Academic Papers
( Protein Purifier System, Transporter -NaMATE1) Lijing Chang, et al. Complete biosynthesis of nicotine. Cell. 2026.
2026-08-17 13:48
( Protein Purifier System, Transporter -NaMATE1) Lijing Chang, et al. Complete biosynthesis of nicotine.
Cell. 2026. https://www.cell.com/cell/fulltext/S0092-8674(26)00335-1
Highlights
• Information-theory-guided omics identifies glycosylation in nicotine biosynthesis
• NAMN hydrolase supplies nicotinic acid via an NAD-cycle-independent route
• A vacuolar five-component metabolon channels nicotine biosynthesis and transport
• MATE transporter enables efficient nicotine engineering in heterologous plants
Summary
Nicotine, tobacco’s addictive and potent insecticidal alkaloid, has shaped human history, agriculture, and the plants that produce it. However, the enzymatic steps and reaction mechanisms involved in nicotine biosynthesis remain elusive. Here, we reveal that the final coupling reaction is stabilized by glycosylation via a uridine diphosphate (UDP)-glycosyltransferase, reduced and activated by an A622, condensed through a stereoselective intermolecular Mannich-like reaction, sequentially oxidized by a berberine bridge enzyme-like (BBL), and finally deglycosylated by a β-glucosidase to yield nicotine. A 5-component metabolon assembles at vacuolar membranes to channel both nicotine biosynthesis and its transport. We reconstituted this metabolon both in vitro and heterologously in vivo. Abrogating any of these components depletes nicotine accumulations. A multidrug and toxic compound extrusion (MATE) transporter is essential for efficiently engineering nicotine production in heterologous plant species, which confers pest resistance. This work completes the nicotine biosynthesis pathway and provides critical insights into the intermolecular Mannich-like reaction, a fundamental mechanism for scaffold formation in many plant alkaloids......
The NaMATE1 gene with a C-terminal Strep tag was cloned into a modified BacMam expression vector. A total of 100 mL of HEK293 cells at a density of 3 x 106 cells/mL were transfected with 100 μg of the NaMATE1-containing plasmid. After culturing at 37 °C for 12 h, sodium butyrate (final concentration 10 mM) and glucose (0.2 g) were added to enhance protein expression. The cells were then cultured at 30 °C for an additional 48 h prior to harvesting. To purify the NaMATE1 protein, cells were homogenized in buffer A (50 mM Tris pH 7.4, 150 mM NaCl, protease inhibitors, and ribonuclease) using a Dounce homogenizer. Membranes were collected by centrifugation at 18,000 rpm for 1 h, resuspended in buffer A, and solubilized at 4 °C for 2 h with 1.5% n-dodecyl β-D-maltoside (DDM; Bluepus)/0.2% cholesteryl hemisuccinate (CHS; Anatrace) mixture. After centrifugation at 18,000 rpm for 50 min, the supernatant was mixed with prewashed Streptactin Beads 4FF (SA053100, Smart-Lifesciences) and incubated at 4 °C for 2 h. The slurry was loaded onto a gravity column (Bio-Rad) and the resin was extensively washed with buffer B (20 mM Tris pH 7.4, 150 mM NaCl and 0.05% DDM/0.005% CHS). The protein was eluted using buffer C (20 mM HEPES pH 7.4, 150 mM NaCl and 0.025% DDM/0.0025% CHS) supplemented with 7.5 mM dethiobiotin. The eluted protein was concentrated and further purified through gel filtration using a Superose 6 increase column (Cytiva) in buffer C on a protein purifier system (Inscinstech Co., Ltd.).
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