Overexpression of Coptis japonica Norcoclaurine 6- O -Methyltransferase Overcomes the Rate-Limiting Step in Benzylisoquinoline Alkaloid Biosynthesis in Cultured Eschscholzia californica

Abstract

Benzylisoquinoline alkaloids are one of the most important secondary metabolite groups, and include the economically important analgesic morphine and the antimicrobial agent berberine. To improve the production of these alkaloids, we investigated the effect of the overexpression of putative rate-limiting step enzymes in benzylisoquinoline alkaloid biosynthesis. We introduced two O-methyltransferase [Coptis japonica norcoclaurine 6-O-methyltransferase (6OMT) and 3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase (4'OMT)] expression vectors into cultured California poppy cells to avoid the gene silencing effect of endogenous genes. We established 20 independent lines for 6OMT transformants and 15 independent lines for 4'OMT transformants. HPLC/liquid chromatography-mass spectrometry (LC-MS) analysis revealed that the overexpression of C. japonica 6OMT was associated with an average alkaloid content 7.5 times greater than that in the wild type, whereas the overexpression of C. japonica 4'OMT had only a marginal effect. Further characterization of 6OMT in California poppy cells indicated that a 6OMT-specific gene is missing and 4OMT catalyzes the 6OMT reaction with low activity in California poppy, which supports the notion that the 6OMT reaction is important for alkaloid biosynthesis in this plant species. We discuss the importance of 6OMT in benzylisoquinoline alkaloid biosynthesis and the potential for using a rate-limiting step gene to improve alkaloid production.

Characterization of Plant Functions Using Cultured Plant Cells, and Biotechnological Applications

“…82) This robust metabolism should help plant cells, to adapt to biochemical changes, and cultured plant cells are a suitable system for testing evolutional diversification, since they have strong potential for somaclonal variation. 78,86) These results also suggest that enzymes in secondary metabolism might have broad substrate-specificity and high adaptability to metabolic modification induced by spontaneous genetic modification. Cultured cells with a modified metabolic pathway might be useful in tracing the evolution of metabolism.…”

“…4A), whereas overexpression of C. japonica 4 0 OMT had only a marginal effect. 78) Overexpression of 4 0 OMT in a homologous host, the C. japonica plant, however, resulted in a marked increase in the amount of alkaloids, whereas transgenic C. japonica plants that overexpressed Cj4 0 OMT showed slower growth than the wild type. 79) Many experiments have indicated that the overexpression of other biosynthetic enzymes in various host plants has various effects, and thus suggest that more intensive studies of metabolic flow in various types of target-plant cells, are essential for continued progress in metabolic engineering, and especially to improve yields, through overexpression of rate-limiting enzymes.…”

Improvement of Benzylisoquinoline Alkaloid Productivity by Overexpression of 3′-Hydroxy-<i>N</i>-methylcoclaurine 4′-<i>O</i>-Methyltransferase in Transgenic <i>Coptis japonica</i> Plants

“…Inc., Osaka, Japan) at 20°C in the dark. After preculture, the segments were co-cultured with the A. tumefaciens strain LBA4404 harboring pBHE4′ (vector for overexpression of Cj4′OMT 12) ) for 2 d in WPGN1K2 liquid medium containing 2% sucrose; then bacteria was eliminated on WPGN1K2 medium containing 500 mg/L claforan (Sanofi Aventis, Tokyo, Japan). After 1 to 2 months, the segments which formed calli and adventitious roots were cultured on WPGN1K2 medium containing 25 mg/L hygromycin at 20°C in the dark.…”

“…11) Overexpression of Cj6OMT in Ec-cultured cells also resulted in an effective increase of benzophenanthridine alkaloids. 12) On the other hand, suppression of the biosynthetic pathway showed more prominent and complicated results. RNAi suppression of PsCOR resulted in the surprising accumulation of reticuline, which is 7 enzymatic steps upstream of COR.…”

Molecular Cloning of anO-Methyltransferase from Adventitious Roots ofCarapichea ipecacuanha

“…While some OMTs in isoquinoline alkaloid biosynthesis have been reported to have strict substrate specificity and reaction specificity, 9) a recent re-investigation of some enzymes showed that 3 0 -hydroxy-N-methylcoclaurine 4 0 -O-methyltransferase (4 0 OMT) not only contributes to 4 0 -Omethylation but also to the 6-O-methylation of norcoclaurine. 17) Furthermore, characterization of a chimeric enzyme of norcoclaurine 6-O-methyltransferase and 4 0 -OMT showed that their substrate specificity and reaction specificity can be modified by substitution in the Nterminal region, which might be involved in substrate binding. 18) IpeOMT2 has glutamic acid, asparagine, and methionine at positions 5, 63, and 121 respectively, while CiOMT1 has glutamine, lysine, and leucine at these positions.…”