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.

Reticuline is a key compound in the biosynthetic pathway for isoquinoline alkaloids in plants, which include morphine, codeine and berberine. We established cultured California poppy (Eschscholzia californica) cells, in which berberine bridge enzyme (BBE) was knocked down by RNA interference, to accumulate the important key intermediate reticuline. Both BBE mRNA accumulation and enzyme activity were effectively suppressed in transgenic cells. In these transgenic cells, end-products of isoquinoline alkaloid biosynthesis, such as sanguinarine, were considerably reduced and reticuline was accumulated at a maximum level of 310 mug/g-fresh weight. In addition, 1 g-fresh weight of these cells secreted significant amounts of reticuline into the medium, with a maximum level of 6 mg/20 mL culture medium. These cells also produced a methylated derivative of reticuline, laudanine, which could scarcely be detected in control cells. We discuss the potential application of RNAi technology in metabolic modification and the flexibility of plant secondary metabolism.

1. Pharmacokinetic properties of difloxacin have been studied in pig and chicken after intravenous and oral administration. 2. The serum concentrations of difloxacin in pig and chicken after intravenous administration were best described by a two-compartment open model, giving distribution half-lives of 0.50 and 0.66 h and elimination half-lives of 7.92 and 4.10 h for pig and chicken respectively. The steady-state distribution volumes were 1.70 and 3.06 l/kg for pig and chicken respectively. 3. After oral administration of 5 mg/kg to pig and chicken, the serum concentrations reached maximal levels of 3.61 and 0.96 microg/ml respectively at 1.25 and 1.40 h. The elimination half-lives were 11.8 and 7.35 h for pig and chicken respectively. 4. The bioavailabilities of difloxacin were calculated as 93.7 (pig) and 86.9% (chicken).

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.

Higher plants produce diverse classes of metabolites. Metabolic engineering offers tremendous potential to improve the production and quality of these chemicals. This report summarizes the possibility of using metabolic engineering in benzylisoquinoline alkaloid biosynthesis. Benzylisoquinoline alkaloids, such as morphine, sanguinarine, and berberine, are synthesized from tyrosine via reticuline in Magnoliaceae, Ranunculaceae, Berberidaceae, Papaveraceae, and many other species. The early pathway from tyrosine to reticuline is common among many plant species, whereas there is more diversity in late pathways. This review describes several strategies to improve the yield and quality of benzylisoquinoline alkaloids. First, the overexpression of a rate-limiting enzyme in an early pathway to increase the overall alkaloid yield is discussed. Second, the introduction of a new branch into the pathway has been shown to produce novel metabolites. Finally, the possibility of accumulating a pathway intermediate by the knock-down of a key step is examined. Further metabolic modification is also discussed, since the latter two modifications may lead to the production of novel compound(s) from an accumulated intermediate through metabolic activation. These metabolic changes could be further modified to increase chemical diversity through somatic variation in cell culture. Besides this direct metabolic engineering with isolated biosynthetic genes, the regulation of biosynthetic activity with transcription factors and/or with reconstruction of the entire biosynthesis will also be discussed for the next generation of metabolite production.