양귀비 현탁 배양 내에서의 모르핀계 알칼로이드 생산을 위한 생합성 경로의 조절

Abstract

Plants have been very important for a long time not only as food but also as chemicals including pharmaceuticals, flavors and insecticides. Though the chemically synthetic productions have many advantages about cost, plants are still a source of chemical compounds which are too complex and expensive to produce by general methods. Generally, we have been interest to the secondary metabolites among the plant products. During several decades, many plants have been cultured and many secondary metabolites were detected and analyzed. The plant cell culture as an alternative source of secondary products has been developed by many reasons. Plant cell culture is independent from seasonal variation and outer diseases, a consistent quality and quantity. The most popular techniques for increasing secondary metabolites were cell selection and elicitation. In chapter II, our target calli obtained from callus induction have been used for screening of high-producing strains. After 6 months of subculture, the suspension cells of P. somniferum and E. californica has showed higher and uniform metabolites product than cells from induction time. By adding elicitors to these cells, we desired to improve the production of our target secondary metabolites. The best timing to elicitor treatment was day 5 after subculture as the early exponential phase of biomass growth. In addition, we investigated how gene expressions were changed in biosynthetic pathway. Secondary metabolites production associated with enzymatic reactions could be influenced by elicitation and accumulated as DNA expression level was increased. But unfortunately benzo[c]phenanthridine alkaloids, which had antimicrobial activity, were only accumulated by elicitation instead of morphinan alkaloids. Agrobacterium tumefaciens mediated transformation protocol for stable integration of foreign genes into opium poppy was established in chapter III. Before transformation, we selected non-producing cell line to reduce sanguinarine production and to enhance efficiency of transformation. pCAMBIA1304 vector was integrated successfully and we checked that expression level of DNA, RNA and protein was increased in our cell line by southern blotting, PCR, RT-PCR, histochemical assay and fluorescence assay. There was no difference between sanguinarine concentration of untransformed calli and that of transformants in our cell line because of using dummy vector. From this study, the useful protocol was set up for future studies on increasing secondary metabolites productivity of opium poppy by transformation. (S)-Reticuline represents a major branch point intermediate in plant secondary metabolism from which a wide variety of benzylisoquinoline alkaloids metabolites can be derived. The 1,2-dehydroreticuline synthase (1,2-DHRS), which is the first enzyme in the branches of benzylisoquinoline alkaloids and morphinan alkaloids may be a key enzyme of particular interest. In chapter IV, we have demonstrated biotransformation to produce (S)-reticuline compound by modulation of biosynthetic pathway in cell-free system. (S)-reticuline as substrate must be prepared essentially with 1,2-DHRS enzyme assay for purifying and separating the target enzyme. For retaining this metabolite, which is maintained with a small quantity in plant or cell and easily converted to (S)-scoulerine by enzyme reaction, we suggested sequential biotransformation system from dopamine to (S)-reticuline via (S)-norlaudanosoline and (S)-3’-hydroxy-N-methylcoclaurine by using two different types of Aspergillus niger protein and Eschscholtzia californica protein. These results suggest that our combination system may be very useful for synthesizing benzylisoquinoline alkaloids with cell-free system that express the desired biosynthetic genes. Sequentially, we tried to separate 1,2-DHRS partially by desalting column and SDS-PAGE, and moreover confirmed existence of our target enzyme by HPLC-DAD-ESI/MS. Putative sequences of protein by MALDI-TOF MS-MS were investigated and matched with stearoyl-acyl carrier protein desaturase (Sesamum indicum). Our ultimate goal, to produce morphinan alkaloids in plant cell culture, has not been accomplished until now. These data served the clues to characterize 1,2-dehydroreticuline synthase for further research. If morphinan alkaloids will be produced in plant cell culture, it will be conformed that these approaches would be a powerful tool for enhancing the productivity of low yield alkaloids or no accumulation alkaloids.