Stable Isotopic Labeling of Dimethylallyl Pyrophosphate (DMAPP) Reveals Compartmentalization of Isoprenoid Biosynthesis during Sporulation in <i>Bacillus subtilis</i>

Isoprenoids are essential metabolites whose biosynthesis originates from two five-carbon (C₅) isomers, isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). Although these isomers serve as the core substrates for isoprenoid biosynthesis, tracking their independent incorporation into downstream metabolites is difficult due to the lack of available chemical tools. To address this issue, we have developed a cell-permeant, stable isotope-labeled analog of DMAPP that employs self-immolating esters to mask the β-phosphate, allowing for efficient cellular uptake and direct forward isotopic labeling of downstream products of DMAPP in <i>Bacillus subtilis</i>. We demonstrate that ¹³C-labeled, ester-protected DMAPP (¹³C₃ SIE-DMAPP) achieves significant incorporation into menaquinone-7 (MK-7), regardless of inhibition of endogenous production of IPP and DMAPP. By knocking out isopentenyl pyrophosphate isomerase (IPPI) expression, we achieve specific ¹³C isotopic labeling exclusively at DMAPP-derived positions of MK-7 and demonstrate that IPPI overexpression enables <i>B. subtilis</i> to utilize DMAPP as its sole isoprenoid source. Finally, we tracked DMAPP incorporation during the transition from vegetative growth to sporulation, revealing compartment-specific isotope labeling patterns that underscore the metabolic independence of isoprenoid metabolism in the mother cell and endospore during sporulation in <i>B. subtilis</i>. The introduction of this stable isotope-labeled DMAPP facilitates the tracking of DMAPP-derived metabolites in native biological contexts and opens new avenues for studying prenyl metabolism, terpene biosynthetic pathways, and the regulatory mechanisms governing cellular isoprenoid pools.