Anoectochilus roxburghii is a traditional Chinese herb with high medicinal value, with main bioactive constituents which are flavonoids. It commonly associates with mycorrhizal fungi for its growth and development. Moreover, mycorrhizal fungi can induce changes in the internal metabolism of host plants. However, its role in the flavonoid accumulation in A. roxburghii at different growth stages is not well studied. In this study, combined metabolome and transcriptome analyses were performed to investigate the metabolic and transcriptional profiling in mycorrhizal A. roxburghii (M) and non-mycorrhizal A. roxburghii (NM) growth for six months. An association analysis revealed that flavonoid biosynthetic pathway presented significant differences between the M and NM. Additionally, the structural genes related to flavonoid synthesis and different flavonoid metabolites in both groups over a period of six months were validated using quantitative real-time polymerase chain reaction (qRT-PCR) and high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS). The results showed that Ceratobasidium sp. AR2 could increase the accumulation of five flavonol-glycosides (i.e., narcissin, rutin, isorhamnetin-3-O-beta-d-glucoside, quercetin-7-O-glucoside, and kaempferol-3-O-glucoside), two flavonols (i.e., quercetin and isorhamnetin), and two flavones (i.e., nobiletin and tangeretin) to some degrees. The qRT-PCR showed that the flavonoid biosynthetic genes (PAL, 4CL, CHS, GT, and RT) were significantly differentially expressed between the M and NM. Overall, our findings indicate that AR2 induces flavonoid metabolism in A. roxburghii during different growth stages, especially in the third month. This shows great potential of Ceratobasidium sp. AR2 for the quality improvement of A. roxburghii.

Oral administration of probiotics represents a promising therapeutic strategy for ulcerative colitis (UC), offering the potential to modulate gut immunity and restore mucosal homeostasis. However, the clinical efficacy of conventional probiotics is fundamentally limited by their susceptibility to the pathological oxidative stress within the inflamed intestine, which severely compromises their viability and function. To overcome this critical barrier, we developed a bioorthogonal engineering strategy to construct a robust probiotic-based biohybrid, Lr-DRA. This was achieved by metabolically labeling Lactobacillus reuteri (Lr) with azido groups, followed by site-specific, covalent conjugation of a potent phenolic antioxidant, DBCO-modified rosmarinic acid (DRA). This design ensures the stable integration of therapeutic components without impairing probiotic vitality. In a murine model of dextran sulfate sodium (DSS)-induced colitis, orally administered Lr-DRA demonstrated superior therapeutic efficacy. The surface-anchored DRA exerted protective effects by neutralizing oxidative stress. This protective action significantly enhanced the survival and colonization of the probiotic carrier Lr. Consequently, Lr-DRA treatment markedly alleviated clinical symptoms, suppressed pro-inflammatory cytokines, restored intestinal barrier integrity (as evidenced by upregulated tight junction proteins Zonula Occludens-1 [ZO-1] and Occludin), and promoted mucosal healing. This work establishes a versatile and modular platform for fabricating advanced living therapeutics. By simply varying the probiotic chassis and the conjugated bioactive payload, this strategy can be readily adapted to develop targeted treatments for a broad spectrum of gastrointestinal and metabolic disorders.

Oral probiotic-based therapy has emerged as a promising solution with multifaceted benefits for immunosuppression treatment. However, their widespread and clinical utility is severely limited by the poor viability of probiotics under harsh gastrointestinal conditions in the intestine. To address these challenges, a probiotic-based biohybrid (Lr@DGN) was bio-orthogonally fabricated by covalently anchoring the prebiotic β-glucan (GN) to the probiotic Limosilactobacillus reuteri (Lr). Upon oral administration, Lr@DGN colonized intestines with high survival rates, aided by gastrointestinal stress-shielding of GN, leading to immuno-enhancing effects through combining GN and live Lr. Consequently, in a Cy-induced immunosuppression mouse model, oral administration of Lr@DGN significantly mitigated body weight loss, restored the key immune organ indexes (thymus and spleen), ameliorated Cy-induced damage to the small intestine, enhanced the intestinal immune response, and elevated the serum levels of immunoglobulins IgG and IgA. By integrating the effects of a prebiotic shield and a live probiotic, this biohybrid system offers a promising and translatable approach for managing immunodeficiency and related disorders.