- Japanese Society for Plant Biotechnology
- Plant Biotechnology (ISSN:13424580)
- vol.31, no.5, pp.511-518, 2014-12-25 (Released:2015-02-27)
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.