Lacto-N-fucopentaose I (LNFP I), a fucosylated neutral human milk oligosaccharide (HMO) with diverse biological functions, was biosynthesized through metabolic engineering in Escherichia coli BL21star (DE3). A de novo pathway was constructed by chromosomal integration of three key enzymes: lgtA (β-1,3-N-acetylglucosaminyltransferase), wbdO (β-1,3-galactosyltransferase), and galE (UDP-galactose-4-epimerase), generating a plasmid-free strain that achieved a lacto-N-tetraose (LNT) titer of 109.80 g/L in a 5 L bioreactor, the highest yield reported to date. Subsequent screening identified α-1,2-fucosyltransferase (FutC) from Helicobacter pylori as the optimal catalyst for LNFP I biosynthesis. Multidimensional optimization strategies were systematically implemented, including copy number balancing of rate-limiting transferases, promoter-RBS engineering, enhanced intracellular cofactor regeneration, and knockout of competing pathways. Fed-batch fermentation under optimized conditions yielded 77 g/L LNFP I with 93.05% LNT-to-LNFP I conversion efficiency, representing both the highest reported titer and precursor utilization efficiency for LNFP I.

Keywords: Escherichia coli; human milk oligosaccharide; lacto-N-fucopentaose I; lacto-N-tetraose; metabolic engineering.

乳糖-N-岩藻五糖I（LNFP I）是一种岩藻糖化的中性人乳低聚糖（HMO），具有多种生物学功能。通过代谢工程在大肠杆菌BL21star (DE3) 中生物合成该化合物。构建了一个新的途径，通过染色体整合三个关键酶：lgtA（β-1,3-N-乙酰氨基葡萄糖转移酶）、wbdO（β-1,3-半乳糖转移酶）和galE（尿苷二磷酸半乳糖-4-差向异构酶），生成了一个不含质粒的菌株，在5升生物反应器中实现了乳糖-N-四糖（LNT）滴度为109.80 g/L，这是迄今为止报道的最高产量。随后的筛选鉴定出幽门螺杆菌来源的α-1,2-岩藻糖转移酶（FutC）是LNFP I生物合成的最佳催化剂。系统地实施了多维优化策略，包括限速转移酶拷贝数平衡、启动子-RBS工程、增强胞内辅因子再生以及竞争途径敲除。在优化条件下进行补料分批发酵可获得77 g/L LNFP I，并且LNT向LNFP I的转化效率达到93.05%，这是迄今为止报道的最高滴度和前体利用率。

关键词：大肠杆菌；人乳低聚糖；乳糖-N-岩藻五糖I；乳糖-N-四糖；代谢工程。