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

Abstract

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-Relative to the unfed control, feeding of labeled 15 N-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-15 N]-L-lysine into anabasine, indicating the main biosynthetic pathway of Δ 1 -piperideine intermediate in tobacco is asymmetrically processes. In contrast, the expression of La-L/ODC showed the symmetric labeling of 15 N atom into anabasine, implying the occurrence of free cadaverine, which is produced by La-L/ODC enzyme, during the biosynthesis of Δ 1 -piperideine intermediate. No considerable incorporation of 15 N into other tobacco alkaloids such as, nicotine, anatabine, and anatalline, was detected. Detailed analysis using ultra-high resolution mass spectrometry indicated that two 15 N atoms were incorporated into anabasine in La-L/ODC-expressing lines after feeding [ε-15 N]-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.

Metabolic diversification of nitrogen‐containing metabolites by the expression of a heterologous lysine decarboxylase gene in Arabidopsis

“…Feeding with AL, EL or NL resulted in 17 (RPLC mode) and 23 (HILIC mode) specific metabolite features being labeled in both AL‐ and EL‐treated DC29 plants (Tables ; Table S5). Previously, feeding AL or EL to the hairy roots of Nicotiana tabacum harboring La‐L/ODC resulted in a similar labeling ratio of anabasine, an Lys‐derived alkaloid (Bunsupa et al ., ), which confirmed that anabasine was derived from cadaverine. Our results also show similar 15 N incorporation levels for the majority of specific metabolite features labeled in AL‐ and EL‐treated DC29 plants, suggesting that these metabolites are derived from cadaverine.…”

“…Next, in order to evaluate the incorporation of exogenously applied l ‐lysine into specific metabolite features, the percentage isotope enrichment factors (%EFs) were calculated using the following formula: %EF = [(intensity of M + 1)/(sum of intensities of M and M + 1)] × 100, where M and M + 1 represent the monoisotopic mass of an unlabeled metabolite and a labeled metabolite with a mass shift of 0.997 Da, respectively. The %EF values were further corrected by taking into account the natural abundance of 15 N isotopolog peaks, as described previously (Campbell, ; Bunsupa et al ., ). Around 92.8–95.4% of l ‐lysine was labeled in DC29 treated with either AL or EL, whereas l ‐lysine was not labeled in DC29 treated with NL (Figure c).…”

“…The percentage isotope enrichment factors (%EF) were calculated using the following formula: %EF = [(intensity of M + 1)/(sum of intensities of M and M + 1)] 9 100, where M and M + 1 represents a monoisotopic mass of an unlabeled metabolite and a labeled metabolite with a mass shift of 0.997 Da, respectively. The %EF values were further corrected using the peak areas of 15 N isotopolog peaks in NL, as described previously (Campbell, 1974;Bunsupa et al, 2014).…”

Molecular Evolution and Functional Characterization of a Bifunctional Decarboxylase Involved in Lycopodium Alkaloid Biosynthesis

“…Anabasine is composed of two rings, a piperidine ring derived from Lys and a pyridine ring derived from nicotinic acid. The two rings in nicotine are a pyrrolidine ring derived from Orn and a pyridine ring (Bunsupa et al, 2014). A negative correlation between LcL/ODC transcript and nicotine was found, but the nicotine levels did not decrease significantly.…”

Tropane and Granatane Alkaloid Biosynthesis: A Systematic Analysis

“…The decarboxylation of lysine (25) described in Hypothesis I would be performed by a P. granatum lysine decarboxylase (LDC). The presence of a lysine decarboxylase in the synthesis of anabasine (8) in Nicotiana species has been studied by the incorporation of [ 15 N]-lysine into anabasine (8) [124]. The methylation of cadaverine (26) would be achieved with the help of a P. granatum cadaverine N-methyltransferase (CMT).…”