Differential patterns of dehydroabietic acid biotransformation by Nicotiana tabacum and Catharanthus roseus cells

The aim of this study was to use whole cell catalysts as tools for modification of selected resin acids in order to obtain value-added functional derivatives. The enzymatic bioconversion capacities of two plant species were tested towards dehydroabietic acid. Dehydroabietic acid (DHA) is an abundant resin acid in conifers, representing a natural wood protectant. It is also one of the constituents found in by-products of the kraft chemical pulping industry. DHA was fed to tobacco (Nicotiana tabacum) and Madagascar periwinkle (Catharanthus roseus) plant cell and tissue cultures and bioconversion product formation was monitored using NMR analysis. Both plant species took up DHA from culture medium, and various types of typical detoxification processes occurred in both cultures. In addition, diverse responses to DHA treatment were observed, including differences in uptake kinetics, chemical modification of added substrate and changes in overall metabolism of the cells. Interestingly, Catharanthus roseus, a host species for pharmaceutically valuable terpenoid indole alkaloids, exhibited a very different bioconversion pattern for exogenously applied DHA than tobacco, which does not possess a terpenoid indole pathway. In tobacco, DHA is readily glycosylated in the carbonyl group, whereas in periwinkle it is proposed that a cytochrome P450-catalyzed enzymatic detoxification reaction takes place before the formation of glycosylated product.     

Enantioselectivity in the biotransformation of bicyclic monoterpene alcohols with the cultured suspension cells of Nicotiana tabacum

The enantiomeric pairs of borneol, isoborneol, and isopinocampheol were incubated with the cultured suspension cells of Nicotiana tabacum. One of each enantiomeric pair was preferentially transformed to the corresponding carbonyl compound. It was thus demonstrated that the cultured cells have the ability to discriminate between the enantiomers of the bicyclic terpene alcohols.     

Suspension cultured transgenic cells of Nicotiana tabacum expressing tryptophan decarboxylase and strictosidine synthase cDNAs from Catharanthus roseus produce strictosidine upon secologanin feeding

A transgenic cell suspension culture of Nicotiana tabacum L. `Petit Havana' SR1 was established expressing tryptophan decarboxylase and strictosidine synthase cDNA clones from Catharanthus roseus (L.) G. Don under the direction of cauliflower mosaic virus 35S promoter and nopaline synthase terminator sequences. During a growth cycle, the transgenic tobacco cells showed relatively constant tryptophan decarboxylase activity and an about two- to sixfold higher strictosidine synthase activity, enzyme activities not detectable in untransformed tobacco cells. The transgenic culture accumulated tryptamine and produced strictosidine upon feeding of secologanin, demonstrating the in vivo functionality of the two transgene-encoded enzymes. The accumulation of strictosidine, which occurred predominantly in the medium, could be enhanced by feeding both secologanin and tryptamine. No strictosidine synthase activity was detected in the medium, indicating the involvement of secologanin uptake and strictosidine release by the cells. Received: 25 February 1996 / Revision received: 16 August 1996 / Accepted: 30 September 1996     

Microplate enzyme-coupled assays of mevalonate and phosphomevalonate kinase from Catharanthus roseus suspension cultured cells

A microplate assay for mevalonate and 5-phosphomevalonate kinase activities has been developed using an enzyme-coupled system of pyruvate kinase and lactate dehydrogenase. Mevalonate and 5-phosphomevalonate kinase activities were measured in crude and partially purified enzyme preparations from Catharanthus roseus suspension-cultured plant cells. The assay was validated with respect to protein and substrate concentration. Mevalonate and 5-phosphomevalonate kinase showed Michaelis-Menten kinetics with respect to ATP and their specific substrates; the apparent Km values of mevalonate kinase for ATP and mevalonate were 210 and 65 microM, respectively, and of 5-phosphomevalonate kinase for ATP and 5-phosphomevalonate were 0.41 and 0.4 mM, respectively. Considering mevalonate kinase, the relative standard deviation of enzyme activity within a determination (n = 3) is always less than 2.5% and in between determinations (n = 9) is less than 2%. The method can be used in a continuous assay as well as in a discontinuous assay.     

Serpentine release by suspension cultures of Catharanthus roseus

Summary Suspension cultures ofCatharanthus roseus filtered (with or without a vacuum) and resuspended in fresh or spent medium will release serpentine into the medium. This treatment is associated with small increases in pH and conductivity of the medium. The released serpentine quickly disappears, and is probably taken up by the cells.     

Biosynthesis of brassinosteroids in cultured cells of Catharanthus roseus

Precursor administration experiments with 2H-labeled 6-oxocampestanol, 6-deoxocastasterone and 6alpha-hydroxycastasterone in cultured cells of Catharanthus roseus were performed and the metabolites were analyzed by GC-MS. [2H6]Cathasterone was identified as a metabolite of [2H6]6-oxocampestanol, whereas [2H6]6alpha-hydroxycastasterone and [2H6]castasterone were identified as metabolites of [2H6]6-deoxocastasterone, and [2H6]castasterone was identified as a metabolite of [2H6]6alpha-hydroxycastasterone, indicating that 6-deoxocastasterone is converted to castasterone via 6alpha-hydroxycastasterone. In addition, 6-deoxocathasterone, a putative biosynthetic intermediate in the late C6-oxidation pathway, was identified as an endogenous brassinosteroid. These studies provide further evidence supporting our proposed biosynthetic pathways for brassinolide.     

Ketoisophorone transformation by Marchantia polymorpha and Nicotiana tabacum cultured cells

Stereospecific olefin (C=C) and carbonyl (C=O) reduction of the readily available prochiral compound ketoisophorone (2,2,6-trimethyl-2-cyclohexene-1,4-dione) (1) by Marchantia polymorpha and Nicotiana tabacum cell suspension cultures produce the chiral products (6R)-levodione (2), (4R,5S)-4-hydroxy-3,3,5-trimethylcyclohexanone (3), and (4R,6R)-actinol (4) as well as the minor components (4R)-hydroxyisophorone (5) and (4S)-phorenol (6).     

Glucosylation of phenols and phenylalkyl alcohols using cultured plant cells

Cultured plant cells of Eucalyptus perriniana can convert phenol and phenylalkyl alcohols [C(6)H(5)(CH(2))(n)OH, n=0-3] into the corresponding beta-D-glucopyranosides in a good yield. The cells preferentially glucosylated phenylmethanol (n=1, 59% yield) rather than phenol (n=0, 49%), 2-phenylethanol (n=2, 38%), and 3-phenylpropan-1-ol (n=3, 20%). On the other hand, 2-, 3-, and 4-hydroxyphenylmethanols were also glucosylated to (hydroxymethyl)phenyl beta-D-glucopyranosides and (hydroxyphenyl)methyl beta-D-glucopyranosides by cultured E. perriniana cells.     

Glycosylation of capsaicin and 8-nordihydrocapsaicin by cultured cells of Catharanthus roseus

The glycosylation of capsaicin and 8-nordihydrocapsaicin was investigated using cultured cells of Catharanthus roseus. In addition to capsaicin 4-O-beta-d-glucopyranoside (170 microg/g fr. wt of cells), the biotransformation products, capsaicin 4-O-(6-O-beta-D-xylopyranosyl)-beta-D-glucopyranoside (116 microg/g fr. wt of cells) and capsaicin 4-O-(6-O-alpha-L-arabinopyranosyl)-beta-D-glucopyranoside (83 microg/g fr. wt of cells), were isolated from the cell suspension after three days of incubation with capsaicin. Two other compounds, 8-nordihydrocapsaicin 4-O-(6-O-beta-D-xylopyranosyl)-beta-D-glucopyranoside (171 microg/g fr. wt of cells) and 8-nordihydrocapsaicin 4-O-(6-O-alpha-L-arabinopyranosyl)-beta-D-glucopyranoside (122 microg/g fr. wt of cells), together with the known 8-nordihydrocapsaicin 4-O-beta-D-glucopyranoside (204 microg/g fr. wt of cells) were also isolated from the cell suspension after incubation with 8-nordihydrocapsaicin.     

Immobilization of cultured Catharanthus roseus cells using a fibreglass substratum

Summary Cultured Catharanthus roseus cells were immobilized using geometrically identical needled fibreglass mats prepared with a range of surface coatings. The phenyl (PS), polyglycol (PG), aldehyde (CHO), alkyl (CTMS), and silanol (AW) coatings, along with the untreated glass (HC) surface, produced surfaces with a range of surface tensions. The immobilization efficiency of the substratum, measured as the percentage of cells immobilized, increased with increasing substratum surface tension in the order PS < PG < CHO < CTMS < AW < HC. The dependence of immobilization efficiency on substratum surface tension can be described using a thermodynamic model of adhesion that considers the extent of plant cell adhesion to be a function of the surface tensions of the substratum, the suspending liquid, and the plant cells. In addition, this dependence also demonstrates the fundamental role of adhesion in the immobilization process involving a glass fibre matrix. However, cell entrapment processes are also implicated. The untreated glass fibre substratum (HC), which demonstrated the greatest immobilization efficiency, was used for further characterization of the immobilization strategy. Maximum inoculum biomass was determined to be approximately 1.9 g cells (fresh weight)/g substratum (dry weight) to achieve greater than 90% immobilization efficiency. The growth rate of immobilized cultures was slower than suspension cultures, probably due to mass transfer limitations. Production of the indole alkaloids, tryptamine, catharanthine, and ajmalicine, was also suppressed relative to suspension-cultured cells. These results are considered in relation to other immobilization strategies and their apparent effects on cellular processes. Offprint requests to: F. Dicosmo     

Noninvasive NMR studies of metabolism in cultured Catharanthus roseus cells

Summary Nuclear magnetic resonance (NMR) spectroscopy provides a unique modality for the study of tissue-cultured plant cells. One of its major attractions is that it allows noninvasive studies of plant material. In addition, it can provide insight into the pH in the vacuole and cytoplasm, and into the compartmentalization of certain metabolites. In this review we show how phosphorus-31 NMR is used to study intracellular pH, phosphate uptake and storage, and energy metabolism in suspension cells of Catharanthus roseus. In addition, multinuclear NMR studies of the uptake of ammonium and the gradients of K⁺ over the membrane are discussed as well. The use of two-dimensional NMR for the study of whole cell extracts is also described. Finally, we show how nitrogen-14 and nitrogen-15 NMR are used to obtain information about the assimilation of inorganic sources in developing carrot somatic embryos. These NMR studies provide a unique insight into the metabolism of tissue-cultured plant cells.     

Gibberellin A12 Is Converted to Gibberellin A53 in Cultured Cells of Nicotiana tabacum and Catharanthus roseus

The metabolism of GA₁₂ and its precursors was investigated incultured cells of seven cell lines of Nicotiana tabacum andthree cell lines of Catharanthus roseus using ^l4C-labeled substrates.The presence of a metabolic pathway from ent-7-hydroxykaurenoicacid to GA₅₃ via GA₁₂-aldehyde and GA₁₂ was demonstrated inthe cultured cells. GA₁₂ was effectively converted to GA₅₃ incells of BY-2, 2b-4, 2b-13 and CG from N. tabacum. By contrast,GA₅₃ was not converted to any other GAs in all of the linesof cells examined. The metabolism of C₁₉-GAs was also examinedusing ³H-labeled substrates. The conversion of GA₂₀ to GA₂₉and GA, and of GA₄ to GA₃₄ occurred more efficiently in cellsfrom C. roseus than in cells from N. tabacum. However, 13-hydroxylationof GA₄ and GA₉ was not observed in any of the cell culturesexamined. Among the various metabolites, GA₅₃, GA₂₉ and GA₃₄were identified by full-scan GC/MS. (Received December 20, 1990; Accepted May 27, 1991)     

Analysis of the nucleotide pool during growth of suspension cultured cells of Nicotiana tabacum by high performance liquid chromatography

In suspension cultured cells of Nicotiana tabacum L. cv. Wisconsin 38 the .concentration of 18 nucleotides and 3 nucleosides have been determined using a new procedure developed for the extraction, purification and HPLC separation of these compounds from plant tissue. The different nucleotide pools increase in size immediately at the onset of the batch culture and reach distinct maxima at the very beginning of the proliferative phase. The main component are the uracil nucleotides with UDP-sugars as the predominant fraction followed by the adenine nucleotides; the energy charge is maintained at a high and constant value throughout the whole culture time. During the growth interval the increases in the nucleotide pools reveal that the cell proliferation phase is followed by an extensive phase of cell elongation. Whereas the concentration of the total nucleotides varies by a factor of about 3 along the growth curve, the ratio of uracil to adenine nucleotides is kept fairly constant indicating regulatory mechanisms for correlation of the individual nucleotide pools.     

Stereoselectivity of the reduction of carvone and dihydrocarvone by suspension cells of Nicotiana tabacum

The biotransformation of foreign substrates with suspension cells of Nicotiana tabacum was tested with (4R)-(?)- and (4S)-(+)-carvones, (     

Cryopreservation of periwinkle,Catharanthus roseus cells cultured in vitro

A procedure for prolonged cryogenic storage of periwinkle cell cultures is described. Cells derived from periwinkle, Catharanthus roseus (L.) G. Don, and subcultured as suspension in 1-B5C nutrient medium have been frozen, stored in liquid nitrogen (–196°C) for 11 weeks, thawed and recultured. Maximal survival was achieved when 3–4 day-old cells precultured for 24 h in nutrient medium with 5% DMSO were frozen at slow cooling rates of 0.5 or 1°C/min prior to storage in liquid nitrogen. The only loss in viability of cells occurred subsequent to treatment with DMSO. Abbreviations: DMSO, dimethylsulfoxide; 2,4-D, 2,4-dichlorophenoxyacetic acid; TTC, triphenyltetrazolium chloride.NRCC No. 20082     

Formation of catharanthine,akuammicine and vindoline in Catharanthus roseus suspension cells

Catharanthine and akuammicine, together with ajmalicine and strictosidine, were isolated from a culture strain of Catharanthus roseus suspension cells. The biosynthetic capability of the cultured cells to produce akuammicine, catharanthine and vindoline was confirmed by feeding experiments with dl-tryptophan-[3-¹⁴C] to yield the radioactive alkaloids.     

Effects of Sugars on the Glucosylation of Exogenous Hydroquinone by Catharanthus roseus Cells in Suspension Culture

The effects of sugars on the glucosylation of exogenous hydroquinone(HQ) was investigated by supplying individual sugars simultaneouslywith HQ to a suspension culture of Catharanthus roseus cells.The production of arbutin was enhanced as much as 2- to 3-foldby sucrose or glucose at concentrations of up to 6%, with theenhancement being directly dependent on the concentration ofthe sugar. The exogenously added sugar was not metabolized andremained unchanged. Sorbitol also promoted the production ofarbutin in a similar manner. Sucrose improved the viability of cells and, in cultures suppliedwith sucrose and HQ, the activity of UDP-glucose: hydroquinoneglucosyltransferase increased over a much longer period of timethan that in control cultures supplemented with HQ only. (Received December 11, 1989; Accepted March 26, 1990)