Designing Engineered Live Functional Microbes with Defined Rhamnose-Containing O-Antigens for Mucosal Immunity Enhancement

• A landscape of rhamnose-containing glycan structures across the gut microbiota was mapped using a glycomics-database-mining approach. • A glycomics-guided and modeling-supported approach was used to design an engineered E. coli W3110 platform with structurally defined surface O-antigens. • The α-L-Rha(1→3)-α-L-Rha motif co-presented with GlcNAc was identified as an effective glycan configuration associated with enhanced anti-Rha immune responses. • Oral administration of the engineered strain increased serum anti-Rha IgG levels in mice, demonstrating targeted and measurable mucosal immunomodulation. The era of precision nutrition demands next-generation functional ingredients capable of modulating host physiology with high specificity and efficacy. Microbial surface glycans are critical food-derived bioactive molecules that play a pivotal role in regulating gut immunity. Significantly, anti-rhamnose (anti-Rha) antibodies are naturally abundant in humans, serving as a critical component of innate-like mucosal immune defense and playing an essential role in maintaining host immune homeostasis. However, the precise structural relationship between these glycans and targeted immune responses remains a critical "black box", limiting the development of truly precise functional foods. This study aims to utilize cutting-edge microbial engineering to develop a proof-of-concept platform for evaluating structurally defined microbial glycans with immunomodulatory potential, thereby enhancing mucosal immune defense. We integrated computational glycomics and structural modeling to precisely identify the specific glycan motif required for high-efficiency stimulation of anti-Rha antibody responses: the co-presentation of the α-L-Rha(1→3)-α-L-Rha unit with N -acetylglucosamine (GlcNAc). Subsequently, we leveraged advanced synthetic biology techniques to genetically engineer a safe and highly efficient biomanufacturing prototype platform (e.g., Escherichia coli W3110 used as a model strain) to biosynthesize and display this defined, highly immunoactive repeat unit, thereby establishing a prototype fermentation framework for evaluating defined glycan structures. In vivo evaluation demonstrated that oral administration of this engineered strain significantly elevated both systemic and mucosal anti-Rha antibody titers in mice—up to ∼3.5-fold enhancement relative to controls ( p < 0.01). Importantly, cytokine profiling did not reveal evidence of pronounced pro-inflammatory immune activation (e.g., TNF-α, IL-6), consistent with a controlled immune response. These findings highlight the potential of precision microbial engineering design in creating highly potent, functionally orally available immunomodulators. This engineered microbial platform provides a conceptual framework for the rational design of engineered microbes that may be further adapted to food-grade hosts in future studies.