底盘细胞选择与适配性差、副产物多及内毒素污染等问题，需通过理性设计酶元件、优化安全底盘、动态调控代谢网络及强化发酵纯化工艺等策略协同攻关。

关键词: 母乳寡糖, 乳糖-N-三糖, 乳糖-N-新四糖, 乳糖-N-四糖, 生物合成