HMOs, a family of unconjugated glycans unique to human breast milk, rank as the third most prevalent solid component following lactose and lipids, exhibiting remarkable structural heterogeneity (Ballard and Morrow, 2013; Huang et al., 2017; Walker, 2010). Concentrations reach 20–25 g/L in colostrum and stabilize at 10–15 g/L in mature milk-levels exceeding other mammalian milks by orders of magnitude, substantiating breastfeeding's nutritional supremacy (Bode, 2012). Structurally, HMOs derive from five core monosaccharides: d-glucose, D-galactose, N-acetylglucosamine, L-fucose, and N-acetylneuraminic acid, with over 200 distinct structures characterized to date (Amano et al., 2009; Ninonuevo et al., 2006; Pfenninger et al., 2008; Urashima et al., 2013).

These multifunctional molecules have garnered global research interest as pivotal immunomodulators and prebiotics. Their indigestibility enables selective stimulation of beneficial gut microbiota (e.g., Bifidobacterium spp. and Lactobacilli), while simultaneously modulating intestinal epithelial responses, exhibiting antiviral properties, and promoting neuroimmune development (Autran et al., 2018; Ayechu-Muruzabal et al., 2018; Craft et al., 2018; He et al., 2014; Li et al., 2020; Morrow et al., 2004; Ramani et al., 2018; Wang, 2009; Zhang et al., 2022c). These collective mechanisms underpin infant health optimization. Emerging evidence extends their therapeutic potential to geriatric care through gut microbiota-T cell axis regulation, metabolic disorder mitigation, and osteoarthritis prevention (Jeon et al., 2018; Li et al., 2025; Park et al., 2025).

LNnT, a neutral tetrasaccharide comprising D-glucose, two D-galactose units, and N-acetylglucosamine units, serves as the architectural core for ∼10 % of HMOs (Chen, 2015; Han et al., 2012). This scaffold enables biosynthesis of complex fucosylated/sialylated derivatives through sequential glycosylation (Yang et al., 2024). LNnT's regulatory approvals underscore its clinical significance: U.S. FDA GRAS status (2015), EU novel food authorization (2015), FSANZ (Food Standards Australia New Zealand) endorsement of microbial-derived LNnT (2019), and China NHC approval as infant formula fortificant (2023). LNnT exhibits diverse biologically relevant properties that support its utility in nutrition and therapeutics, with experimental evidence spanning in vitro and in vivo systems. In vitro, it demonstrates favorable safety and tolerability, while also supporting gut microbial health by promoting the growth of beneficial Bifidobacterium species, shaping adult gut microbiota composition, and inhibiting the development of necrotizing enterocolitis (NEC) (Elison et al., 2016; Terrazas et al., 2001). In murine models, LNnT exerts notable in vivo effects: it stimulates Gr1+ cell proliferation, enhances anti-inflammatory cytokine secretion, and suppresses naive CD4+ T cell proliferation, which collectively supports positive type 2 immune response induction, strengthens anti-inflammatory capacity, and promotes angiogenesis and cutaneous wound healing (Farhadihosseinabadi et al., 2020a; Farhadihosseinabadi et al., 2020b; Hoeflinger et al., 2015). Beyond immunomodulatory and gut-related benefits, LNnT acts as an analog of the receptor molecule Galβ1,4GlcNAcβ1,3-Gal to inhibit Streptococcus pneumoniae adhesion to human nasopharyngeal epithelial cells, and it also exhibits antiviral activity—specifically, it is efficiently recognized by the VP8* domain of rotavirus P[11], with the crystal structure of the P[11] VP8*/LNnT complex elucidating their molecular interaction and confirming this antiviral mechanism (Andersson et al., 1983; Hu et al., 2015).

Biosynthetically, LNnT production requires coordinated action of β-1,3-N-acetylglucosaminyltransferase (β3GNT, EC 2.4.1.149) and β-1,4-galactosyltransferase (β4GalT, EC 2.4.1.22), employing UDP-GlcNAc and UDP-Gal as donors with lactose as acceptor (Fig. 1). Despite pathway elucidation in Neisseria meningitidis, its pathogenicity precludes industrial use (Wakarchuk et al., 1996). Current metabolic engineering platforms in Escherichia coli, Bacillus subtilis, and yeast strains address key bottlenecks in nucleotide sugar supply and pathway optimization, enabling microbial production of various HMOs including 2’-FL, LNT, and LNnT (Faijes et al., 2019; Parschat et al., 2020; Zhu et al., 2022). However, a dedicated review systematically analyzing modular engineering strategies (enzyme evolution, flux balancing, chassis optimization) specifically for LNnT bioproduction remains conspicuously absent.

This work provides the hierarchical analysis of LNnT metabolic engineering paradigms, critically evaluating: (1) E. coli-centric pathway optimization strategies; (2) alternative GRAS microbial platforms. We further propose an innovation roadmap integrating enzyme design with dynamic pathway control to advance LNnT manufacturing toward sustainable food additive production.