Rosa Cusidó | Universitat de Barcelona (original) (raw)

Papers by Rosa Cusidó

Phytochemistry, 2015

The response of two Taxus cell systems to the action of cyclodextrin (CD) and coronatine (CORO), ... more The response of two Taxus cell systems to the action of cyclodextrin (CD) and coronatine (CORO), supplied to the culture medium either separately or together, was studied. Two-stage Taxus globosa and Taxus media cell cultures were established and the elicitors were added at the beginning of the second stage. Growth, taxane production, and the expression of known taxol biosynthetic genes, including the recently characterized CoA ligase gene, were studied. Although CORO reduced the growth capacity of both cell lines, CD apparently counteracted this negative effect. Taxane production was significantly enhanced by the simultaneous addition of CD and CORO to the medium. The total taxane production in the T. media cell line was more than double that of T. globosa, but in the latter more than 90% of the taxanes produced were excreted to the medium. Individual taxane patterns also differed: at the height of production, the main taxanes in T. globosa cultures were cephalomannine and 10-deacetyltaxol, and in T. media, taxol and baccatin III. The low transcript levels of taxane biosynthetic genes found in T. globosa cells mirrored the lower taxane production in these cultures, while a high expression was strongly correlated with a high taxane production in T. media.

In Vitro Cellular & Developmental Biology - Plant, 2015

ABSTRACT The detection of taxol and related taxanes in Corylus avellana has generated considerabl... more ABSTRACT The detection of taxol and related taxanes in Corylus avellana has generated considerable interest, particularly for in vitro cell cultures. Cell suspensions are a sustainable and rational option for obtaining a continuous and reliable source of secondary metabolites in large-scale processes. We therefore focused our study on the main factors that affect the growth of C. avellana cell suspensions as a key approach to improving culture productivity. In this work, calli were successfully induced from C. avellana seeds, leaves, and stems, and the efficiency of different sterilization methods was analyzed. The effects of the basal medium, carbon source, and the type and quantity of plant growth regulators on culture growth were studied. A fractional factorial design allowed us to reduce the number of experiments and analyze all the combinations in one run, thereby reducing time, variability, and costs. Statistical analysis (ANOVA) revealed that 1-naphthaleneacetic acid (NAA) and sucrose are mandatory for the growth of C. avellana cell suspension cultures, with no interactions detected between the parameters analyzed, while growth did not depend on the addition of cytokinins. The secondary metabolism was not inhibited, detecting 1175.45 ng/L of baccatin III and traces of taxol, deacetyltaxol, and cephalomannine. Additionally, prompted by the high growth rate of the C. avellana calli, we assayed a new cold-temperature-based method to maintain a stock of calli using half-strength Murashige and Skoog solid medium, concluding that up to 5 mo at 4°C is optimal to ensure white friable calli upon regrowth at 25°C.

Plant Biotechnology Journal, 2015

We tested the effect of the presence in the culture medium of chitosan, vanadyl sulfate or methyl... more We tested the effect of the presence in the culture medium of chitosan, vanadyl sulfate or methyl jasmonate on growth and ginsenoside production of three stable hairy root lines of Panax ginseng C.A. Meyer showing different morphological phenotypes C-M, HR-M and T-M. The response depended upon line phenotype, specificity of the elicitor and the stage of growth at which the

Methyl jasmonate and cyclodextrins are proven effective inducers of secondary metabolism in plant... more Methyl jasmonate and cyclodextrins are proven effective inducers of secondary metabolism in plant cell cultures. Cyclodextrins, which are cyclic oligosaccharides, can form inclusion complexes with nonhydrophilic secondary products, thus increasing their excretion from the producer cells to the culture medium. In the present work, using a selected Taxus x media cell line cultured in a two-stage system, the relationship between taxane production and the transcript profiles of several genes involved in taxol metabolism was studied to gain more insight into the mechanism by which these two elicitors regulate the biosynthesis and excretion of taxol and related taxanes. Gene expression was not clearly enhanced by the presence of cyclodextrins in the culture medium and variably induced by methyl jasmonate, but when the culture was supplemented with both elicitors, a synergistic effect on transcript accumulation was observed. The BAPT and DBTNBT genes, which encode the last two transferases involved in the taxol pathway, appeared to control limiting biosynthetic steps. In the cell cultures treated with both elicitors, the produced taxanes were found mainly in the culture medium, which limited retroinhibition processes and taxane toxicity for the producer cells. The expression level of a putative ABC gene was found to have increased, suggesting it played a role in the taxane excretion. Taxol biosynthesis was clearly increased by the joint action of methyl jasmonate and cyclodextrins, reaching production levels 55 times higher than in nonelicited cultures.

Plant Biotechnology Journal, 2014

Small peptides play important roles in the signalling cascades that steer plant growth, developme... more Small peptides play important roles in the signalling cascades that steer plant growth, development and defence, and often crosstalk with hormonal signalling. Thereby, they also modulate metabolism, including the production of bioactive molecules that are of high interest for human applications. Yew species (Taxus spp.) produce diterpenes such as the powerful anticancer agent paclitaxel, the biosynthesis of which can be stimulated by the hormone jasmonate, both in whole plants and cell suspension cultures. Here, we identified Taximin, as a gene encoding a hitherto unreported, plant-specific, small, cysteine-rich signalling peptide, through a transcriptome survey of jasmonate-elicited T. baccata suspension cells grown in two-media cultures. Taximin expression increased in a coordinated manner with that of paclitaxel biosynthesis genes. Tagged Taximin peptides were shown to enter the secretory system and localize to the plasma membrane. In agreement with this, the exogenous application of synthetic Taximin peptide variants could transiently modulate the biosynthesis of taxanes in T. baccata cell suspension cultures. Importantly, the Taximin peptide is widely conserved in the higher plant kingdom with a high degree of sequence conservation. Accordingly, Taximin overexpression could stimulate the production of nicotinic alkaloids in Nicotiana tabacum hairy root cultures in a synergistic manner with jasmonates. In contrast, no pronounced effects of Taximin overexpression on the specialized metabolism in Medicago truncatula roots were observed. This study increases our understanding of the regulation of Taxus diterpene biosynthesis in particular and plant metabolism in general. Ultimately, Taximin might increase the practical potential of metabolic engineering of medicinal plants.

Journal of Biotechnology, 2015

The growing demand for the antitumorous agent paclitaxel and the difficulty in increasing its pro... more The growing demand for the antitumorous agent paclitaxel and the difficulty in increasing its production by genetic engineering has prompted a search for new sources of taxanes. It has been reported that taxanes can be extracted from the angiosperm Corylus avellana L. Our aim was to improve taxane production by scaling up the process from mL-level to benchtop bioreactors, optimizing culture conditions and comparing the effect of two elicitors, 1 μM coronatine (Cor) and 100 μM methyl jasmonate (MeJA). Orbitally shaken flask cultures achieved a maximum fresh cell weight of 11.54 gDCW/L under control conditions, and MeJA- and Cor-treatment produced a statistically significant reduction in growth to 4.28 gDCW/L and 5.69 gDCW/L, while increasing the taxane content 3- and 27-fold, respectively. The enhancing effect of these elicitors on taxane production, despite affecting growth, was confirmed in orbitally shaken TubeSpin Bioreactors 50, where the highest taxane content (8583.3 μg/L) was obtained when 1μM Cor was used and elicitation took place at a packed cell volume of 50%. Two benchtop stirred bioreactors, BIOSTAT B plus and UniVessel SU, were compared, the latter providing a higher biomass of C. avellana cell suspension cultures. Transferring the established optimum culture conditions for taxane production to the UniVessel SU resulted in a total taxane content of 6246.1 μg/L, a 10-fold increase compared with shake flask experiments.

Process Biochemistry, 2011

Planta Medica, 2008

We tested the capacity of Galphimia glauca cells to produce galphimine-B (G-B) when under the eff... more We tested the capacity of Galphimia glauca cells to produce galphimine-B (G-B) when under the effects of a two-stage culture system: cell immobilization in Ca2+-alginate beads and culture scale-up from shake-flask to two different types of bioreactor (stirred and airlift). In the shake-flask culture, using optimum media for cell growth (first stage) and G-B production (second stage), the G-B yield was similar in both immobilised and free cells. However, while the free cells accumulated G-B within cytoplasmatic compartments, where it could not be recovered without cell disruption, immobilized cells excreted up to 100 % of the G-B produced. Immobilized cells grown in bioreactors running for 14 days with growth medium and an additional 26 days with production medium in batch mode showed a high G-B yield. The stirred bioreactor was the most efficient with a G-B content in the culture medium of 1381 microg.L (-1) at day 24 of culture.

Plant Cell, Tissue and Organ Culture (PCTOC), 2011

Phytochemistry, 2002

Putrescine:SAM N-methyltransferase (PMT) catalyses the N-methylation of the diamine putrescine to... more Putrescine:SAM N-methyltransferase (PMT) catalyses the N-methylation of the diamine putrescine to form N-methylputrescine, the first specific precursor of both tropane and pyridine-type alkaloids, which are present together in the roots of Duboisia plants. The pmt gene of Nicotiana tabacum was placed under the regulation of the CaMV 35S promoter and introduced into the genome of a scopolamine-rich Duboisia hybrid by a binary vector system using the disarmed Agrobacterium tumefaciens strain C58C1 carrying the rooting plasmid pRiA4. The presence of the foreign gene in kanamycin-resistant hairy roots and its overexpression were confirmed by polymerase chain reaction and Northern blot analysis respectively. The N-methylputrescine levels of the resulting engineered hairy roots increased (2-4-fold) compared to wild type roots, but there was no significant increase in either tropane or pyridine-type alkaloids.

Journal of Plant Physiology, 2007

Journal of Plant Physiology, 2013