LDOB: multi-dimensional programmable lactose-derived oligosaccharide biosensors

Human milk oligosaccharides (HMOs) are the third most abundant solid constituent of breast milk and exert anti-infective, prebiotic and immunomodulatory functions. Microbial de novo synthesis is currently the only scalable route for commercial HMOs production, yet the absence of rapid and universal analytical tools has become a major bottleneck for strain improvement. Exploiting the strict stoichiometry between lactose consumption and HMOs biosynthesis, we constructed a set of Escherichia coli-based lactose-derived oligosaccharide biosensors (LDOB)-genetically engineered whole-cell systems that translate lactose concentration into inversely correlated biomass and fluorescence read-outs, enabling real-time screening of HMO-producing strains without downstream metabolite processing. The sensing circuit integrates multidimensional negative-feedback modules-encompassing multi-repression, targeted protein degradation and an occluded ribosome binding site (oRBS)-endowing LDOB with a wide dynamic range, straightforward operability, excellent compatibility, and substantial application potential. Validation experiments demonstrated that LDOB signals exhibited strong consistency with the yields of key HMOs, including 2'-fucosyllactose (2'-FL) and lacto-N-neotetraose (LNnT). Furthermore, coupling LDOB with fluorescence-activated droplet sorting (FADS) enabled high-throughput screening of evolved strains, resulting in remarkable titer enhancements of 42.8 % for 2'-FL and 86.4 % for LNnT, respectively. Collectively, these findings fully confirm the superiority of LDOB in monitoring HMOs synthesis and screening high-yield HMOs-producing strains, providing a valuable tool for advancing HMOs biomanufacturing.