Stable reconstruction of the breviscapine biosynthetic pathway enables accumulation of a putative scutellarin-related product and reshapes endogenous metabolism in tobacco

Breviscapine, a medicinal flavonoid extract from Erigeron breviscapus , is widely used for the treatment of cardiovascular and cerebrovascular disorders, yet its industrial production remains dependent on plant extraction, which is constrained by unstable metabolite accumulation and limited biomass productivity. Here, the breviscapine biosynthetic genes EbF6H , EbFSII , and EbF7GAT were stably co-expressed in Nicotiana tabacum to reconstruct the pathway in a heterologous plant host. Transgenic tobacco accumulated a putative scutellarin-related product at estimated concentrations of 0.678 ± 0.257 and 7.863 ± 0.987 mg scutellarin equivalents g -1 DW in leaves and flowers, respectively. Apigenin-7- O -glucuronide concentrations were 0.066 ± 0.013 and 0.255 ± 0.140 mg ·g -1 DW in the corresponding tissues. Values represent descriptive means ± SD across three independent transgenic lines, with one targeted measurement per line per tissue. Neither target analyte was detected in WT samples under the analytical conditions used. Integrated metabolomic analysis revealed substantial metabolic divergence between transgenic and wild-type plants, characterized by enhanced flavonoid accumulation and tissue-dependent changes in nicotine-associated metabolism. Transcriptomic profiling further showed coordinated up-regulation of endogenous phenylpropanoid and flavonoid biosynthetic genes, including PAL , C4H , 4CL , CHS , CHI , F3H , FNS , and FLS , suggesting activation of endogenous flavonoid metabolism. Together, these results establish stable tobacco as a proof-of-concept platform for producing glucuronidated medicinal flavonoids with no major morphological or reproductive abnormalities under the tested greenhouse conditions; they also demonstrate that multigene pathway reconstruction is accompanied by broader remodeling of endogenous specialized metabolism. Notably, the estimated concentration of the putative scutellarin-related product in transgenic flowers numerically overlapped with the lower part of published scutellarin concentration ranges for natural E. breviscapus populations, although direct cross-study comparison is limited by the provisional product identity and differences in sampling and analytical conditions. Further pathway and host optimization will be required for industrial scale-up.