Nicotine, a potent insecticidal alkaloid unique to the nightshade family, has been employed in agriculture as a pesticide since 1690. It also has therapeutic potential for neurological disorders such as Alzheimer’s disease, Parkinson’s disease, and depression. Despite its profound influence on human history, agriculture, and plant evolution, however, the final steps of nicotine biosynthesis have remained unclear until now.

In a study published in Cell, a research group led by Prof. LI Dapeng from the Center for Excellence in Molecular Plant Sciences of the Chinese Academy of Sciences elucidated the complete biosynthetic pathway of nicotine in the wild tobacco species Nicotiana attenuata using an information theory-guided multidimensional omics approach. The results revealed a five-component dynamic metabolon—a temporary cluster of enzymes working together to carry out a series of chemical reactions in a cell—that orchestrates the final condensation steps of nicotine synthesis and its transport.

Researchers first discovered a nicotine-free mutant, ao2, in wild tobacco, leading to the identification of the NaAO2 gene, which is essential for the formation of nicotine‘s pyridine ring. By constructing a multi-scale co-association network integrating genomics, transcriptomics, and untargeted structural metabolomics at the population, individual, and single-cell levels, they identified a suite of components involved in nicotine assembly, including a glycosyltransferase, a reductase, berberine bridge enzyme-like enzymes, β-glucosidases, and a MATE transporter.

Moreover, researchers found that plants employed an elegant and cryptic “glycosylation/deglycosylation” strategy for the final coupling reaction of nicotine’s 5- and 6-membered nitrogen-containing heterocyclic rings. This process required two cyclic cation intermediates stabilized by glycosylation via a UDP-glycosyltransferase (NaUGT1), reduced and activated by an isoflavone reductase-like enzyme (NaA622), condensed through a stereoselective intermolecular Mannich-like reaction and sequentially oxidized by a berberine bridge enzyme-like enzyme (NaBBL1/2), and finally deglycosylated by a β-glucosidase (NaBGL1/2) to yield chirally pure nicotine, which was then stored in the vacuole via a MATE transporter (NaMATE1).

This “nicotine synthase” process was carried out by a dynamic metabolon assembled at the vacuolar membrane. This protein complex assembly enabled efficient substrate channeling, prevented the accumulation of toxic intermediates, and elegantly circumvented the “autotoxicity dilemma” inherent in plant defense.

This study completes the nicotine biosynthesis pathway and offers critical insights into the intermolecular Mannich-like reaction, a fundamental mechanism for scaffold formation in many plant alkaloids. The discovery of this naturally occurring multifunctional metabolon establishes a new synthetic biology paradigm for the scalable production of high-value natural products with defined stereochemistry.