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1.
Catharanthus roseus plants produce many pharmaceutically important indole alkaloids, of which the bisindole alkaloids vinblastine and vincristine
are antineoplastic medicines and the monoindole alkaloids ajmalicine and serpentine are antihypertension drugs. C. roseus cell cultures have been studied for producing these medicines or precursors catharanthine and vindoline for almost four decades
but so far without a commercially successful process due to biological and technological limitations. The research thus focused
on the one hand on engineering the bioreactor process on the other engineering the cell factory itself. This review mainly
summarizes the progress made on biochemical engineering aspects of C. roseus cell cultures in bioreactors in the past decades and metabolic engineering of indole alkaloid production in recent years.
The paper also attempts to highlight new strategies and technologies to improve alkaloid production and bioreactor performance.
Perspectives of metabolic engineering to create new cell lines for large-scale production of indole alkaloids in bioreactors
and effective combination of these up- and down-stream processing are presented. 相似文献
2.
Hiroshi Sudo Takashi Yamakawa Mami Yamazaki Norio Aimi Kazuki Saito 《Biotechnology letters》2002,24(5):359-363
A large-scale culture of hairy root of Ophiorrhiza pumila using a modified 3 l bioreactor was established. The hairy roots, incited by infection of Agrobacterium rhizogenes were grown in the bioreactor equipped with a stainless net. The final concentration of camptothecin was 0.0085% fresh wt of tissue, and the total production of camptothecin, an anti-neoplastic quinoline alkaloid, reached 22 mg over 8 weeks' culture in the reactor. Approx. 17% (3.6 mg) of the total camptothecin produced was excreted into the culture medium. 相似文献
3.
Cultures of Solanum aviculare hairy roots were established after transformation with Agrobacterium rhizogenes A4. High levels of steroidal alkaloids measured as solasodine equivalents were produced in shake-flasks and bioreactor, even though relatively low concentrations are found in roots in vivo. In shake flasks the maximum alkaloid yield was 32 mg g-1 dry weight; in a 3-1 air-driven bioreactor the yield was 29 mg g-1. These yields represent a 5-fold increase over previous reports for in vitro production, and are comparable with levels found in the aerial parts of intact S. aviculare plants. Production of steroidal alkaloids was growth-associated. High sugar levels at stationary phase and insensitivity to increased levels of medium components suggest that root cultures were limited by oxygen mass-transfer. In Petri-dish culture with and without exogenous gibberellic acid, root length and number of root tips increased exponentially; growth proceeded with a constant length per root tip of about 35 mm. Addition of gibberellic acid enhanced growth but reduced the specific steroidal-alkaloid level. Taking into account both growth and alkaloid yield, accumulation of steroidal alkaloids was improved by about 40% at gibberellic-acid concentrations of 10 and 100 g l-1. 相似文献
4.
Frédérique Garnier Philippe Label Didier Hallard Jean-Claude Chénieux Marc Rideau Saïd Hamdi 《Plant Cell, Tissue and Organ Culture》1996,45(3):223-230
Cytokinins play a critical role in several aspects of plant growth, metabolism and development. We previously reported that adding cytokinins to the culture medium of a suspension-cultured cell line of periwinkle increased the accumulation of indole alkaloids, and our aim was to compare the effect of exogenously-applied cytokinins with that of elevated levels of endogenous cytokinins on indole alkaloid production. We used an Agrobacterium tumefaciens strain yielding a plasmid with the isopentenyl transferase gene under control of its own promoter. Co-culture of suspension cells with the bacteria caused a severe stress response leading to cell necrosis. Therefore, we failed to transform this material but we succeeded in transforming periwinkle cotyledons. We verified that callus cultures generated from the isopentenyl transferase-transgenic cotyledons accumulated high cytokinin concentrations. Treating normal callus cultures (generated from untransformed cotyledons) with cytokinins enhanced their alkaloid production. By contrast, the enhanced concentration of endogenous cytokinins in transgenic calli did not increase indole alkaloid production, and thus did not mimic the effect of exogenously-applied cytokinins. Hypothesis to explain this discrepancy are discussed.Abbreviations 2,4-d
2,4-dichlorophenoxyacetic acid
- DW
dry cell mass
-
ipt
isopentenyl transferase gene 相似文献
5.
Centaurea calcitrapa suspension cultures were grown either in Erlenmeyer flasks or in a mechanically stirred bioreactor. Its rheological behaviour,
when fitted to the Oswald–de Waele model (power law), showed pseudoplastic characteristics in both cases. The flow behaviour
index (n) decreased over the course of a growth cycle and the consistency index (K) increased, reached a value of 1.81 N sn m−2 run on 2 l bioreactor. Bioreactor cultivation of C. calcitrapa cells at different agitation rates (30, 60, 100 and 250 rpm), highlighted the influence of shear forces on cell viability
loss (90–34%) and phenol accumulation (74–140 μg l−1), due to increased stirring speeds. Analysis of these results suggests that this cell line is shear-sensitive. An empirical
exponential correlation was defined between apparent viscosity and biomass concentration, under the studied conditions, giving
the possibility to estimate the prevailing broth regime and to optimize bioreactor design.
Revisions requested 10 October 2005; Revisions received 19 December 2005 相似文献
6.
Fumihiko Sato Norimatsu Takeshihta Hiroyuki Fujiwara Yasuyuki Katagiri Liping Huan Yasuyuki Yamada 《Plant Cell, Tissue and Organ Culture》1994,38(2-3):249-256
Several cell lines of Coptis japonica with different alkaloid productivities were characterized to obtain information on how a high metabolite production is established. High and low metabolite producing cells, except those from one cell line, showed similar growth kinetics and a similar pattern of nutrient uptake. Amino acid contents, especially that of tyrosine, differed between cell lines, but no correlation was found between the amino acid or tyrosine levels and alkaloid production. Since the addition of tyrosine did not increase the production of berberine, this primary substrate is apparently not the limiting factor for high production in cultured Coptis cells. The addition of berberine to the medium revealed that low-producing cells also have the ability to store alkaloid, and that low productivity is not due to decomposition of alkaloids which have been produced. The direct measurement of the biosynthesis of berberine using 14C-tyrosine clearly showed that high-producing cells had a higher biosynthetic activity of berberine from tyrosine than low-producing cells. The measurement of enzyme activities in berberine biosynthesis indicated that the early steps of berberine biosynthesis are important in the increased production of berberine. 相似文献
7.
Raffaella Filippini Rosamaria Caniato Francesca Dalla Vecchia Elsa M. Cappelletti Lucia Puricelli Anna Piovan 《Plant biosystems》2013,147(2):179-184
ABSTRACT A new protocol to obtain an embryogenic cell line from cultured seedling explants of Catharanthus roseus is described. In order to assess the relationship between tissue differentiation and secondary metabolite biosynthesis, the biosynthetic capabilities (alkaloid production) of an embryogenic cell line and two non-embryogenic C. roseus strains were comparatively examined. Faster cell growth rate was associated with higher alkaloid production in the embryogenic cell line. The kinetics of ajmalicine and serpentine production by the three cell lines is also reported. 相似文献
8.
Summary Growth and alkaloid production of surface-immobilized C. roseus cells were studied in a 2-1 bioreactor. Media designed to maximize cell growth or alkaloid production were employed. Nitrate and carbohydrate consumption rates as well as growth rates and biomass yields of immobilized cultures were equal or somewhat lower than for cell suspension cultures. Respiration rate (O2 consumption and CO2 production rates) of immobilized C. roseus cell cultures was obtained by on-line analysis of inlet and outlet gas composition using a mass spectrometer. Respiration rate increased during the growth phase and decreased once the nitrogen or the carbon source was depleted from the medium. The respiration rate of immobilized C. roseus cells resembled rates reported in the literature for suspension cultures.
Offprint requests to: Denis Rho 相似文献
9.
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. 相似文献
10.
Aggregation of baby hamster kidney (BHK) cells cultivated in perfusion mode for manufacturing recombinant proteins was characterized. The potential impact of cultivation time on cell aggregation for an aggregating culture (cell line A) was studied by comparing expression profiles of 84 genes in the extracellular adhesion molecules (ECM) pathway by qRT‐PCR from 9 and 25 day shake flask samples and 80 and 94 day bioreactor samples. Significant up‐regulation of THBS2 (4.4‐ to 6.9‐fold) was seen in both the 25 day shake flask and 80 and 94 day bioreactor samples compared to the 9 day shake flask while NCAM1 was down‐regulated 5.1‐ to 8.9‐fold in the 80 and 94 day bioreactor samples. Subsequent comparisons were made between cell line A and a non‐aggregating culture (cell line B). A 65 day perfusion bioreactor sample from cell line B served as the control for 80 and 94 day samples from four different perfusion bioreactors for cell line A. Of the 84 genes in the ECM pathway, four (COL1A1, COL4A1, THBS2, and VCAN) were consistently up‐regulated in cell line A while two (NCAM1 and THBS1) were consistently down‐regulated. The magnitudes of differential gene expression were much higher when cell lines were compared (4.1‐ to 44.6‐fold) than when early and late cell line B samples were compared (4.4‐ to 6.9‐fold) indicating greater variability between aggregating and non‐aggregating cell lines. Based on the differential gene expression results, two mechanistic models were proposed for aggregation of BHK cells in perfusion cultures. Biotechnol. Bioeng. 2013; 110: 483–490. © 2012 Wiley Periodicals, Inc. 相似文献
11.
P. Morris K. Rudge R. Cresswell M. W. Fowler 《Plant Cell, Tissue and Organ Culture》1989,17(2-3):79-90
Three unselected cell lines of C. roseus maintained on a growth-associated alkaloid production medium were studied over a period of 2 to 5.5 years for the stability of alkaloid production (serpentine and ajmalicine). Large fluctuations in the total alkaloid content of 20-day-old cells were found for all three cell lines at each subculture over a two-year period. Growth rates increased during prolonged subculture and one cell line became unproductive after five years culture. By selection of small autofluorescent aggregates, high alkaloid production was restored in this cell line, while the parent line was found to be unresponsive to alkaloid induction treatments. The instability in both alkaloid production and spectrum and the loss of alkaloid productivity are discussed in relation to the selection pressures present during long-term maintenance of cell suspension cultures.Abbreviations 2,4-D
2,4-dichlorophenoxyacetic acid
- nHS
n-heptane sulphonate 相似文献
12.
Martina Klöppinger Georg Fertig Elisabeth Fraune Herbert G. Miltenburger 《Cytotechnology》1990,4(3):271-278
The aim of our study was to establish an efficient system for thein vitro production of the insect pathogenic Autographa californica nuclear polyhedrosis virus in a Spodoptera frugiperda cell line. We optimized cultivation conditions for cell proliferation as well as for virus replication in a 1.5 litre stirred tank bioreactor. Cell and virus propagation were found to be optimal at a constant oxygen tension of 40%. In order to provide sufficient nutrients during virus synthesis filtration and perfusion devices were connected to the bioreactor. A virus production procedure in a repeated batch mode by using a two stage bioreactor system is described. Stage I was optimized for cell production and stage II for virus production.Abbreviations Ac-NPV
Autographa californica Nuclear Polyhedrosis Virus
- BV
Baculovirus
- MOI
Multiplicity Of Infection
- ECV
Extracellular Virus 相似文献
13.
Ignacio E. Maldonado-Mendoza Terasa R. Del Ayora-Talavera Victor M. Loyola-Vargas 《In vitro cellular & developmental biology. Plant》1992,28(2):67-72
Summary Tropane alkaloid production was studied in different root cultures ofDatura stramonium. Cultured roots were obtained with 10−6
M of indolbutyric acid. Their doubling times were from 6 to 19 days. Hyoscyamine content varied from 0.17 to 0.62% dry weight,
and scopolamine content from 0.08 to 0.33% dry weight, depending on the lines. A comparison of the bioproductivity of these
compounds in the pot-grown plant roots showed that it was two to three orders lower than cultured roots, and it increased
one order of magnitude considering the productivity on the whole plant. Bioproductivity, growth capacity and alkaloid production
stability during subsequent transfers (more than 2 yr) are reported. Only one root line (N5) showed excretion of the alkaloids
to the culture medium. Characterization of three selected lines (N1, N5, and N9) showed that the highest alkaloid production
is reached at the stationary phase of growth, with the exception of line N9. 相似文献
14.
《Enzyme and microbial technology》1987,9(10):619-624
A culture of Catharanthus roseus has been developed that is capable of growth-linked serpentine formation. Two separate cell lines of this culture, C87 and C87N, were grown in air-lift bioreactors of 7, 30, and 80 liter working volume. Good growth was obtained with both cell lines in all vessels, with better growth rates at the higher volumes. In contrast, serpentine formation was very low when either cell line was grown in any of the vessels when compared with shake flasks. The reason for this loss of alkaloid formation does not appear to be associated with either bioreactor type or cell line. 相似文献
15.
Benoît Dubuis Oemer M. Kut Jiri E. Prenosil 《Plant Cell, Tissue and Organ Culture》1995,43(2):171-183
A novel bubble free loop fluidized bed reactor for plant cell cultures was developed and tested usingCoffea arabica as a model cell line. The effects of main operational parameters like morphology and size of inoculum, oxygen supply as well as recirculation of sparingly soluble gases on cell growth and alkaloid production rates in this reactor were studied and the results were compared with standard shake flask experiments. By on-line monitoring of biomass and oxygen uptake rates the main kinetic parameters for cell growth and alkaloid production were evaluated. It was demonstrated that the novel reactor is easy to run and is particularly adequate for measuring kinetic parameters necessary for scale up. 相似文献
16.
Summary An endo-pectate lyase (PL; EC 4.2.2.2), originally cloned fiom the phytopathogenic bacterium Erwinia chrysanthemi EC16, was expressed in recA
–
E. coli strain DK1, purified to a single band by isoelectric focusing and used to induce berberine production in established plant suspension cultures of Thalictrum minus L. subsp. saxatile. Addition of 10–9M pectate lyase c (PLc) stimulated berberine production and enhanced secretion of the alkaloid into the medium. A lower concentration of PLc, 10–11M, stimulated a transient two-fold increase in cell growth rate relative to untreated cultures. Parallel changes in L-phenylalanine ammonia lyase (PAL; EC 4.3.1.5) activity with the rate of berberine synthesis and the inverse relationship between cell growth and berberine synthesis imply that berberine synthesis is stress-related in this cell line. 相似文献
17.
Effects of sucrose, inoculum density, auxins, and aeration volume on ceil growth ofGymnema sylvestre
Eun Jung Lee Mohammad Mobin Eun Joo Hahn Kee Yoeup Paek 《Journal of Plant Biology》2006,49(6):427-431
To improve the cell protocol forCymnema sylvestre, we investigated the influence of initial sucrose concentration, inoculum density, and optimal concentrations of auxins (IBA
and NAA) in flask cultures, as well as the role of aeration volume in bioreactor cultures. Cell growth was enhanced 9-fold
when the medium was supplemented with 3% sucrose versus a sucrose-free environment. Increasing the inoculum density to 60
g (wet weight) L-1, but no further, greatly improved the growth of these cultures. All concentrations of IBA proved inhibitory while supplementation
with 5 nig L-1 NAA was associated with significantly higher dry-cell weights. In our bioreactor cultures, a step-wise increase in aeration
volume from 0.05 to 0.40 wm was optimal for cell growth. Although biomass (i.e., fresh weight) accumulated in the bioreactor
up until Day 20, the dry-cell weights increased 10-fold, but only through Day 15. The internal dynamics of our culture media
indicated that sucrose was preferentially utilized and that its concentration steeply decreased at the log phase. In contrast,
both glucose and fructose supplies were exhausted only at the beginning of the declining phase. Our findings suggest that
a 15-d culture period is optimal for G.sylvestre cell growth in a bioreactor. 相似文献
18.
Frick S Chitty JA Kramell R Schmidt J Allen RS Larkin PJ Kutchan TM 《Transgenic research》2004,13(6):607-613
The berberine bridge enzyme cDNA bbe from Papaver somniferumL. was transformed in antisense orientation into seedling explants of the industrial elite line C048-6-14-64. In this way, 84 phenotypically normal T0 plants derived from embryogenic callus cultures were produced. The selfed progeny of these 84 plants yielded several T1 plants with an altered alkaloid profile. One of these plants T1-47, and its siblings T2-1.2 and T2-1.5 are the subject of the present work. The transformation of these plants was evaluated by PCR, and northern and Southern hybridisation. The transgenic plants contained one additional copy of the transgene. The alkaloid content in latex and roots was determined with HPLC and LC-MS. We observed an increased concentration of several pathway intermediates from all biosynthetic branches, e.g., reticuline, laudanine, laudanosine, dehydroreticuline, salutaridine and (S)-scoulerine. The transformation altered the ratio of morphinan and tetrahydrobenzylisoquinoline alkaloids in latex but not the benzophenanthridine alkaloids in roots. The altered alkaloid profile is heritable at least to the T2 generation. These results are the first example of metabolic engineering of the alkaloid pathways in opium poppy and, to our knowledge, the first time that an alkaloid biosynthetic gene has been transformed into the native species, followed by regeneration into a mature plant to enable analyses of the effect of the transgene on metabolism over several generations. 相似文献
19.
Raisa Vermasvuori Jani Koskinen Katri Salonen Noora Sirén Jan Weegar John Dahlbacka Nisse Kalkkinen Niklas von Weymarn 《Biotechnology progress》2009,25(1):95-102
Three popular expression host systems Escherichia coli, Pichia pastoris and Drosophila S2 were analyzed techno‐economically using HIV‐1 Nef protein as the model product. On scale of 100 mg protein, the labor costs corresponded to 52–83% of the manufacturing costs. When analyzing the cost impact of the different phases (strain/cell line construction, bioreactor production, and primary purification), we found that with the microbial host systems the strain construction phase was most significant generating 56% (E. coli) and 72% (P. pastoris) of the manufacturing costs, whereas with the Drosophila S2 system the cell line construction and bioreactor production phases were equally significant (46 and 47% of the total costs, respectively). With different titers and production goal of 100 mg of Nef protein, the costs of P. pastoris and Drosophila S2 systems were about two and four times higher than the respective costs of the E. coli system. When equal titers and bioreactor working volumes (10 L) were assumed for all three systems, the manufacturing costs of the bioreactor production of the P. pastoris and Drosophila S2 systems were about two and 2.5 times higher than the respective costs of the E. coli system. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 相似文献
20.
Kang-Ik Jo You-Jung Lee Bo-Kyung Kim Bo-Hwa Lee Chung-Han Chung Soo-Wan Nam Sung-Koo Kim Jin-Woo Lee 《Biotechnology and Bioprocess Engineering》2008,13(2):182-188
Optimal conditions for pilot-scale production of the carboxymethylcellulase (CMCase) by Bacillus amyloliquefaciens DL-3 were investigated. The best carbon and nitrogen sources for the production of CMCase by B. amyloliquefaciens DL-3 were found to be rice hull and peptone and their optimal concentrations were 5.0 and 0.20% (w/v), respectively. Optimal
temperature and initial pH for the production of CMCase were 37°C and 6.8. Optimal agitation speed and aeration rate for the
production of CMCase were 300 rpm and 1.0 vvm in a 7 L bioreactor, which were different from those for the cell growth of
B. amyloliquefaciens DL-3. The highest productions of CMCase by B. amyloliquefaciens DL-3 from 5.0% (w/v) rice hull as a carbon source under optimal conditions in a 7 or 100 L bioreactor were 220 and 367 U/mL,
respectively. 相似文献