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: 3.17). 01/2013; 250:371-380. DOI: 10.1007/s00709-012-0423-5
Source: PubMed

ABSTRACT 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.

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The availability and quality of wild berry material is strongly affected by seasonal variations, human resources and both chemical and biological pollutants. Here we describe for the first time the industrial scale biotechnological production of cloudberry (Rubus chamaemorus) cells of consistent quality and defined chemical composition. Callus was initiated from sterile cuts of the wild cloudberry leaves. Bright yellow, soft callus of uniform quality was selected by regular sub-culturing, and a stable line successfully used for suspension culture was obtained within 18 months. The cultivation process of the cell culture was initiated in 250 ml shake flasks and stepwise expanded to a stirred tank bioreactor of 300 l working volume. Each growth period in flasks and fed-batch cultures in bioreactors was 10 ± 3 days. Viability of the cultures was approximately 90 % throughout the cultivation processes. The 300 l culture was harvested by pressure filtration and the biomass was freeze-dried. The 300 l yields of fresh cells and freeze-dried material were 19 kg and 2 kg, respectively. The unusual phenolic profile of cloudberry cells including flavanols, as well as their fatty acid composition with a high proportion of α-linolenic acid and high protein content, make them a unique and interesting alternative for industrial applications for various industrial fields.This article is protected by copyright. All rights reserved
    Engineering in Life Sciences 07/2014; · 1.89 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Research on transcriptional regulation of terpenoid indole alkaloid (TIA) biosynthesis of the medicinal plant, Catharanthus roseus, has largely been focused on gene function and not clustering analysis of multiple genes at the transcript level. Here, more than ten key genes encoding key enzyme of alkaloid synthesis in TIA biosynthetic pathways were chosen to investigate the integrative responses to exogenous elicitor ethylene and copper (Cu) at both transcriptional and metabolic levels. The ethylene-induced gene transcripts in leaves and roots, respectively, were subjected to principal component analysis (PCA) and the results showed the overall expression of TIA pathway genes indicated as the Q value followed a standard normal distribution after ethylene treatments. Peak gene expression was at 15-30 μM of ethephon, and the pre-mature leaf had a higher Q value than the immature or mature leaf and root. Treatment with elicitor Cu found that Cu up-regulated overall TIA gene expression more in roots than in leaves. The combined effects of Cu and ethephon on TIA gene expression were stronger than their separate effects. It has been documented that TIA gene expression is tightly regulated by the transcriptional factor (TF) ethylene responsive factor (ERF) and mitogen-activated protein kinase (MAPK) cascade. The loading plot combination with correlation analysis for the genes of C. roseus showed that expression of the MPK gene correlated with strictosidine synthase (STR) and strictosidine b-D-glucosidase(SGD). In addition, ERF expression correlated with expression of secologanin synthase (SLS) and tryptophan decarboxylase (TDC), specifically in roots, whereas MPK and myelocytomatosis oncogene (MYC) correlated with STR and SGD genes. In conclusion, the ERF regulates the upstream pathway genes in response to heavy metal Cu mainly in C. roseus roots, while the MPK mainly participates in regulating the STR gene in response to ethylene in pre-mature leaf. Interestingly, the change in TIA accumulation does not correlate with expression of the associated genes. Our previous research found significant accumulation of vinblastine in response to high concentration of ethylene and Cu suggesting the involvement of posttranscriptional and posttranslational mechanisms in a spatial and temporal manner. In this study, meta-analysis reveals ERF and MPK form a positive feedback loop connecting two pathways actively involved in response of TIA pathway genes to ethylene and copper in C. roseus.
    Protoplasma 10/2014; · 3.17 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: A vincamine positive hairy root clone of Vinca minor with integration of all the three- rolA, rolB and rolC genes and its subsequently raised cell suspensions were treated with culture filtrate of four endophytic fungi namely Chaetomium globosum; Aspergillus niveoglaucus; Paecilomyces lilacinus and Trichoderma harzianum. Addition of 10 % v/v T. harzianum culture filtrate boosted maximum biomass accumulation and highest total alkaloid content (TAC) both in the hairy roots and in the cell suspensions. Treated (10 % v/v T. harzianum) hairy roots registered growth index (GI) of 640 ± 28.3 and TAC of 3.6 ± 0.1 dry wt% in comparison to non-treated roots (GI = 420 ± 14.2; TAC = 3.1 ± 0.3 dry wt%). Treated cell suspensions showed GI of 600 ± 19.1 and TAC of 1.67 ± 0.03 dry wt% in comparison to non-treated cell suspensions (GI = 250 ± 12.6; TAC = 1.15 ± 0.02 dry wt%). The hairy roots and the cell suspensions were successfully up-scaled in the 5 l stirred tank bioreactor with respective GI of 850.0 and 654.0 under optimized conditions. On Real time (qPCR) analysis, treated hairy roots showed fourfold to sixfold enhanced tryptophan decarboxylase (TDC) transcript level [relative quantity value (RQ) = 4.64 ± 0.30 (shake flask); RQ = 5.95 ± 0.31 (bioreactor)] while treated cell suspensions showed only two fold increase in TDC transcript [RQ = 2.1 ± 0.26 (shake flask); RQ = 2.5 ± 0.21 (bioreactor)]. Similarly, fivefold to sixfold [RQ = 5.6 ± 0.20 (shake flask); RQ = 6.7 ± 0.49 (bioreactor)] and threefold to fourfold [RQ = 3.5 ± 0.18 (shake flask); RQ = 3.8 ± 0.68 (bioreactor)] increased transcript of strictosidine synthase (STR) in treated hairy roots and cell suspensions, respectively was observed. Treated shake flask roots showed 0.002 dry wt% vincamine that was further enhanced in the bioreactor grown treated roots (0.015 dry wt%). No vincamine was detected in the shake flask culture or in the bioreactor grown cell suspensions.
    Plant Cell Tissue and Organ Culture 04/2014; · 2.61 Impact Factor