Tryptophan over-producing cell suspensions of Catharanthus roseus (L) G. Don and their up-scaling in stirred tank bioreactor: Detection of a phenolic compound with antioxidant potential
Department of Plant Biotechnology, Central Institute of Medicinal and Aromatic Plants (CIMAP), Council of Scientific and Industrial Research, PO CIMAP, Kukrail Picnic Spot Road, Lucknow, 226015, India.Protoplasma (Impact Factor: 2.65). 01/2013; 250:371-380. DOI: 10.1007/s00709-012-0423-5
Five cell suspension lines of Catharanthus roseus resistant to 5-methyl tryptophan (5-MT; an analogue of tryptophan) were selected and characterized for growth, free tryptophan content and terpenoid indole alkaloid accumulation. These lines showed differential tolerance to analogue-induced growth inhibition by 30 to 70 mg/l 5-MT supplementation (LD(50) = 7-15 mg/l). Lines P40, D40, N30, D50 and P70 recorded growth indices (i.e. percent increment over the initial inoculum weight) of 840.9, 765.0, 643.9, 585.7 and 356.5 in the absence and, 656.7, 573.9, 705.8, 489.0 and 236.0 in the presence of 5-MT after 40 days of culture, respectively. A corresponding increment in the free tryptophan level ranging from 46.7 to 160.0 μg/g dry weight in the absence and 168.0 to 468.0 μg/g dry weight in the presence was noted in the variant lines. Higher tryptophan accumulation of 368.0 and 468.0 g/g dry weight in lines N30 and P40 in 5-MT presence also resulted in higher alkaloid accumulation (0.65 to 0.90 % dry weight) in them. High-performance liquid chromatography (HPLC) analysis of the crude alkaloid extracts of the selected lines did not show the presence of any pharmaceutically important monomeric or dimeric alkaloids except catharanthine in traces in the N30 line that was also unique in terms of a chlorophyllous green phenotype. The N30 line under optimized up-scaling conditions in a 7-l stirred tank bioreactor using Murashige and Skoog medium containing 2 mg/l α-naphthalene acetic acid and 0.2 mg/l kinetin attained 18-folds biomass accumulation within 8 weeks. Interestingly, the cell biomass yield was enhanced to 30-folds if 30 mg/l 5-MT was added in the bioreactor vessel one week prior to harvest. Crude alkaloid extract of the cells grown in shake flask and this bioreactor batch also showed the formation of yellow-coloured crystals which upon (1)HNMR and ESI-MS analysis indicated a phenolic identity. This crude alkaloid extract of bioreactor-harvested cells containing this compound at 50 μg/ml concentration registered 65.21, 17.75, 97.0, 100 % more total antioxidant capacity, reducing power, total phenolic content, and ferric-reducing antioxidant power, respectively, when compared with that of extracts of cells grown in shake flask cultures. The latter, however, showed 57.47 % better radical scavenging activity (DPPH) than the bioreactor-harvested cells.
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- "Exogenous feeding of tryptophan is also not tolerated by the cells as it down-regulates several other essential metabolic pathways by stimulating the activity of another key enzyme chorismate mutase. The 5-methyltryptophan (5MT) tolerance, on the other hand, allows higher tryptophan accumulation due to a relaxed feedback mechanism for anthranilate synthase and hence such tolerance can lead to accumulation of more tryptophan and plants survive and continue to grow (Verma et al. 2013). Plants tolerate the growth inhibitory effect of 5MT (an analogue of tryptophan) by over producing free tryptophan in the amino acid pool. "
ABSTRACT: Sixty six plants raised via direct shoot bud organogenesis from pre-plasmolysed leaf explants of Catharanthus roseus were assessed under in vivo conditions for their physio-morpho traits, tryptophan metabolism, genetic fidelity and alkaloid profile. Morphologically all plants were parent like except the three morpho-types i.e. broad cup-shaped leaves, dwarf phenotype and enlarged pink corona. Vindoline was detected in most of the plants in substantial amount while ajmalicine, ajmaline and catharanthine were detected in few plants only. More than eightfold increased vindoline (0.08 % dry wt.) was showed by plant no. 1, 4 and 39. Except plant no. 54, which accumulated the highest alkaloid content of 3.09 % dry wt. with tryptophan content of 0.0283 % dry wt., majority of the plants showed a negative correlation between total alkaloid content and tryptophan accumulation and strong positive correlation between tryptophan content and 5-methyltryptophan tolerance. ISSR and RAPD profile of seventeen randomly selected, field established plants was generated by using 10 ISSR and 60 RAPD primers. In RAPD, a total of 753 bands were detected, out of which 624 (82.87 %) were monomorphic. In ISSR profiling, a total of 205 bands were detected out of which 200 bands were monomorphic (97.56 %) and only 5 bands were found to be polymorphic. Highly significant data for the monomorphic banding pattern across all the three genotypes (p < 0.01) was observed. Principal components, AMOVA analysis and multivariate analysis using Nei and Li’s coefficient further validate the monomorphism. Thirteen plants having superior traits were grown in Random Block Design in field. The relevance of physiological and epigenetic changes occurred in the light of morpho-types and alkaloid profile of directly regenerated plants during in vitro to in vivo acclimatization in C. roseus is discussed.
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ABSTRACT: Ten 5-methyltryprophan (5-MT)-resistant multiple shoot culture lines in three genotypes of Catharanthus roseus were selected in vitro. The variant shoot lines displayed a differential threshold tolerance limit against the analogue stress, ranged from 20 to 70 mg/l 5-MT in the medium. The lines tolerant to 40 mg/l 5-MT stress were most stable and fast proliferating. All the selected lines in the presence of 5-MT stress recorded increased level of tryptophan in their free amino acid pool. Highest tryptophan accumulation occurred in lines P40, P30, D40, and N40 (i.e., 296.5, 241.0, 200.6, and 202.0 μg/g dry wt., respectively). A concomitant increase in the total alkaloid content (2.3-3.8 % dry wt.) under the analogue stress was also noticed in these lines when compared to 1.0-1.58 % dry wt. in the respective wild-type shoot maintained on a stress-free medium. The HPLC analysis of the alkaloid extracts of the 5-MT-tolerant lines grown under analogue stress also revealed vindoline as a major constituent with maximum accumulation in lines N40, N30, D30, D40, and P40 (0.046, 0.032, 0.034, and 0.022 % dry wt., respectively). The rooted shoots of 5-MT-tolerant lines were successfully acclimatized under glasshouse environment wherein they grew normally and set seeds. Flowering twigs or leaves excised from 1-year-old glasshouse-grown plants of 5-MT variant lines upon postharvest in vivo elicitation with 30 mg/l 5-MT or 5.0 mg/l tryptophan registered an eight-to-tenfold increment in their vindoline content within 24-48 h.
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ABSTRACT: Vinca minor is the sole source of vincamine, an alkaloid known to be used in a variety of cerebral disorders. Three stable variant shoot lines (V10, V20 and V30) with tolerance thresholds of 10, 20 and 30 mg/l 5-methyltryptophan (5-MT; analogue of tryptophan), respectively, were selected. These lines showed twofold to threefold increase in tryptophan content and 1.5- to 2-fold increment in the total alkaloids in comparison to the wild line shoots. A maximum of 16-fold enhancement in vincamine production was recorded in V30 line followed by eightfold in V20 line. Inter simple sequence repeat (ISSR)-PCR amplification of all the three lines showed total of 65 bands; out of which 60 were monomorphic (92.3 %) and 5 were polymorphic (7.7 %). Tryptophan being a limiting factor in the indole alkaloid pathway plays a crucial role in modulating the flux towards vincamine production and its over-production positively resulted into enhanced vincamine production.
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