Plant alkaloids, one of the largest groups of natural products, provide many pharmacologically active compounds. Several genes in the biosynthetic pathways for scopolamine, nicotine, and berberine have been cloned, making the metabolic engineering of these alkaloids possible. Expression of two branching-point enzymes was engineered: putrescine N-methyltransferase (PMT) in transgenic plants of Atropa belladonna and Nicotiana sylvestris and (S)-scoulerine 9-Omethyltransferase (SMT) in cultured cells of Coptis japonica and Eschscholzia californica. Overexpression of PMT increased the nicotine content in N. sylvestris, whereas suppression of endogenous PMT activity severely decreased the nicotine content and induced abnormal morphologies. Ectopic expression of SMT caused the accumulation of benzylisoquinoline alkaloids in E. californica. The prospects and limitations of engineering plant alkaloid metabolism are discussed.berberine ͉ nicotine ͉ polyamine ͉ sanguinarine ͉ scopolamine H igher plants constitute one of our most important natural resources. They provide not only foodstuffs, fibers, and woods, but many chemicals, such as oils, flavorings, dyes, and pharmaceuticals. Although plants are renewable resources, some species are becoming more difficult to obtain in sufficient amounts to meet increasing demands. Destruction of natural habitats and technical difficulties in cultivation also are driving the drastic reductions in plant availability. For example, it is claimed that the demand for paclitaxel, a potent anticancer compound, could endanger forests of Taxus brevifolia (Pacific yew) because of the low paclitaxel content (40-100 mg͞kg of bark) in and slow growth of the trees (1).For many natural chemicals it is possible to synthesize alternatives from petroleum, coal, or both. The economic limitations of chemical synthesis and the pollution that accompanies this type of chemical synthesis, however, have led to the development of cell culture and molecular engineering of plants for the production of important and commodity chemicals. Plant cell and organ culture offer promising alternatives for the production of chemicals because totipotency enables plant cells and organs to produce useful secondary metabolites in vitro (2). Cell culture is also advantageous in that useful metabolites are obtained under a controlled environment, independent of climatic changes and soil conditions. In addition, the products are free of microbe and insect contamination. Fermentation technology also can be used to produce desired metabolites and can be optimized to maintain high and stable yields of known quality by cellular and molecular breeding techniques to further improve productivity and quality. After extensive empirical trials, some metabolites are now being produced by large-scale cell culture (e.g., shikonin and berberine; ref. 2), but the numbers of compounds that are producible commercially by cell culture technology are still very few. The main limitations are low productivity and the necessity of the down-stream pr...
S-Adenosyl-L-methionine:coclaurine N-methyltransferase (CNMT) converts coclaurine to N-methylcoclaurine in isoquinoline alkaloid biosynthesis. The N-terminal amino acid sequence of Coptis CNMT was used to amplify the corresponding cDNA fragment and later to isolate full-length cDNA using 5-and 3-rapid amplification of cDNA ends (RACE). The nucleotide sequence and predicted amino acid sequence showed that the cDNA encoded 358 amino acids, which contained a putative S-adenosyl-L-methionine binding domain and showed relatively high homology to tomato phosphoethanolamine-N-methyltransferase. A recombinant protein was expressed in Escherichia coli, and its CNMT activity was confirmed. Recombinant CNMT was purified to homogeneity, and enzymological characterization confirmed that Coptis CNMT has quite broad substrate specificity, i.e. not only for 6-O-methylnorlaudanosoline and norreticuline but also for 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline. The evolution of N-methyltransferases in secondary metabolism is discussed based on sequence similarity. S-Adenosyl-L-methionine (AdoMet)1 :coclaurine N-methyltransferase (CNMT) (1-3) catalyzes the transfer of a methyl group from S-adenosyl-L-methionine to the amino group of the tetrahydrobenzylisoquinoline alkaloid coclaurine. This is a unique N-methyltransferase in the biosynthesis of benzylisoquinoline alkaloids (Fig. 1). This enzyme is thought to be important because N-methylation of coclaurine strongly enhances the 4Ј-O-methylation activity of 3Ј-hydroxy-N-methylcoclaurine 4Ј-O-methyltransferase and enables the sequential metabolic conversion of substrates (4, 5). Furthermore, the enzymatic activity at this important step is rather low relative to the entire biosynthetic pathway (1, 6 -8). Thus, we purified CNMT and characterized its properties. Previous studies have clearly indicated that CNMT is non-stereospecific and has broad substrate specificity; Coptis enzyme methylated even simple dihydroxyisoquinoline alkaloids.Whereas several O-methyltransferases have been characterized at the molecular level (4, 9 -12), there have been very few molecular studies of N-methyltransferases in secondary metabolite biosynthesis in plants (13,14). The differences in the primary structures, including the S-adenosyl-L-methionine binding site of NMTs and OMTs reported to date, suggest that molecular isolation of CNMT based on the structural similarity of methyltransferases would be very difficult. Thus, we adapted the conventional strategy to isolate cDNA based on the amino acid sequence of purified enzyme. Whereas our purified CNMT fraction still contained two protein bands of about 45 kDa, careful inspection of the chromatographic behavior of the proteins and enzyme activity suggested that the slow moving 45-kDa polypeptide would encode CNMT (1). Based on this observation, we determined the N-terminal amino acids, isolated the corresponding cDNA fragment, and finally the fulllength cDNA. The nucleotide sequence of cDNA suggested that the deduced amino acid sequence showed some simi...
To identify all of the O-methyltransferase genes involved in isoquinoline alkaloid biosynthesis in Coptis japonica cells, we sequenced 1014 cDNA clones isolated from high-alkaloidproducing cultured cells of C. japonica. Among them, we found all three reported O-methyltransferases and an O-methyltransferase-like cDNA clone (CJEST64). This cDNA was quite similar to S-adenosyl-L-methionine:coclaurine 6-O-methyltransferase and S-adenosyl-L-methionine:isoflavone 7-O-methyltransferase. As S-adenosyl-L-methionine:columbamine O-methyltransferase, which catalyzes the conversion of columbamine to palmatine, is one of the remaining unelucidated components in isoquinoline alkaloid biosynthesis in C. japonica, we heterologously expressed the protein in Escherichia coli and examined the activity of columbamine O-methyltransferase. The recombinant protein clearly showed O-methylation activity using columbamine, as well as (S)-tetrahydrocolumbamine, (S)-, (R,S)-scoulerine and (R,S)-2,3,9,10-tetrahydroxyprotoberberine as substrates. This result clearly indicated that EST analysis was useful for isolating the candidate gene in a relatively well-characterized biosynthetic pathway. The relationship between the structure and substrate recognition of the O-methyltransferases involved in isoquinoline alkaloid biosynthesis, and a reconsideration of the biosynthetic pathway to palmatine are discussed.
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