Indole alkaloid production by hairy root cultures of Catharanthus roseus

Hairy root cultures of Catharanthus roseus were established by infection with six different Agrobacterium rhizogenes strains. Two plant varieties were used and found to exhibit significantly different responses to infection. Forty-seven hairy root clones derived from normal plants and two derived from the flowerless variety were screened for their growth and indole alkaloid production. The growth rate and morphological appearance showed wide variations between the clones. The alkaloid spectra observed were qualitatively but not quantitatively very similar to that of the corresponding normal plant roots. No vindoline or deacetyltransferase activity could be detected in any of the cultures studied. O-acetylval-lesamine, an alkaloid which has not been previously observed in C. roseus was identified from extracts of hairy root clone No. 8. Two root clones were examined for their growth and alkaloid accumulation during a 26-day culture period. Alkaloid accumulation parallelled growth in both clones with ca. 2 mg ajmalicine and catharanthine per g dry weight being observed.Dedicated to Dr. Friedrich Constabel on the occasion of his 60th birthday     

Catharanthine and ajmalicine synthesis in Catharanthus roseus hairy root cultures

Two year old, transformed root cultures of Catharanthus roseus accumulate ajmalicine and catharanthine (0.57 and 0.36 mg g^-1 DW, or 7.0 and 3.0 mg l^-1, respectively). Changes in the concentration of the medium components, as well as the addition of hydrolytic enzymes and biotic elicitors, were used as strategies to increase these alkaloid yields. Regarding the components of the medium, the results obtained, when sucrose was raised from 3 to 4.5%, are noteworthy. The nitrogen source induced differential responses in the individual alkaloid yields. No net change in the alkaloid content was observed either with changes in the concentration of vitamins or macro-and micronutrients. Though the root culture only shows a limited response to elicitors, Aspergillus treatment and the use of macerozyme increased the accumulation of ajmalicine selectively, while the addition of methyl jasmonate increased the yield of both alkaloids.Abbreviations MeJa methyl jasmonate - mU milliunits     

Studies on the optimization of growth and indole alkaloid production by hairy root cultures of Catharanthus roseus

Catharanthus roseus hairy root cultures, genetically transformed with Agrobacterium rhizogenes, produce a wide variety of indole alkaloids. The effect of sucrose, phosphate, nitrate, and ammonia concentrations on growth and indole alkaloid production of C. roseus hairy root cultures were studied by using statistical experimental designs and linear regression analysis. Contradictory effects of these nutrients on growth and indole alkaloid production were found. The maximal growth was obtained by having 77. 8 mg NaH(2)PO(4) . H(2)O/L and 1. 311 g KNO(3)/L in the medium, whereas the specific production of alkaloids was highest at the lowest levels of all the nutrients studied. The maximal dry weight was obtained with high values of sucrose and ammonia, but clear optimum concentrations could not be found. When having enough nutrients to support reasonable growth, it appeared difficult to affect the specific alkaloid production rates considerably. The growth (dry wt.) with the optimized nutrient concentrations in the medium was more than 50% better than in the control medium with about the same alkaloid production.     

Determination of metabolic rate-limitations by precursor feeding in Catharanthus roseus hairy root cultures

Precursors from the terpenoid and tryptophan branches were fed to Catharanthus roseus to determine which of the two branches limits metabolic flux to indole alkaloids. The feeding of tryptophan at 17 days of the culture cycle produced auxin-like effects. Addition of low levels of auxin or tryptophan resulted in significant increases in flux to the indole alkaloids. Conversely, feeding higher levels of auxin or tryptophan resulted in increased branching and thickening of the hairy root cultures. A dramatic reduction in flux to the alkaloids was also observed. However, feeding tryptamine or terpenoid precursors had no effect. Therefore, neither pathway tested revealed to be rate-limiting during the late growth phase. Feeding of either geraniol, 10-hydroxygeraniol, or loganin at 21 days each resulted in significant increases in the accumulation of tabersonine. The addition of tryptophan or tryptamine had no effect during the stationary phase of the growth cycle. Thus, during the early stationary phase of growth the terpenoid pathway appears to be rate-limiting. Combined elicitation with jasmonic acid and feeding either loganin or tryptamine did not further enhance the accumulation of indole alkaloids.     

Bioreactor studies on hairy root cultures of Catharanthus roseus: Comparison of three bioreactor types

Summary Hairy roots of Catharanthus roseus were cultivated in three different types of bioreactors. The best growth and indole alkaloid production was achieved in an airsparged bioreactor with no other mixing. In the stirred bioreactor or in the bioreactor with medium circulation the roots did not grow, suggesting that hairy roots of C. roseus are more sensitive to stress than root cultures of many other plant species.     

Long-term maintenance of a transgenic Catharanthus roseus hairy root line

Stably transformed transgenic hairy root cultures have the potential to be a valuable production platform for a variety of secondary metabolites. This study reports that a transgenic hairy root culture of Catharanthus roseus has been stably maintained for over 4.5 years. This culture carries a transgene that expresses the green fluorescent protein under the control of the glucocorticoid-inducible promoter. Genomic PCR confirmed the presence of the GFP insert within the hairy roots, and induction with dexamethasone caused a significant (p < 0.02) increase in GFP levels.     

Jasmonate-induced epoxidation of tabersonine by a cytochrome P-450 in hairy root cultures of Catharanthus roseus

Methyl jasmonate, a chemical inducer of secondary metabolism, was shown to promote tabersonine 2 biosynthesis in hairy root cultures of Catharanthus roseus. Tabersonine 6,7-epoxidase activity was detected in total protein extract of jasmonate-induced hairy root cultures using labeled 14C-tabersonine 2. This enzyme converted tabersonine 2 to lochnericine 3 by selective epoxidation at positions 6 and 7 via a reaction dependent on NADPH and molecular oxygen. Carbon monoxide, clotrimazole, miconazole, and cytochrome C were shown to be strong inhibitors of the enzyme. The activity was found in microsomes, indicating that tabersonine 6,7-epoxidase was a cytochrome P-450-dependent monooxygenase.     

The effect of temperature on hairy root cultures of Catharanthus roseus: Growth,indole alkaloid accumulation and membrane lipid composition

Cultivation of Catharanthus roseus hairy root cultures at different temperatures was found to have an effect on growth rate and indole alkaloid content as well as lipid composition. When lowering the temperature, the roots responded by increasing the degree of unsaturation of cellular lipids, which was mainly due to an increased proportion of linolenic acid in the main lipid classes. The modifications in lipid composition were obviously necessary for the roots to retain the proper cell membrane fluidity at each temperature. Despite of changes in membrane lipids, no effect on the distribution of indole alkaloids between the roots and the medium could be detected. Instead, the level of alkaloid accumulation showed a clear increase with lowering temperature.Abbreviations PC phosphatidylcholine - PE phosphatidylethanolamine - PI phosphatidylinositol - PS phosphatidylserine - PG phosphatidylglycerol - CL cardiolipin - DGD digalactosyldiglyceride - MGD monogalactosyldiglyceride - NL neutral lipids - DU degree of fatty acid unsaturation     

Alkaloid production by transformed root cultures of Catharanthus roseus

Transformed roots of Catharanthus roseus were obtained following infection of detached leaves with Agrobacterium rhizogenes. Roots would not grow in full strength Gamborg's B5 medium but would grow satisfactorily if the medium was diluted to one half strength. Little alkaloid appeared in the growth medium but root tissue contained a high level and wide variety of alkaloids. Ajmalicine, serpentine, vindolinine and catharanthine were prominent components. Vinblastine could also be detected by a combination of HPLC and radioimmunoassay, though at a level of only 0.05g/g dry weight.Abbreviations B5 Gamborg's B5 nutrient salts - LC/MS combined liquid chromatography/mass spectrometry - FW fresh weight - Kb kilobase     

Production of indole alkaloids by selected hairy root lines of Catharanthus roseus

Hairy root cultures of Catharanthus roseus were established by infection of seedlings with Agrobacterium rhizogenes 15834. Approximately 150 transformants from four different. C. roseus cultivars were screened for desirable traits in growth and indole alkaloid production. Five hairy root clones grew well in liquid culture with doubling times similar to those reported for cell suspensions. Fast growing clones had similar morphologies, characterized by thin, straight, and regular branches with thin tips. The levels of key alkaloids, ajmalicine, serpentine, and catharanthine, in these five clones, also compared well with literature data from cell suspensions, yet HPLC and GC-MS data indicate the presence of vindoline in two clones at levels over three orders of magnitude greater than the minute amounts reported in cell culture. These results suggest that further optimization may result in hairy roots as a potential source of vindoline and catharanthine, the two monomers necessary to synthesize that antineoplastic drug, vinblastine. (c) 1993 John Wiley & Sons, Inc.     

Transient studies of light-adapted cultures of hairy roots of Catharanthus roseus: growth and indole alkaloid accumulation

Cultures of C. roseus transgenic ("hairy") root clones LBE-6-1 and LBE-4-2 were adapted with periodic daily illumination to investigate the effect of light on growth and nutrient utilization, and the accumulation of the indole alkaloids. Light-adapted roots appeared green and had radially thickened morphology compared with dark-grown controls. Their growth rates were higher than dark-grown controls, with 45% lower doubling times: LBE-6-1, 3.6 days; LBE-4-2, 2.8 days. Relative to dark-grown controls, light-adapted growth increased the biomass (DW) of LBE-6-1 by 25%, but had no effect on the DW of LBE-4-2. The macronutrients NH4+, NO3-, Pi, and sugars, were depleted completely by light-adapted root cultures in that order. The specific and total levels of the indole alkaloid serpentine was enhanced and of tabersonine was lowered in both root clones, while the overall trends of growth and non-growth association of tabersonine and serpentine, respectively, remained unaltered by light adaptation. Ajmalicine accumulation was enhanced in LBE-6-1, but lowered in LBE-4-2; its accumulation was growth-associated in dark-grown LBE-6-1, but appeared non-growth associated in light-adapted cultures. The accumulation of tabersonine-related compounds, lochnericine, and h?rhammericine exhibited growth-associated trends, and were either negatively affected or unaffected by light adaptation of LBE-6-1. Neither vindoline nor its precursor, deacetylvindoline, was detected.     

Nutrient consumption and alkaloid accumulation in a hairy root line of Catharanthus roseus

A hairy root line from Catharanthus roseus was cultured in a 14 l bioreactor. Nitrate and phosphate uptakes were similar to the same root line cultured in a 250 ml Erlenmeyer flask. However, sucrose consumption rate was slower in roots cultured in the bioreactor. These results show that it is feasible to upgrade this hairy root line to bioreactor level and, although carbon utilization has to be improved, ajmalicine and catharanthine were still produced and were retained within the biomass tissue.     

Development of a kinetic metabolic model: application to Catharanthus roseus hairy root

A kinetic metabolic model describing Catharanthus roseus hairy root growth and nutrition was developed. The metabolic network includes glycolysis, pentose-phosphate pathway, TCA cycle and the catabolic reactions leading to cell building blocks such as amino acids, organic acids, organic phosphates, lipids and structural hexoses. The central primary metabolic network was taken at pseudo-steady state and metabolic flux analysis technique allowed reducing from 31 metabolic fluxes to 20 independent pathways. Hairy root specific growth rate was described as a function of intracellular concentration in cell building blocks. Intracellular transport and accumulation kinetics for major nutrients were included. The model uses intracellular nutrients as well as energy shuttles to describe metabolic regulation. Model calibration was performed using experimental data obtained from batch and medium exchange liquid cultures of C. roseus hairy root using a minimal medium in Petri dish. The model is efficient in estimating the growth rate.     

Immunocytolocalization of tryptophan decarboxylase in Catharanthus roseus hairy roots

We investigated the intracellular distribution of tryptophan decarboxylase (TDC) (EC 4.1.1.28) in Catharanthus roseus hairy roots using immunofluorescence and immunogold techniques. TDC was detected by immunofluorescence localization in the cytosol and in the apoplastic region of the meristematic cells of the roots, with a slight enrichment in the epidermal cells of the root cap and in the meristematic region. In the enlargement zone, TDC was localized only in the first three layers of the cortex. In the maturation zone, the enzyme was not present. Immunogold studies confirmed that the enzyme was localized in the cytosol of the meristematic region, and intense gold labeling was found in the apoplastic zone. A protein fraction isolated from the apoplastic zone and assayed for TDC activity showed high activity.     

Effects of buffered media upon growth and alkaloid production of Catharanthus roseus hairy roots

The influence of buffered media upon the growth and alkaloid productivity of Catharanthus roseus hairy root culture was examined. As expected, the buffers minimized shifts in the pH of the media and had slightly negative effects upon growth. The growth of the hairy roots remained optimal in unbuffered media. The specific yield of lochnericine was significantly lower in response to the addition of buffers, while tabersonine was significantly higher. In contrast, the specific yields of ajmalicine, serpentine, and horhammericine remained unchanged.     

Development of two stage culture process by optimization of inorganic salts for improving catharanthine production in hairy root cultures of Catharanthus roseus

The effects of the concentrations of inorganic salts in Schenk and Hildebrandt (SH) medium on catharanthine production in hairy root cultures of Catharanthus roseus were investigated. The inorganic salt components could be categorized into four groups. The first group (nitrate) supported both the growth of and catharanthine production by hairy roots with incremental increases in the concentration. The second (ammonium and phosphate) yielded contradictory effects with respect to growth and production. The third (borate and molibdate) inhibited both growth and production, while the fourth (potassium iodide, sulfate, and iron) did not exhibit any significant effects. Through optimization of the concentrations of inorganic salts in the medium, a two stage process of hairy root cultures with different media for growth and production was developed which enabled us to enhance the volumetric yield of catharanthine up to 60.5 mg/l. This productivity was 5.4 times higher than that of a one stage culture in the original SH medium.