CONTRATADOS
GIULIETTI Ana Maria
congresos y reuniones científicas
Título:
A Metabolic Engineering Strategy to Improve Anthraquinone Production in Morinda citrifolia Plant Cell Cultures
Autor/es:
CARLA QUEVEDO; PERASSOLO M,; GIULIETTI A M; RODRIGUEZ TALOU, J
Reunión:
Simposio; 14th International Biotechnology Symposium and Exhibition.; 2010
Resumen:
Plant cell suspension cultures are attractive alternatives for large scale production of secondary metabolites with therapeutic properties like Anthraquinones (AQs). Morinda citrifoliaMorinda citrifolia is an example of this, since it is able to produce these metabolites. AQs are anthracenes derivatives. The A and B rings are synthesized from isochorismate and o-succinylbenzoate whereas the C ring is originated from the 2-C-methyl-D-erythritol 4-phosphate pathway (MEP). 1-deoxy-D-Xylulose-5-phosphate synthase (DXS), the first enzyme in the MEP pathway, catalyzes the conversion of pyruvate and glyceraldehydes-3-phosphate into 1-deoxy-DXylulose- 5-phosphate.The aim of this work was to overexpress the DXS in M. citrifolia cell suspension cultures in order to enhance the production of AQs and to evaluate the behaviour of the transgenic lines obtained after the addition of substrates like pyruvate and production of AQs and to evaluate the behaviour of the transgenic lines obtained after the addition of substrates like pyruvate and M. citrifolia cell suspension cultures in order to enhance the production of AQs and to evaluate the behaviour of the transgenic lines obtained after the addition of substrates like pyruvate and -ketoglutarate-ketoglutarate Methods: An expression vector carrying the dxs gene fromAn expression vector carrying the dxs gene from Catharanthus roseus and direct transformation of M. citrifolia cell suspension cultures with Agrobacterium. tumefaciens strain was carried out. The transgenic lines were treated with 10mM of both pyruvate and -ketoglutarate. To determine the levels of relative carried out. The transgenic lines were treated with 10mM of both pyruvate and -ketoglutarate. To determine the levels of relative suspension cultures with Agrobacterium. tumefaciens strain was carried out. The transgenic lines were treated with 10mM of both pyruvate and -ketoglutarate. To determine the levels of relative carried out. The transgenic lines were treated with 10mM of both pyruvate and -ketoglutarate. To determine the levels of relative and direct transformation of M. citrifolia cell suspension cultures with Agrobacterium. tumefaciens strain was carried out. The transgenic lines were treated with 10mM of both pyruvate and -ketoglutarate. To determine the levels of relative carried out. The transgenic lines were treated with 10mM of both pyruvate and -ketoglutarate. To determine the levels of relative grobacterium. tumefaciens strain was carried out. The transgenic lines were treated with 10mM of both pyruvate and -ketoglutarate. To determine the levels of relative-ketoglutarate. To determine the levels of relative dxs gene expression, Real Time PCR assays were performed and AQs content was determined spectophotometrically content was determined spectophotometrically gene expression, Real Time PCR assays were performed and AQs content was determined spectophotometrically Results: The dxs transformed cell lines showed highermRNAlevels and reached amaximum of 2.16 fold after 6 days of culture when compared to controls lines. Transgenic cells lines showed significantly higher levels of AQs (21% and 30% after 3 and 6 days of culture (p<0,01) compared control cell lines. With respect to the addition of pyruvate and -ketoglutarate, we observed that the transgenic line overexpresing DXS showed an increase of AQs content (50% at 6 days of culture) when a combination of both substrates were used line overexpresing DXS showed an increase of AQs content (50% at 6 days of culture) when a combination of both substrates were used and reached amaximum of 2.16 fold after 6 days of culture when compared to controls lines. Transgenic cells lines showed significantly higher levels of AQs (21% and 30% after 3 and 6 days of culture (p<0,01) compared control cell lines. With respect to the addition of pyruvate and -ketoglutarate, we observed that the transgenic line overexpresing DXS showed an increase of AQs content (50% at 6 days of culture) when a combination of both substrates were used line overexpresing DXS showed an increase of AQs content (50% at 6 days of culture) when a combination of both substrates were used The dxs transformed cell lines showed highermRNAlevels and reached amaximum of 2.16 fold after 6 days of culture when compared to controls lines. Transgenic cells lines showed significantly higher levels of AQs (21% and 30% after 3 and 6 days of culture (p<0,01) compared control cell lines. With respect to the addition of pyruvate and -ketoglutarate, we observed that the transgenic line overexpresing DXS showed an increase of AQs content (50% at 6 days of culture) when a combination of both substrates were used line overexpresing DXS showed an increase of AQs content (50% at 6 days of culture) when a combination of both substrates were used -ketoglutarate, we observed that the transgenic line overexpresing DXS showed an increase of AQs content (50% at 6 days of culture) when a combination of both substrates were used Discussion: The present metabolic engineering and precursor addition strategies showed to be a powerful tool to improve secondary metabolite production in plant cell bioprocesses addition strategies showed to be a powerful tool to improve secondary metabolite production in plant cell bioprocesses The present metabolic engineering and precursor addition strategies showed to be a powerful tool to improve secondary metabolite production in plant cell bioprocesses