Elucidation of the Hydroxylation Sequence in 1-DNJ Biosynthesis through Stable Isotope Tracing and Enzymatic Characterization.

1-Deoxynojirimycin (DNJ) is the primary bioactive polyhydroxy alkaloid responsible for the antidiabetic properties of mulberry leaves. While the upstream biosynthesis is characterized, the downstream hydroxylation logic and transient intermediates remain elusive. Here, we combined stable isotope tracing with in vivo and in vitro enzymatic assays to delineate this hydroxylation sequence. Stable isotope feeding experiments identified piperidine as a key precursor and pinpointed C-7 as a critical secondary hydroxylation site. We demonstrated that (2R,4R)-2-methylpiperidine-4-ol (2) and (2S,4R)-2-(hydroxymethyl)piperidine-4-ol (4) are successive intermediates of the first two steps, occurring via sequential C-4 and C-7 hydroxylation. Furthermore, using mulberry leaf crude enzyme and recombinant C-5 hydroxylase, we deduced that 4 is subsequently converted to fagomine (FGM) via an endogenous C-3 hydroxylation activity, followed by C-5 modification to yield DNJ. Collectively, this study clarifies the sequential logic of the downstream hydroxylation (C-4→C-7→C-3→C-5), providing a foundational framework for the rational engineering and heterologous biosynthesis of DNJ.