P.somniferum cell culture

The release of sanguinarine from the cells into the medium after elicitation was used as the basis for the design of an industrial process for production of the alkaloid 72,99,487,492,493). As the cells of P. somniferum remain viable after a 72-hr exposure to a fungal elicitor, it is possible to recycle the cells after induction of alkaloid biosynthesis. Because about 40-60% of the alkaloid is released to the medium, a simple change of medium is sufficient to collect the alkaloid and to recover viable cells. By a repeating sequence of elicitation and medium replenishment, a semicontinuous production process was obtained. Such a process was also developed for alkaloid production in Catharanthus roseus, Ruta grave-olens, and P. somniferum cell cultures using different elicitors (99). 

Acknowledgements. This work was supported by NSF Grant PCM 8011471 and NIH Training Grant 5 T32 HL-07050-09. We thank Elizabeth M. Coscia and Micheal D. Rush for their able assistance. Dr. Alan Kinnersley for P. somniferum cell cultures, and Schering, AG for Renografin. 

Salutaridinol 7-O-acetyltransferase was purified to apparent electrophoretic homogeneity from P. somniferum cell suspension cultures and the amino acid sequence of ten endoproteinase Lys-C-generated peptides was determined.28 A comparison of these amino acid sequences with those available in the GenBank/EMBL sequence databases indicated no relevant similarity to known proteins. The first attempt to isolate a cDNA encoding salutaridinol 1-0-... 

Many researchers have so far investigated tissue culture of P. somniferum [131, ref. cited therein], and most cultured P. somniferum cells, either as callus or cell suspensions, readily produced sanguinarine and its analogs [130-137], but rarely, if even, produce morphinan alkaloids [138]. Kamo et al. [139], Schuchmann and Wellmann [134], and Yoshikawa and Furuya [140] reported the production of morphinan alkaloids in redifferentiated organs, either shoots or somatic embryos, and their results emphasize the importance of the degree of cell differentiation for the biosynthesis of morphinan alkaloids. 

Bioconversion rates can be optimized by using immobilized cells, cell-free preparations, or immobilized (purified) enzymes. Furuya et al. lOI) reported the reduction of codeinone to be more efficient with immobilized P. somniferum cells than with suspended cells. Strictosidine could be produced in large quantities using immobilized strictosidine synthase 102). The potential for this use of plant cell cultures is enormous however, the number of successful applications is still limited. 

The first reported biotransformation by means of cell cultures of P. somniferum was the conversion of thebaine to codeine (453). As P. som-nifemm cell cultures only produced alkaloids derived from (5)-reticuline (e.g., sanguinarine. Fig. 9) but none of the alkaloids derived from (/ )-reticuline, Furuya et al. 461) administered (R,S)-reticuline to the cell suspension cultures. After 3 days the alkaloids were isolated. Two alkaloids derived from (5)-reticuline were identified cheilathifoline and scoulerine. A third alkaloid isolated was identified as pure (/ )-reticuline. Thebaine, morphine, and codeine were not metabolized by these cell cultures. However, the cells were capable of stereospecifically reducing codeinone to codeine. Tam et al. 462) also found the same bioconversion. Furthermore, the conversion of thebaine to neopine was reported. Codeine, neopine, papaverine, and D,L-laudanosoline were not metabolized. Enzymatic reduction of codeinone to codeine was also achieved with cell-free preparations of whole plants of both P. somniferum and P. bracteatum 464). Yeoman and co-workers 108,465) reported the use of in reticulate polyurethane immobilized P. somniferum cells for this bioconversion. 

Lenz, R. and M. H. Zenk, Purification and properties of codeinone reductase (NADPH) from Papaver somniferum cell culture s and differentiated plants. Eur. J. Biochem., 1995. 233(1) p. 132-9. 

Biotransformations of morphinan alkaloids have been reported for plant, fungal, and mammalian enzymatic systems with emphasis on rather specific reactions such as the reduction of ketones, N- and O-demethylation, and perox-idative transformations. Furuya et al. used immobilized tissue culture cells of Papaver somniferum to accomplish the selective reduction of codeinone (135) to codeine (136) (207) (Scheme 30). Suspension cultures of a well-established cell line of P. somniferum were grown for one week as a source of cell mass for immobilization in calcium alginate. The cells continued to live in the alginate matrix for 6 months maintaining their biological activity. The reduction of co-... 

Codeine, 14-hydroxycodeine, neopine, and O-methylflavinantine have been isolated from Papaver bracteatum,146 flavinantine and amurine from Meconopsis cambrica,113 isosinoacutine from Stephania elegans,174 and a new alkaloid, tridictyophylline, for which the structure (96) was determined by X-ray crystallography, from Triclisia dictyophylla.175 Bound morphine, codeine, and thebaine have been found in P. bracteatum and P. somniferum,176 and the effect of the period of maturation of the plants on the yield of these three alkaloids from P. somniferum has been studied.177,178 Codeine has been isolated from cell suspension cultures of P. somniferum under conditions where no morphine, norcodeine, or thebaine could be detected.179... 

Fairbaim and Williamson studied P. bracteatum anatomically and compared it to P. somniferum (18). They found the two plants to be very similar in stmcture. The laticifers of P. bracteatum were usually more closely packed and anastomose more frequently. The subcellular fraction of protoplasts from cultured P. bracteatum cells (organelles sedimenting at 1000 g) was the major site where thebaine and sanguinarine accumulated (19). It also contained dopamine as a precursor and the vacuolar enzyme a-mannosidase. Dopamine also appeared in the supernatant. Dopamine compartmentalization in vacuoles of cultured cells was observed by histofluorescence microscopy. Dopamine, sanguinarine, and thebaine occurred in vacuoles of different densities. This result is consistent with... 

Hutin and co-workers (436,437) reported that usual alkaloid extraction procedures were unable to extract any alkaloid from cell cultures of P. somniferum. By refluxing with hydrochloric acid (1 M, 10 min) or formic acid (3 M, 4 min) morphine and codeine were extracted in detectable amounts. 

Coutts IGC, Hamblin MR, Tinley EJ (1979) The enzymatic oxidation of phenolic tetrahydroiso-quinoline-1-carboxylic acids. J Chem Soc Perkin Trans 1 2744-2750 Davis VE, Cashaw JL, McMurtrey KD, Ruchirawat S, Nimit Y (1982) Metabolism of tetrahydro-isoquinolines and related alkaloids. In Bloom F, Barchas J, Sandler M, Usdin E (eds) Beta-carbolines and tetrahydroisoquinolines. Liss, New York, p 99 Furuya T, Nakano M, Yoshikawa T (1978) Biotransformation of (RS)-reticuline and morphinan alkaloids by cell cultures of Papaver somniferum. Phytochemistry 17 891-893 Gates M (1953) Conversion of codeinone to codeine. J Am Chem Soc 75 4340-4341 Graves JMH, Smith WK (1967) Transformation of pregnenolone and progesterone by cultured plant cells. Nature (London) 214 124 8-1249... 

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