Atropa Belladonna Hairy Roots Research Articles

A new synthetic biology approach for the production of curcumin and its glucoside in Atropa belladonna hairy roots

Curcumin has ignited global interest as an elite drugable molecule, owing to its time-honoured pharmacological activities against diverse human ailments. Limited natural accessibility and poor oral bioavailability caused major hurdles in the curcumin-based drug development process. We report the first successful testimony of curcumin and its glucoside synthesis in Atropa belladonna hairy roots (HR) through metabolic engineering. Re-routing the inherent biosynthetic precursors of the phenylpropanoid pathway of A. belladonna by heterologous expression of key curcumin biosynthetic pathway genes (i.e., Diketide-CoA synthase-DCS and Curcumin synthase-CURS3) and glucosyltransferase gene (CaUGT2) resulted in the production of curcumin and its glucoside in HR clones. Under shake-flask cultivation, the PGD2-HR1clone bearing DCS/ CURS3 genes showed the maximum curcumin yield (180.62 ± 4.7 μg/g DW), while the highest content of curcumin monoglucoside (32.63 ± 2.27 μg/g DW) along with curcumin (67.89 ± 2.56 μg/g DW) were noted in the PGD3-HR3 clone co-expressing DCS/CURS3 and CaUGT2 genes. Bioreactor up-scaling showed yield improvements in the PGD2-HR1 (2.3 fold curcumin) and the PGD3-HR3 clone (0.9 and 1.65 folds of curcumin-monoglucoside and curcumin respectively). These findings proved the advantageous use of HR cultures as the production source for curcumin and its glucoside, which remained unexplored so far.

Mechanism exploration on nitrogen metabolism and secondary metabolism in Atropa belladonna hairy roots treated with yeast extract

In order to study the mechanism of nitrogen metabolism and secondary metabolism in Atropa belladonna hairy roots treated with yeast extract, yeast extract（YE） was added to the culture medium. Then the changes of physiological and biochemical indexes of A. belladonna hairy roots after treatment with YE were detected. The results are as follows，the activity of key enzymes of nitrogen metabolism changed differently. Compared with the control group (CK), the activity of nitrate reductase (NR) and glutamine synthetase (GS) were significantly increased, while the activity of glutamate dehydrogenase (GDH) was not changed significantly. The content of nitrate nitrogen, ammonium nitrogen had a significant decrease,but the content of soluble protein, free amino acid, total nitrogen are significantly more than CK. Moreover, YE treatment led to the increase of the content precursor amino acids (ornithine and arginine) and precursor putrescine in secondary metabolic pathways of A. belladonna. The expression level of gene putrescine N-methyl transferase (pmt), tropinone reductase-I (trI) and hyoscyamine 6-β-hydroxylase(h6h) all increased in a different rate caused by YE treatment, which eventually led to the increase of the yield of tropane alkaloids. The yield of hyoscyamine and scopolamine were 3.09 and 1.85 folds than that of CK after 16 days treatment time. The results indicated that YE can induce more synthesis of tropane alkaloids by increasing the activity of key enzymes in nitrogen metabolism to provide more synthetic materials for secondary metabolism, meanwhile it regulated the expression level of some genes of key metabolic enzyme to accelerate secondary metabolism.

Removal of Phenol by A. belladonna L. Hairy Root

Phenolic compounds that present in the several industries are harmful and dangerous for human health. In this study we have studied the potential of Atropa belladonna hairy roots in phenol removal of wastewater. The optimal conditions for the removal process were evaluated using different phenol (10–500 mg.1−1) and H2O2 (1–15 Mm) concentrations. In the presence of H2O2, Roots were able to remove phenol concentrations up to 500 mg.1−1. in the wide range of pH (4–9), reaching high removal efficiency. When roots were re-used for five consecutive cycles, phenol removal efficiency decreased from 98–62%, in the last cycle. After the removal process, the solutions were obtained from the experiment were estimated for their toxicity using a test with Lactaca sativa L. seeds. Results showed that the treated solution was less toxic than the parent solution.

Atropa belladonna Hairy Roots: Orchestration of Concurrent Oxidation and Reduction Reactions for Biotransformation of Carbonyl Compounds

The biotransformation potential of a selected Atropa belladonna hairy root clone (AB-09) had been evaluated with regard to three different aromatic carbonyl compounds, i.e., 3,4,5-trimethoxybenzaldehyde (1), 3,4,5-trimethoxyacetophenone (2), and 3,4,5-trimethoxy benzoic acid (3). The results demonstrated for the first time the untapped potentials of the selected hairy root clone to perform simultaneous oxidation (34.49%) and reduction (32.68%) of 3,4,5-trimethoxy benzaldehyde (1) into 3,4,5-trimethoxy benzoic acid (3), and 3,4,5-trimethoxy benzyl alcohol (4), respectively, without any intermediate separation or addition of reagents. The same hairy root clone also demonstrated reduction (<5%) of a 3,4,5-trimethoxyacetophenone (2) into a secondary alcohol, i.e., 1-(3,4,5-trimethoxyphenyl) ethanol (5), while in the case of aromatic carboxylic acid substrate (3), no biotransformation could be obtained under the similar conditions. The current observations revealed oxidation and reduction of the formyl group of the aromatic ring, and only reduction of the carbonyl group of acetophenone through the specific hairy root clone. The concurrent oxidation and reduction reactions by the selected hairy root clone highlight the importance of this study, which, as per our observations, is the first of its kind relating the hairy root culture of A. belladonna.

Effects of Medium Components and Shear Conditions on the Formation and Growth of Adventitious Bud Derived from Hairy Roots of Atropa belladonna L.

Effects of Medium Components and Shear Conditions on the Formation and Growth of Adventitious Bud Derived from Hairy Roots of Atropa belladonna L.

Salicylic acid carboxyl methyltransferase induced in hairy root cultures of Atropa belladonna after treatment with exogeneously added salicylic acid.

In Atropa belladonna hairy roots, exogeneously added salicylic acid (SA) is converted to methyl salicylate (MSA) through the reaction, which might be catalysed by S-adenosyl-L-methionine: salicylic acid carboxyl methyltransferase (SAMT). Here we cloned a cDNA for A. belladonna SAMT (AbSAMT1), which consisted of 357 aa residues. It was expressed in E. coli, and the recombinant AbSAMT1 showed SAMT activity. When A. belladonna hairy roots were exposed to a high concentration of SA, AbSAMT1 mRNA begins to be expressed 12 h after the exposure, and steady expression continued over 144 h.

The effect of nitrate and ammonium concentrations on growth and alkaloid accumulation of Atropa belladonna hairy roots

The growth, the alkaloid production, as well as the scopolamine/hyoscyamine ratio of two clones of belladonna hairy roots were studied. The effects of nitrate and ammonium concentrations on these cultures were investigated. A rise in ammonium concentration caused the decline of the hairy roots, while nitrate had a marked effect on the alkaloid content. The alkaloid production obtained with 15.8 mM of NO 3 − and 20.5 mM of NH 4 + was 1.2–1.4 times higher than that obtained when the roots were grown in the standard Murashige and Skoog medium (MS medium, 39.5 mM of NO 3 − and 20.5 mM of NH 4 +). The nitrate and ammonium concentrations in the culture medium also had a strong influence on the scopolamine/hyoscyamine ratio. When nitrate or ammonium concentrations were raised, that ratio also was increased 2–3-fold. The hairy root clones originating from transformations with two distinct strains of Agrobacterium had similar responses.

Tropane alkaloid production by hairy roots of Atropa belladonna obtained after transformation with Agrobacteriumrhizogenes 15834 and Agrobacterium tumefaciens containing rolA, B, C genes only

Atropa belladonna leaf disks were infected by a wild strain Agrobacterium rhizogenes 15834 harboring the Ri-TL-DNA and by a disarmed Agrobacterium tumefaciens strain harboring a construction with only rolABC and npt II genes. Thirteen root lines were established and examined for their growth rate and alkaloid productivity to evaluate the possible role of rol genes in morphological differentiation and in tropane alkaloid formation. A great diversity has been observed in the growth rate of these 13 root lines. The root biomass increased up to 75 times. The total alkaloid contents were similar in the root lines obtained by infection with A. rhizogenes 15834 and A. tumefaciensrolABC. The last ones accumulated between 4 (1.1 mg g−1 DW) and 27 (8 mg g−1 DW) times more alkaloids than the intact roots (0.3 mg g−1 DW). This work has shown that the rolABC genes were sufficient to increase tropane alkaloid production in A. belladonna hairy root cultures.

Hairy root culture in a liquid-dispersed bioreactor: characterization of spatial heterogeneity.

A liquid-dispersed reactor equipped with a vertical mesh cylinder for inoculum support was developed for culture of Atropa belladonna hairy roots. The working volume of the culture vessel was 4.4 L with an aspect ratio of 1.7. Medium was dispersed as a spray onto the top of the root bed, and the roots grew radially outward from the central mesh cylinder to the vessel wall. Significant benefits in terms of liquid drainage and reduced interstitial liquid holdup were obtained using a vertical rather than horizontal support structure for the biomass and by operating the reactor with cocurrent air and liquid flow. With root growth, a pattern of spatial heterogeneity developed in the vessel. Higher local biomass densities, lower volumes of interstitial liquid, lower sugar concentrations, and higher root atropine contents were found in the upper sections of the root bed compared with the lower sections, suggesting a greater level of metabolic activity toward the top of the reactor. Although gas-liquid oxygen transfer to the spray droplets was very rapid, there was evidence of significant oxygen limitations in the reactor. Substantial volumes of non-free-draining interstitial liquid accumulated in the root bed. Roots near the bottom of the vessel trapped up to 3-4 times their own weight in liquid, thus eliminating the advantages of improved contact with the gas phase offered by liquid-dispersed culture systems. Local nutrient and product concentrations in the non-free-draining liquid were significantly different from those in the bulk medium, indicating poor liquid mixing within the root bed. Oxygen enrichment of the gas phase improved neither growth nor atropine production, highlighting the greater importance of liquid-solid compared with gas-liquid oxygen transfer resistance. The absence of mechanical or pneumatic agitation and the tendency of the root bed to accumulate liquid and impede drainage were identified as the major limitations to reactor performance. Improved reactor operating strategies and selection or development of root lines offering minimal resistance to liquid flow and low liquid retention characteristics are possible solutions to these problems.

Investigation of liquid-solid hydrodynamic boundary layers and oxygen requirements in hairy root cultures.

Rates of oxygen uptake, growth and alkaloid production by hairy roots in submerged culture were investigated using a recirculation reactor allowing operation at high liquid velocities for removal of hydrodynamic boundary layers. Measurements were performed at dissolved oxygen tensions of 31-450% air saturation. Critical oxygen concentrations for Atropa belladonna hairy roots were above air saturation, viz. 100-125% air saturation for oxygen uptake and 150% air saturation for growth, demonstrating that these roots cultivated in reactors with air sparging are oxygen-limited. The critical oxygen tension for oxygen uptake by Solanum aviculare hairy roots was 75% air saturation. Both the specific oxygen uptake rate and specific growth rate of A. belladonna hairy roots were dependent on the mass (g dry weight) of roots present; even in the absence of boundary layers, growth did not remain exponential over the entire culture period. Cryo-scanning electron microscopy showed that hairy roots grown submerged in liquid medium were covered with thick layers of hydrated mucilage and root hairs, representing a significant additional barrier to oxygen transfer. Roots protruding out of the liquid medium showed no evidence of mucilage accumulation. The specific oxygen demand of A. belladonna root tips was 3.3-11.5 times higher than for the remainder of the roots, the ratio increasing as the dissolved oxygen tension was reduced. Specific growth rates, biomass yields from sugar, and atropine levels were maximum at around 150% air saturation, but decreased significantly with oxygen concentrations above ca. 200%.

Isolation and identification of littorine from hairy roots of Atropa belladonna.

A hairy root clone (M8) of Atropa belladonna, producing high levels of tropane alkaloids, was established by transformation with Agrobacterium rhizogenes (MAFF 03-01724). Littorine, an intermediate of tropane alkaloids, was detected by high-performance liquid chromatography and gas chromatography-mass spectrometry in the alkaloid fraction of the hairy roots and identified by nuclear magnetic resonance analysis. Littorine was also detected in the non-transformed root culture of A. belladonna.

Application of membrane tubing aeration and perfluorocarbon To improve oxygen delivery to hairy root cultures

Growth and atropine production by Atropa belladonna hairy roots were studied in bioreactor cultures using porous polypropylene membrane tubing as a supplementary aeration device and with FC-43 perfluorocarbon emulsion added to the medium. Both these treatments were applied to improve oxygen transfer to the roots. Membrane tubing aeration allowed direct delivery of oxygen within the root clump, thus overcoming mass transfer resistances associated with poor intraclump penetration of liquid convective currents. Combined air sparging and membrane tubing aeration in a gas-driven bioreactor supported biomass levels 32-65% higher than sparging only of air or oxygen-enriched air at the same total gas flow rate. The optimal air flow rate ratio between the sparger and membrane tubing giving the maximum final biomass concentration was 0.2:0.4 L min-1. Intraclump dissolved oxygen tensions at high biomass densities were generally greater using air delivered by combined sparger-membrane tubing aeration than with sparging only of air or oxygen-enriched air. Specific atropine levels were not significantly affected by membrane tubing aeration. Indicators of anaerobic metabolism, such as lactic acid, ethanol, and ADH activity levels, were not significantly different in sparged and membrane-aerated systems; A. belladonna hairy roots also did not produce aerenchyma in response to oxygen limitations. Addition of perfluorocarbon emulsion to Murashige and Skoog medium in sparged stirred tank bioreactors did not improve growth, even when the emulsion was continuously recycled for re-aeration in an external vessel. Perfluorocarbons are associated with enhancement of gas-liquid oxygen transfer, so their ineffectiveness in this work most likely reflects the dominance of liquid-solid transfer resistances in hairy root cultures. The results of this investigation highlight the importance of developing new approaches for site-directed aeration of hairy root cultures, targeting oxygen delivery into the zones of highest root density.

The extent to which external oxygen transfer limits growth in shake flask culture of hairy roots

This study demonstrates the effects of oxygen limitations on growth of Atropa belladonna hairy roots in 250-mL shake flasks. Exponential growth was observed only for the first 6-8 d culture; after this time, growth continued with declining specific rate. Increasing the shaking speed and decreasing the medium volume improved biomass production and, to some extent, the specific growth rate of the roots. Direct enhancement of oxygen delivery, either by periodic exposure of the roots to air or oxygen enrichment of the atmosphere, also increased growth rates and/or final biomass levels. These results show that oxygen limitations are likely to affect biomass production and kinetic measurements in shake flask cultures of hairy roots.

Effect of inoculum size on growth of Atropa belladonna hairy roots in shake flasks

The initial specific growth rate of Atropa belladonna hairy roots in shake flasks was found to vary with the number of inoculum root tips. Increasing the number of tips from 3 to 9 reduced growth rates in 50 and 100 ml of medium by up to 40%. These results are interpreted by considering the influence of factors such as medium conditioning and oxygen mass transfer on root growth.

Influence of inoculum morphology on growth of Atropa belladonna hairy roots and production of tropane alkaloids

The specific growth rate of Atropa belladonna hairy roots measured in terms of root length increased by about 25% to 0.49 d-1 when the inoculum consisted of a root with the primary root tip excised. The biomass dry weight produced after 14 d increased by 28%. In contrast, presence of laterals in the inoculum with or without lateral tips excised did not influence the growth rate. Although hyoscyamine was found to accumulate at higher concentrations in the more mature root tissues (2.1 ± 0.2 mg g-1) than in the tips (1.1 ± 0.3 mg g-1), hyoscyamine content in the harvested roots was independent of inoculum morphology.

Oxygen requirements and mass transfer in hairy‐root culture

Oxygen mass transfer in clumps of Atropa belladonna hairy roots was investigated as a function of root density and external flow conditions. Convection was the dominant mechanism for mass transfer into root clumps 3.5 to 5.0 cm in diameter; Peclet numbers inside the clumps ranged from 1.4 x 10(3) to 7.1 x 10(4) for external superficial flow velocities between 0.4 and 1.4 cm s(-1). Local dissolved-oxygen levels and rates of oxygen uptake were measured in aflow chamber and in bubble column and stirred bioreactors. When air was used as oxygen source, intraclump dissolved-oxygen tensions ranged from90% to 100% air saturation at high external flow velocity andlow root density, to less than 20% air saturation in dense root clumps. Specific oxygen-uptake rate declined with increasing root density. When external boundary layers around individual roots were eliminated byforcing liquid through the clumps at superficial velocities between 0.2 and1.0 cm s(-1), internal dissolved-oxygen tension was maintained at 95% to 100% air saturation and rate of oxygen uptake at 1.6 x 10(-6) g g(-1) s(-1) dry weight. Liquid culture of single A. belladonna hairy roots was used to investigate the effect of dissolved-oxygen tensionon root growth and morphology. Total root length and number of root tips increased exponentially at oxygen tensions between 70% and 100%air saturation. Specific growth rate increased with oxygen tension up to 100% air saturation; this result demonstrates that hairy roots aeratedwithout oxygen supplementation are likely to be oxygenlimited. No growth occurred at 50% air saturation. Growth of hairy roots proceeded with an average length per tip of about 1 cm; this value was essentially independent of dissolved-oxygen tension between 70% and 100% air saturation. (c) 1994 John Wiley & Sons, Inc.

Limitations associated with conductivity measurement for monitoring growth in plant tissue culture

The suitability of conductivity measurement for monitoring growth in plant cell culture has been tested using suspended cells and genetically-transformed hairy roots of Atropa belladonna, and aggregated cells of Solanum aviculare. Other researchers have proposed that a constant ratio exists between increase in cell concentration (Δx) and decrease in medium conductivity (ΔC). In all cases studied in this work, Δx/ΔC was not constant over a wide range of cell densities tested in batch culture. With cell suspensions, Δx/ΔC decreased continuously during the growth phase from 3.4 to 2.5 g cm l−1 mS−1. For the hairy roots, the ratio between Δx and ΔC varied by as much as 4-fold during growth. The relationship between conductivity and growth for S. aviculare aggregates was found to vary depending on inoculum density. No simple correlation between conductivity change and cell growth was apparent for the plant-cell systems studied.

Characteristics of growth and tropane alkaloid synthesis in Atropa belladonna roots transformed by Agrobacterium rhizogenes

Growth of Atropa belladonna hairy roots in shake flasks followed first-order kinetics. The specific growth rate was 0.28 d −1, corresponding to a doubling time of 60 h. In airlift reactors, first order kinetics were not observed due to nutrient limitation. Synthesis of tropane alkaloids was growth-associated; no atropine was released into the medium. Sugar concentrations were analyzed during hairy-root culture for calculation of atropine yield. Atropine yields from substrate were 1.4 mg g −1 in shake flasks and 0.46 mg g −1 in airlift reactors. Although levels of atropine in reactor-grown roots (0.37% dry weight) were higher than in shake-flask roots (0.25% dry weight), growth and substrate conversion were better in shake flasks. Activity in the flasks and bioreactors levelled off after 17–20 d, even though 20–22 g l −1 of sugar remained in the medium.