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A metabolic engineering strategy to improve anthraquinone production in Morinda citrifolia plant cell cultures
Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576
[P-P&F.36] A metabolic engineering strategy to improve anthraquinone production in Morinda citrifolia plant cell cultures C. Quevedo, M. Perassolo, A.M. Giulietti, J. Rodriguez Talou ∗ Microbiología Industrial y Biotecnología, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires. Junin 956,1113. Buenos Aires, Argentina, Argentina Keywords: Plant cell culture; metabolic engineering; anthraquinones; Morinda citrifolia Introduction: Plant cell suspension cultures are attractive alternatives for large scale production of secondary metabolites with therapeutic properties like Anthraquinones (AQs). Morinda 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 ␣-ketoglutarate Methods: An 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 10 mM of both pyruvate and ␣-ketoglutarate. To determine the levels of relative dxs gene expression, Real Time PCR assays were performed and AQs content was determined spectophotometrically Results: The dxs transformed cell lines showed higher mRNA levels and reached a maximum 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 Discussion: The present metabolic engineering and precursor addition strategies showed to be a powerful tool to improve secondary metabolite production in plant cell bioprocesses
S485
var groups. However, no method could precisely differentiate the two groups, especially from cured leaf samples. Amplified fragment length polymorphism (AFLP) analysis was introduced to estimate genetic polymorphism of 19 tobacco cultivars grown in Thailand. Thirty-two selective primer-combinations were screened on the genomic DNA extracted from cured leaves. Three primer pairs were selected and resulted in 139 scorable AFLP fragments, of which 103 (74.1%) were polymorphic. Genetic relationship analysis revealed clustering patterns of tobacco samples generally following the cultivar groups. Almost all local cultivars were found closely related to Burley and Turkish types of the imported group, but significantly separated from Virginia type. Our finding therefore should be an important knowledge for further research on cultivar identification and genetic improvement of tobaccos.
doi:10.1016/j.jbiotec.2010.09.741 [P-P&F.38]
doi:10.1016/j.jbiotec.2010.09.740
Brugmansia candida hairy root cultures in bioreactors as an anisodamine production system
[P-P&F.37]
A.B. Cardillo 1,∗ , A.M. Otálvaro 2 , V.D. Busto 1 , J. Rodriguez Talou 1 , M.E. Velásquez 2 , A.M. Giulietti 1
Genetic polymorphism between tobacco cultivar-groups revealed by amplified fragment length polymorphism analysis
1
Jessada Denduangboripant 1,∗ , Tianrat 2 Nantharat
Piteekan 2 , Matchima
1 Department of Biology, Faculty of Science, Chulalongkorn University, Thailand 2 Biotechnology Program, Faculty of Science, Chulalongkorn University, Thailand Keywords: Amplified fragment length polymorphism; Genetic polymorphism; Molecular marker; Tobacco
Tobacco (Nicotina tabacum) has been introduced to Thailand for hundreds of years. All tobaccos cultivated in the country are legally separated to local (or early-imported) and imported culti-
Microbiología Industrial y Biotecnología, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Argentina 2 Ingeniería Química y Ambiental, Universidad Nacional de Colombia, Colombia Keywords: Anisodamine; Hairy roots; Stirred tank bioreactor; Brugmansia candida Brugmansia candida is a South American native plant that produces tropane alkaloids, such as hyoscyamine, anisodamine (6-hydroxyhyoscyamine) and scopolamine. These alkaloids were traditionally used in medicine because of their anticholinergic activity. Recently there were described further therapeutical properties for anisodamine renewing the interest in these alkaloids. Nowadays, these alkaloids are produced by the exploitation of producer plants due to the cost and complexity that chemical synthesis
[P-P&F.36] A metabolic engineering strategy to improve anthraquinone production in Morinda citrifolia plant cell cultures C. Quevedo, M. Perassolo, A.M. Giulietti, J. Rodriguez Talou ∗ Microbiología Industrial y Biotecnología, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires. Junin 956,1113. Buenos Aires, Argentina, Argentina Keywords: Plant cell culture; metabolic engineering; anthraquinones; Morinda citrifolia Introduction: Plant cell suspension cultures are attractive alternatives for large scale production of secondary metabolites with therapeutic properties like Anthraquinones (AQs). Morinda 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 ␣-ketoglutarate Methods: An 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 10 mM of both pyruvate and ␣-ketoglutarate. To determine the levels of relative dxs gene expression, Real Time PCR assays were performed and AQs content was determined spectophotometrically Results: The dxs transformed cell lines showed higher mRNA levels and reached a maximum 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 Discussion: The present metabolic engineering and precursor addition strategies showed to be a powerful tool to improve secondary metabolite production in plant cell bioprocesses
S485
var groups. However, no method could precisely differentiate the two groups, especially from cured leaf samples. Amplified fragment length polymorphism (AFLP) analysis was introduced to estimate genetic polymorphism of 19 tobacco cultivars grown in Thailand. Thirty-two selective primer-combinations were screened on the genomic DNA extracted from cured leaves. Three primer pairs were selected and resulted in 139 scorable AFLP fragments, of which 103 (74.1%) were polymorphic. Genetic relationship analysis revealed clustering patterns of tobacco samples generally following the cultivar groups. Almost all local cultivars were found closely related to Burley and Turkish types of the imported group, but significantly separated from Virginia type. Our finding therefore should be an important knowledge for further research on cultivar identification and genetic improvement of tobaccos.
doi:10.1016/j.jbiotec.2010.09.741 [P-P&F.38]
doi:10.1016/j.jbiotec.2010.09.740
Brugmansia candida hairy root cultures in bioreactors as an anisodamine production system
[P-P&F.37]
A.B. Cardillo 1,∗ , A.M. Otálvaro 2 , V.D. Busto 1 , J. Rodriguez Talou 1 , M.E. Velásquez 2 , A.M. Giulietti 1
Genetic polymorphism between tobacco cultivar-groups revealed by amplified fragment length polymorphism analysis
1
Jessada Denduangboripant 1,∗ , Tianrat 2 Nantharat
Piteekan 2 , Matchima
1 Department of Biology, Faculty of Science, Chulalongkorn University, Thailand 2 Biotechnology Program, Faculty of Science, Chulalongkorn University, Thailand Keywords: Amplified fragment length polymorphism; Genetic polymorphism; Molecular marker; Tobacco
Tobacco (Nicotina tabacum) has been introduced to Thailand for hundreds of years. All tobaccos cultivated in the country are legally separated to local (or early-imported) and imported culti-
Microbiología Industrial y Biotecnología, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Argentina 2 Ingeniería Química y Ambiental, Universidad Nacional de Colombia, Colombia Keywords: Anisodamine; Hairy roots; Stirred tank bioreactor; Brugmansia candida Brugmansia candida is a South American native plant that produces tropane alkaloids, such as hyoscyamine, anisodamine (6-hydroxyhyoscyamine) and scopolamine. These alkaloids were traditionally used in medicine because of their anticholinergic activity. Recently there were described further therapeutical properties for anisodamine renewing the interest in these alkaloids. Nowadays, these alkaloids are produced by the exploitation of producer plants due to the cost and complexity that chemical synthesis