Jacqueline V. Shanks

Effect of Elicitor Dosage and Exposure Time on Biosynthesis of Indole Alkaloids by Catharanthus roseus Hairy Root Cultures

Late exponential phase hairy root cultures of Catharanthus roseus were elicited with pectinase and jasmonic acid. The effects of elicitor concentration and exposure time on growth and levels of several compounds in the indole alkaloid biosynthetic pathway were monitored. Pectinase decreased the fresh weight to dry weight ratio of the roots, while addition of jasmonic acid had no significant effect. Selective effects on indole alkaloid yields were observed upon addition of elicitors. An increase of 150% in tabersonine specific yield was observed upon addition of 72 units of pectinase. Transient studies at the same level demonstrated possible catabolism as serpentine, tabersonine, and lochnericine levels decreased immediately after elicitation. The levels of these compounds recovered back to control levels or were higher than the control levels after some time. Jasmonic acid was found to be a unique elicitor leading to an enhancement in flux to several branches in the indole alkaloid pathway. Jasmonic acid addition caused an increase in the specific yields of ajmalicine (80%), serpentine (60%), lochnericine (150%), and hörhammericine (500%) in dosage studies. Tabersonine, the likely precursor of lochnericine and hörhammericine, decreased at lower levels of jasmonic acid and then increased with increasing jasmonic acid concentration. Transient studies showed that lochnericine and tabersonine levels go through a maxima, then decrease back to control levels and reduce below control levels, respectively. The yields of ajmalicine, serpentine, and hörhammericine increased continuously after the addition of jasmonic acid. The methods described in this article could generally be used in devising strategies for enhancement in productivity of secondary metabolites and for probing and studying the complex secondary metabolite pathways in plant tissue cultures.

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.

Quantification of metabolites in the indole alkaloid pathways of Catharanthus roseus: Implications for metabolic engineering

In this article, we present a review of the current state of metabolic engineering in Catharanthus roseus. A significant amount of research has contributed to characterization of several individual steps in the biosynthetic pathway of medicinally valuable alkaloids. However, knowledge of the regulation of these pathways is still sparse. Using hairy root cultures, we studied the responses of alkaloid metabolism to environmental stimulation such as light and elicitation. Through precursor feeding studies, the putative rate-limiting steps of the terpenoid pathway in hairy root cultures also have been examined. Relating this knowledge to specific events at the molecular level, and the cloning of corresponding genes are the next key steps in metabolic engineering of the C. roseus alkaloids. Copyright 1998 John Wiley & Sons, Inc.

Phytotransformation of trinitrotoluene (TNT) and distribution of metabolic products in Myriophyllum aquaticum

Dissolved [U-ring-C]2,4,6-trinitrotoluene, when exposed to Myriophyllum aquaticum plants, was transformed with approximately 30% of products recovered in the aqueous medium, regardless of incubation period. The remaining fraction was retained inside of the plant and was distributed throughout various anatomical structures. After five days of incubation the plant associated fraction became increasingly resistant to extraction with methanol. The plant associated accumulation of C was highest in roots.

Statistical design of the effect of inoculum conditions on growth of hairy root cultures of Catharanthus roseus

The factors in the inoculum conditions that influence the growth rate of hairy roots of Catharanthus roseus in liquid culture were investigated in a randomized two-level, three-factor statistical design. The three variables were the number of root tips, the length of root tips, and the initial volume of media. The experiments and analysis demonstrated the effect of inoculum conditions on hairy root growth, with the length of hairy root tips being the dominant variable without any clonal variability. The best growth rates (doubling times) were obtained with an inoculum of 5 hairy root tips, each 35–40 mm long in 50 mL media: 3.7 and 3.1 days for C. roseus hairy root clones LBE-6-1 and LBE-4-2, respectively.

Influence of Aeration on Cytoplasmic pH of Yeast in an NMR Airlift Bioreactor

Nuclear magnetic resonance spectroscopy has been increasingly pursued as a tool for noninvasive, real‐time studies of metabolic processes of cell suspensions in bioreactors. One acute challenge in NMR bioreactor design has been supplying enough oxygen for cell respiration in a suspension that contains sufficient cells for NMR signal detection. The use of cytoplasmic pH as an intracellular marker of adequate oxygenation was evaluated from 31P NMR spectra of the yeast Saccharomyces cerevisiae at several cell densities, ranging from low (0.9% (v/v)) to very high (45% (v/v)) cell densities, in an airlift bioreactor. 31P NMR spectra were obtained for derepressed yeast cells prior to, and during, glycolysis under nongrowth conditions. During endogenous respiration, pHcyt can be used as an intracellular marker for aeration for cell densities up to 18% (v/v) based on two criteria: a value of pHcyt at least 0.2 pH units higher under aerobic than anaerobic conditions and an absolute pHcyt value of 7.1−7.2. These results were more conservative than values of the maximum cell density obtained from calculations using kLa and respiration rate estimates and highlight the utility of intracellular measurements in conjunction with engineering design calculations. During glycolysis, pHcyt values were similar under aerobic and anaerobic conditions and hence pHcyt cannot be used as a marker under these conditions. Carbon dioxide in the influent gas was observed to aid cells in maintaining physiological pHcyt at high cell densities.

A Transient Study of Formation of Conjugates during TNT Metabolism by Plant Tissues

ABSTRACT The formation of TNT-derived conjugates was investigated in hairy root tissue cultures of Catharanthus roseus and in aquatic plant systems of Myriophyllum aquaticum. The temporal profiles of four TNT-derived conjugates, TNT-1, 2A-1, TNT-2 and 4A-1, were determined over 3 to 16-day exposure durations. When axenic C. roseus roots were exposed separately to 2,4,6 trinitrotoluene, 2-amino-4,6-dinitrotoluene and 4-amino-2,6-dinitrotoluene, the array and levels of conjugates varied. Exposure of axenic roots to either 4-amino-2,6-dinitrotoluene or 2-amino-4,6-dinitrotoluene resulted in the formation of only 4A-1 and 2A-1, respectively, and not TNT-1 and TNT-2. However, amendment of previously unexposed roots with TNT produced all four conjugates. The conjugates were preferentially accumulated within the biomass phase of root cultures. Significantly, conjugates TNT-1 and TNT-2 were observed in the biomass phase of intact M. aquaticum plants exposed to TNT. The results clearly indicate the presence of com...

Effect of Subculture Cycle on Growth and Indole Alkaloid Production by Catharanthus roseus Hairy Root Cultures

The effect of subculture cycles on Catharanthus roseus hairy root culture growth and indole alkaloid production was investigated by adapting the cultures to 2-, 3-, and 4-week cycles. Conductivity of the culture medium was measured every 2–3 days and the culture fresh weight determined using a conductivity-fresh weight correlation. Cultures were harvested in triplicate at 21, 28, and 35 days and analyzed for final biomass and indole alkaloid content. These experiments demonstrated that the subculture cycle could lead up to a 20% decrease in the doubling times of hairy root cultures when the subculture cycle is changed from 4- to 2-weeks. The 2-week subculture cycle yielded the fastest while the 4-week cycle yielded the slowest specific growth rates. Specific yields of tabersonine decreased from day 21 to 35 and was inversely correlated with biomass content. Total yields of lochnericine were highest in the 2-week cycle indicating a strong correlation to growth rate. Serpentine yields were lowest in the 2-week cycle and highest in the 4-week cycle, indicating inverse proportionality of serpentine accumulation to growth rate.

Effects of initial medium pH on growth and metabolism ofCatharanthus roseus hairy root cultures —A study with31P and13C NMR spectroscopy

SummaryHairy root cultures ofCatharanthus roseus were grown for 26 days in half-strength Gamborgs B5 liquid medium at different initial pH values of 4.2, 5.7, 6.5, and 7.3. Maximum growth was obtained for cultures with an initial medium pH 6.5. The lowest growth rate was found in cultures at initial pH values of 4.2 and 7.3. Roots in cultures at initial pH of 4.2 had a thickened and stunted morphology in contrast to the other cultures. Also, cultures at initial medium pH of 4.2 exhibited an increase in medium pH in the first few days instead of the characteristic acidification. All cultures maintained a cytoplasmic pH of 7.4 throughout the growth cycle. However, vacuolar pH was 5.1–5.2 in cultures of initial pH 4.2, as opposed to 5.4–5.5 for other cultures. Sucrose was hydrolyzed completely to glucose and fructose by day 26 except for cultures at initial pH of 7.3. Glucose was the preferred substrate throughout the growth cycle for cultures with initial pH values of 7.3 and 6.5, after day 20 for an initial pH of 5.7, and after day 26 for pH 4.2.

Quantification of Metabolic Fluxes for Metabolic Engineering of Plant Products

The application of metabolic engineering to plant secondary metabolite products is hindered by a lack of quantitative information on fluxes in the metabolic pathways. Analysis of fluxes in metabolic pathways in response to an environmental or genetic manipulation can help identify rate-limiting steps. Since total productivity is the goal for enhanced production of a valuable secondary metabolite, the product of the biomass productivity times the metabolite specific yield should be optimized. This implies a systems analysis of both primary and secondary metabolic pathways. This chapter will provide an overview of a strategy using HPLC photodiode array analysis, TLC, and MS to obtain temporal concentrations of secondary metabolites, and in situ measurements using31P and13C NMR spectroscopy to monitor primary metabolism and physiology. The model system is hairy root cultures of Catharanthus roseus and indole alkaloid biosynthetic pathways.