Deciphering the 2-MIB production mechanism in Actinobacteria: the interplay of synthesis enzymes, nutrient metabolism, and seasonal regime.

The multifaceted regulatory network by which Actinobacteria transition from microbial quiescence to active 2-methylisoborneol (2-MIB) production in response to environmental cues remains poorly defined. To elucidate this complex system, an integration of field monitoring, laboratory experiments, microbial community analysis, and machine learning was employed to decode the functional network governing 2-MIB biosynthesis. This multi-scale approach revealed a critical physiological decoupling in Actinobacteria: dissolved oxygen (DO) governs cellular proliferation, whereas bioavailable nitrogen, specifically NH4+-N, acts as the direct metabolic trigger for 2-MIB synthesis. This functional specialization was further corroborated by machine learning models, which consistently identified NH4+-N as the dominant predictor of 2-MIB risk when phosphorus was sufficient. Mechanistically, a tight functional coupling was demonstrated between glutamine synthetase and 2-MIB synthase, revealing a dedicated nitrogen assimilation pathway that directly channels NH4+-N into odorant production. The establishment of this NH4+-N driven regulatory axis transforms a complex ecological phenomenon into a quantifiable and predictable process, providing a key for early warning and source water control strategies.