Revisiting anabasine biosynthesis in tobacco hairy roots expressing plant lysine decarboxylase gene by using ¹⁵N-labeled lysine

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Anabasine is an alkaloid found in a small number of Nicotiana species. The components of the anabasine biosynthetic pathway have yet to be identified. Here, we report the reinvestigation of biosynthetic pathways of anabasine and related tobacco alkaloids in genetically engineered cells. Hairy roots of N. tabacum harboring a lysine/ornithine decarboxylase gene from Lupinus angustifolius (La-L/ODC) were fed with labeled [ε-¹⁵N]- or [α-¹⁵N]-L-lysine. Relative to the unfed control, feeding of labeled ¹⁵N-L-lysine greatly enhanced anabasine levels 13.5-fold in La-L/ODC-expressing line compared to 5.3-fold in the control line, suggesting that both LDC activity and substrate supplied are important factors for the efficient production of anabasine. GUS-expressing line showed preferential incorporation of [ε-¹⁵N]-L-lysine into anabasine, indicating the main biosynthetic pathway of Δ¹-piperideine intermediate in tobacco is asymmetrically processes. In contrast, the expression of La-L/ODC showed the symmetric labeling of ¹⁵N atom into anabasine, implying the occurrence of free cadaverine, which is produced by La-L/ODC enzyme, during the biosynthesis of Δ¹-piperideine intermediate. No considerable incorporation of ¹⁵N into other tobacco alkaloids such as, nicotine, anatabine, and anatalline, was detected. Detailed analysis using ultra-high resolution mass spectrometry indicated that two ¹⁵N atoms were incorporated into anabasine in La-L/ODC-expressing lines after feeding [ε-¹⁵N]- or [α-¹⁵N]-L-lysine. Our results not only provide information insight into the biosynthesis of anabasine but also suggest an alternative route for the production of anabasine by genetic engineering.