标题：Improvement of Benzylisoquinoline Alkaloid Productivity by Overexpression of 3′-Hydroxy-N-methylcoclaurine 4′-O-Methyltransferase in Transgenic Coptis japonica Plants
摘要：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 Cj HE4′) by introducing one copy of Cj 4′OMT by Agrobacterium -mediated transformation. The successful overexpression of 4 ′ OMT was confirmed in all tissues of Cj HE4′ by real-time polymerase chain reaction (PCR) analysis. HPLC analysis revealed that the berberine content of Cj HE4′ leaves and roots cultivated for 4 months was increased to 2.7- and 2.0-fold, respectively, compared with non-transgenic wild-type ( Cj WT), and these inductions of alkaloids were stable for at least 20 months. Furthermore, in Cj HE4′ 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 Cj HE4′ resulted in the improvement of berberine yields (1.5-fold), whereas Cj HE4′ showed slower growth than Cj WT. These results indicated that 4′OMT is one of the key-step enzymes in berberine biosynthesis and is useful for metabolic engineering in Cj .
