Biotransformation of cannabinoids by a cell suspension culture of Cannabis sativa L

A cell suspension culture of Cannabis sativa L. is able to convert cannabidiol to bound cannabielsoins and delta-9 tetrahydrocannabinol to cannabicoumaronon. The localization and the mechanism of the bioconversion are discussed.Abbreviations CBD cannabidiol - CBE cannabielsoin - CBon cannabicoumaronon - EHHC hexahydrocannabinol epoxide - EtOAc ethyl acetate - FBS Fast Blue B salt - GLC gas-liquid chromatography - THC delta-9 tetrahydrocannabinol - TLC thin-layer chromatography     

Bioconversion of gitoxigenin by immobilized plant cells in a column bioreactor

Summary Daucus carota cells immobilized in a Ca-alginate performed the bioconversion of gitoxigenin to 5-hydroxygitoxigenin in a column bioreactor. The smooth spherical shape of the alginate beads was preserved for more than three weeks. The bioreactor was functional for more than thirty days as detected by the bioconversion activity. The rate of bioconversion was influenced by means of aeration.Issued as National Research Council of Canada Publication Number 19556     

In vitro culture of cell aggregates of rye (Secale cereale L.) at low densities

The growth of cell aggregates from a rye suspension culture was tested at low density in three culture systems. Mass seeding was the most supportive system, followed by agarose droplets. Microdroplet culture using Cuprak dishes was the least effective system. Growth was stimulated by the presence of a feeder layer of suspension cells but only if the feeder contact with the nursed cells was via a liquid and not a gaseous phase. Plating efficiences were enhanced by the feeder effect, whereas the subsequent growth rates were less affected. The techniques described should prove useful in programs aimed at the in vitro genetic manipulation of rye or other cereals.Abbreviations 2,4-D 2, 4 — dichlorophenoxy acetic acid - PE Plating efficiency     

Directed electrofusion between protoplasts with different responses in a mass fusion system

Protoplasts of Solanum brevidens (leaves) and Nicotiana rustica (suspensions) have been aligned and fused electrically between widely spaced electrodes, and the yield of 1:1 (binary) fusion products in chains of aligned protoplasts has been determined by light microscopy. Leaf protoplasts fuse more easily than protoplasts from suspension cultures (Tempelaar and Jones, 1985), thus electrical parameters and the ratio of leaf: suspension protoplasts can be varied to control the yield of binary and multifusion products. In experiments to determine optimum ratios for electrofusion, up to 60–70% of S. brevidens — N. rustica fusion products were binary at overall fusion frequencies of 40–50%.Fusions in samples of protoplasts with the same characteristics can also be controlled to direct the fusion process towards binary products. However, in this case, at least half of the binary products may be derived from self-fusions.     

Characterization of a monoterpene hydroxylase from cell suspension cultures of Catharanthus roseus (L.) G. Don

Conditions have been established for the optimization of the specific activity of a membrane-bound monoterpene hydroxylase from cell suspension cultures of Catharanthus roseus. In time course studies, the hydroxylase and NADPH-cytochrome c reductase exhibited maximal activities 18–20 days after inoculation, i.e., during early stationary phase. By late stationary phase, enzyme activity had declined. In contrast an enzyme of primary metabolism achieved optimal specific activity by the 12th day and remained constant through day 26, synchronous with general growth. Effects of nutritional and hormonal factors on the specific activity of the hydroxylase and cell growth were evaluated. Inhibitors of hydroxylase activity were also assessed in vitro. A soluble form of the monoterpene hydroxylase has been detected in cultured cells possibly affording a useful source of this enzyme for further purification.     

Protoplast fusion and the cell cycle

Protoplasts isolated from rapidly dividing cell suspension cultures of either Nicotiana sylvestris or tumor derived cultures of Crepis capillaris were fused by PEG or liposome treatments to form homokaryons. Analysis of binucleates by Feulgen microspectrophotometry, and autoradiography, has revealed that whereas fusion products of all cell cycle combinations occur, protoplasts of certain cycle phases participate in fusions more frequently than expected, and there is a slight predominance of like-with-like cycle combinations. It is argued that this tendency towards specificity of fusion may be explained by cycle related variation in surface charge on protoplasts, and the mechanisms of action of the fusogens used.Abbreviations PEG polyethylene glycol - CAPT tumorous cell culture of Crepis capillaris - NS-1 cell culture of Nicotiana sylvestris     

Bioconversion with whole cell penicillin acylase in aqueous two-phase systems

The deacylation of Pen G was carried out by using recombinant E. coli in an aqueous two-phase system consisting of polyethylene glycol and potassium phosphate solution, which partitions the cells to the bottom phase and the products to the top phase. Bioconversion and product separation were carried out in the same reactor. Repeated batch conversion was employed ten times and enzymic activity showed only a slight decline. When pure enzyme was used for bioconversion in an aqueous two-phase system, the decline was fast and bioconversion using whole cell penicillin acylase was better than that obtained using the pure acylase.     

Use of an electrochemical technique to study plasmamembrane redox reactions in cultured cells of Daucus carota L.

By exploiting the electron transfer reactions of ferricyanide-ferrocyanide at a gold electrode, an electrochemical technique was devised and successfully employed for the measurement of extracellular ferricyanide reduction or ferrocyanide oxidation by carrot (Daucus carota L.) cells grown in suspension culture. This technique eliminated problems of cell damage and insensitivity encountered when these activities are measured spectrophotometrically in magnetically stirred cell suspensions. Cells harvested from the mid-exponential phase of culture growth catalysed a rapid reduction of ferricyanide which was accompanied by H⁺ extrusion and was stimulated by ethanol. These cells also oxidised ferrocyanide, which was not associated with H⁺ extrusion. These observations can be explained on the basis of a plasmamembrane located, H⁺-translocating redox system. The electrochemical technique is a useful method of studying this system.     

Biotransformation of tabersonine in cell suspension cultures of Catharanthus roseus.

To investigate the reactions involved in the biosynthesis of vindoline from tabersonine, the bioconversion products formed when the latter compound was fed to cell suspension cultures of Catharanthus roseus were isolated and characterized. Two biotransformation products of tabersonine were isolated and shown to be lochnericine, which is formed by epoxidation of tabersonine at positions 14, 15, and lochnerinine, the 11-methoxylation product of lochnericine. The bioconversion ratio of the main biotransformation product, lochnericine, reached a value of 80.6% within three days.     

Alkaloid production in cell suspension cultures of Thalictrum flavum and T. dipterocarpum

Both cell suspension cultures of Thalictrum flavum and T. dipterocarpum were found to produce berberine (0.3 and 0.4 g/l, respectively) as a main alkaloid. Berberine production in the latter was markedly stimulated by 1-naphthaleneacetic acid in combination with 6-benzylaminopurine, whereas it was rather suppressed by the same auxin in the former. T. flavum cultures accumulated berberine and columbamine in the cells without releasing them into medium. On the other hand, T. dipterocarpum cultures released berberine into medium during the logarithmic growth phase, but thereafter accumulated all the berberine synthesized in the cells.Abbreviations LS Linsmaier and Skoog - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - BA 6-benzylaminopurine - TFG a culture strain of T. flavum ssp. glaucum - TDP a culture strain of T. dipterocarpum     

An assessment of the cultural capabilities of protoplasts of some wild species of linum

Protoplasts of several wildLinum species were isolated enzymatically from roots, hypocotyls and cotyledons of seedlings, and also from theirin vitro grown shoots and cell suspension cultures. When cultured all these protoplasts divided to produce callus but only good plant regeneration capability was evident in the case ofLinum lewissii and to a much lesser extent forL. strictum. Only rhizogenesis was observed withL. alpinum, L. narbonense, L. grandiflorum andL. altaicum. The high plant regeneration capacity ofL. lewissii from protoplast -derived tissues ofin vitro shoots and cell suspension cultures makes this species an attractive experimental system for somatic genetic manipulation.Abbreviations BAP benzylaminopurine - NAA -naphthaleneacetic acid - CPW cell and protoplast wash solution - gFW gram fresh weight On leave from Department of Crop Sciences University of Alexandria Egypt     

Production of L-DOPA from cell suspension culture of Mucuna pruriens f. pruriens

Summary Production of L-DOPA was studied in cell suspension culture of Mucuna pruriens f. pruriens. Suspension culture was established in MSI medium composed of half concentration of Murashige and Skoog's salts and 2% sucrose. A two-stage cell suspension culture was developed for enhanced accumulation of L-DOPA. In the first stage, the culture system was composed of MSI medium without CaCl, which was suitable for cell growth and in the second stage MSI medium containing 42.5 mg.l^–1 KH₂PO₄ and 4% sucrose favoured L-DOPA production. A discernible higher production of L-DOPA was obtained in this two-stage cell suspension culture in comparison to single stage culture.     

Potential of Datura innoxia cell suspension cultures for glucosylating hydroquinone

The efficiency of glucosylation of hydroquinone by Datura innoxia cell suspension cultures was investigated. The yield of arbutin was 2.4 g/l medium when 10 mM of hydroquinone was added to a suspension culture that was then incubated for 24 h, but the yield decreased at a higher concentration. This decrease, which could not be overcome by changing the growth phase or increasing the cell density used, could be avoided by the repeated addition of a low concentration of hydroquinone over 3 days. This increased the yield of arbutin to 4.2 g/l at the usual cell density and to 7.1 g/l at a high density. The kinetics of this reaction were explained by the Michaelis-Menten formula. The theoretical maximum velocity of the arbutin-forming reaction was estimated as 0.77 mg/h/g. The velocity increased linearly up to a cell density of 300 g/l under standard aeration, the theoretical maximum yield of arbutin being calculated to be 5.5 g/l/day.     

The screening of microorganisms for hydroxylation activity: Hydroxylation of diamantanols

Summary 1-and/or 4-diamantanols are hydroxylated by various species of the genus Rhizopus giving rise to diamantandiols. The GLC method revealed both the species specificity of the organism and substrate specificity in the bioconversion. The effectivity of biotransformation by some species of the genus Rhizopus indicates that the procedure could be used for the preparation of some diamantandiols substituted on tertiary carbon atoms.     

Studies on Bacillus stearothermophilus. Part IV. Influence of enhancers on biotransformation of testosterone

The impact of chemical enhancers on the biotransformation of testosterone has been exploited. Application of crude cell concentrates to produce Bacillus stearothermophilus-mediated bioconversion of testosterone at 65 degrees C for 72 h has been examined. After incubation, the xenobiotic substrate was added to the concentrated whole cell suspensions. The enhancer molecules were included in the whole cell suspension. The resultant products, after extraction into an organic solvent, were purified by thin layer chromatography and identification was carried out through spectroscopic data. Five steroid metabolites 9,10-seco-4-androstene-3,9,17-trione, 5alpha-androstan-3,6,17-trione, 17beta-hydroxy-5alpha-androstan-3,6-dione, 3beta,17beta-dihydroxyandrost-4-ene-6-one and 17beta-hydroxyandrost-4,6-diene-3-one were identified as biotransformation products of testosterone. A possible biosynthetic route for these bioconversion products is postulated.     

Specific differences in tolerance to exogenous berberine among plant cell cultures

The tolerance of plant cells to exogenously administered berberine, an antimicrobial isoquinoline alkaloid, was studied using berberine-producing and nonproducing cell suspension cultures. Both Coptis japonica and Thalictrum flavum cells, which have an intrinsic ability to synthesize berberine, took up exogenous berberine from the culture medium by an energy-requiring active transport to accumulate it exclusively in vacuoles. By contrast, T. minus cells, which excrete indigenous berberine mostly into the medium, did not take up exogenously supplied berberine, indicating that the alkaloid transport in this species is unidirectional. No inhibition of cell growth by exogenous berberine was observed in the three berberine-producing cell cultures. On the other hand, a small amount of exogenous berberine strongly inhibited cell growth in the berberine-free cultures of Datura innoxia, Catharanthus roseus, and Paeonia albiflora. The berberine taken up actively by Datura cells could not be transported into vacuoles but was dispersed in the cytoplasm, causing a severe inhibition of cell growth.     

Microencapsulation of mammalian cells in a water-insoluble polyacrylate by coextrustion and interfacial precipitation

A process for the microencapsulation of mammalian cells in a commercially available water-insoluble polyacrylate (EUDRAGIT RL) is described, and the effects of process parameters are outlined The polymer dissolved in diethyl phthalate was pumped along the annulus formed from two concentric needles, while the cell suspension was pumped inside the inner needle Droplets of polymer solution containing cells were blown off the end of the needles by a coaxial air stream. The droplets fell into a corn oil-mineral oil curing bath, in which the solvent was removed from the nascent capsule causing the polymer to precipitate around the cell suspension core. Capsules were washed free of oils and solvent in a fractionated plasma that allowed for quantitative transfer of capsules from the oil phase to an aqueous medium. By appropriate adjustment of the coaxial air flow rate, capsule size could be varied from 250-1000 mum, although the most convenient size was found to be 400-700 mum. Adding Ficoll 400 to the cell suspension to match the density of the suspension to the polymer solution resulted in capsules with a well-centered core but did not affect capsule strength. It appeared that increasing the polymer solution concentration or the polymer to the cell flow rate ratio resulted in an increased capsule strength, although differences in capsule size made unequivocal conclusions difficult. These capsules are of potential use as an artificial pancreas for the treatment of diabetes (with pancreatic islets) or for large-scale tissue culture and the production of bioactive products (e.g., with fibroblasts).     

Steroid modification with immobilized mycelium of Aspergillus phoenicis

Summary The bioconversion of progesterone by Aspergillus phoenicis has been studied. The metabolism in our conditions of experiment gave five important products. About 10 hours were necessary to obtain the highest concentration of 11--hydroxyprogesterone. During this time, 90% of progesterone were transformed at the optimal pH value 2.5. The transformations were tested using different immobilization methods which merits and limitations will be discussed.     

A new bioassay system for screening high berberine-producing cell colonies of Thalictrum minus

A new method of estimating the amount of berberine released from minute cell colonies of Thalictrum minus has been devised to facilitate the selection of high berberine-producing cell lines. In this system, cell aggregates obtained from a cell suspension culture are grown on small pieces of an agar culture medium and the concentration of berberine which has been released from the cells into the agar piece is assayed by the antibacterial activity against Bacillus cereus MT2026. Screening of 1000 cell colonies by the agar piece method has resulted in the isolation of four, high berberine-producing cell lines, although they have been found to be more or less unstable with respect to the biosynthetic capability during successive subcultures.     

Growth of baculovirus-infected insect cells in microcapsules to a high cell and virus density

Summary Insect cells (Spodoptera Frugiperda), infected with a temperature-sensitive mutant (TS10) of theAutographa Californica nuclear polyhedrosis virus (AcNPV), were cultured in multiple membrane alginate-polylysine (PLL) microcapsules. It was possible to obtain intracapsular cell densities of 8× 10⁷ cells/mL of capsules and virus concentrations of up to 10⁹ IFU/mL of capsules. This was higher by a factor of 10 than that which could be achieved by conventional cell suspension culture.