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

Abstract

Coptis japonica (Cj) rhizomes are used as a crude drug for gastroenteritis, since they accumulate antimicrobial berberine. Berberine also shows various useful bioactivities, including cholesterol-lowering activity. Unfortunately, Cj is a slow-growing plant and more than 5 years are required to obtain a crude drug suitable for the Japanese Pharmacopoeia. To improve alkaloid productivity, we overexpressed the 3′-hydroxy-N-methylcoclaurine 4′-O-methyltransferase (4′OMT) gene in Cj. We established the transgenic plant (named CjHE4′) by introducing one copy of Cj4′OMT by Agrobacterium-mediated transformation. The successful overexpression of 4′OMT was confirmed in all tissues of CjHE4′ by real-time polymerase chain reaction (PCR) analysis. HPLC analysis revealed that the berberine content of CjHE4′ leaves and roots cultivated for 4 months was increased to 2.7-and 2.0-fold, respectively, compared with non-transgenic wild-type (CjWT), and these inductions of alkaloids were stable for at least 20 months. Furthermore, in CjHE4′ cultivated for 20 months, the berberine content in medicinal parts, stems and rhizomes was significantly increased (1.6-fold). As a consequence, increased amounts of alkaloids in CjHE4′ resulted in the improvement of berberine yields (1.5-fold), whereas CjHE4′ showed slower growth than CjWT. These results indicated that 4′OMT is one of the key-step enzymes in berberine biosynthesis and is useful for metabolic engineering in Cj.

Microbial production of isoquinoline alkaloids as plant secondary metabolites based on metabolic engineering research

“…For example, the overexpression of C. japonica SMT and 4′OMT using a strong constitutive promoter, Cauliflower mosaic virus 35S, led to an increase in berberine production in C. japonica cells and plants, respectively. 39,40) Similarly, heterologous overexpression of C. japonica 6OMT improved the level of alkaloid productivity in E. californica cells. 41) It is important to note, however, that the overexpression of a biosynthetic enzyme does not always result in a successful increase in the levels of alkaloid production.…”

Green Routes for the Production of Enantiopure Benzylisoquinoline Alkaloids

“…Recently, it has been reported that opium poppy transformed with constitutively expressed gene for codeinone reductase, showed a higher content of morphinan alkaloid [ 37 , 38 ]. Coptis japonica was modified to improve alkaloid yields by overexpressing the 3-hydroxy- N -methylcoclaurine-4- O -methyltransferase (4-OMT) gene, one of the key enzymes in BIAs biosynthesis in this species [ 39 , 40 ]. Overexpression of the Papaver somniferum berberine bridge enzyme (BBE) in Eschscholzia californica led to the accumulation of the end products benzophenanthridine alkaloids compared to controls [ 41 ].…”

“…Although plant engineering has been used to induce secondary metabolites accumulation [ 40 , 48 , 49 , 50 ], it is not easy to achieve the desired products because their biosynthesis are tightly regulated. For example, in most pathways, overexpression of one enzyme results in making subsequent reactions more limiting.…”

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

“…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. 73) Thus, cell culture systems offer another advantage in analyzing metabolic flux, since they are amenable to the application of stable isotopes.…”