- Email: [email protected]
Genome-scale reconstruction of the metabolic network in Chromohalobacter salexigens
New Biotechnology · Volume 25S · September 2009
from their natural source or too complex to be produced by chemical synthesis. Taxol (paclitaxel), a complex polyoxygenated diterpenoid natural product of the pacific yew tree (Taxus brevifolia Nutt.), is used extensively for the treatment of a variety of human cancers. Today Taxol is derived by semisynthesis from other advanced taxoids (obtained from various Taxus sp.) or extracted from plant cell culture material. The various Taxus species produce arrays of chemically similar taxoid structures, and levels of production vary extensively due to genetic, environmental and epigenetic factors. The extraction of specific taxoids from plant material can be hampered by low yields of the desired target compound and the complex background of chemically similar (co-purifying) taxoid structures. The biosynthesis of Taxol involves at least 19 enzyme-catalysed steps starting form geranylgeranyl diphosphate. Several of these steps are now characterized and the underlying biosynthetic genes have been cloned. Thus, the biotechnological production of Taxol or of other advanced taxoids (polyoxygenated taxanes) is now becoming possible, and would offer an economic alternative to current means of production. Furthermore the taxoid synthesis in Taxus sp. represents an anastamosing pathway, therefore metabolic engineering of a heterologous microbial host like yeast would offer the possibility to establish a linear pathway to defined products. However, the host primary metabolism must be able to support the heterologous introduced natural product pathway. For example Saccharomyces cerevisiae does not possess an extensive diterpenoid metabolism, and therefore can offer only very limited amounts of geranylgeranyl diphosphate, the universal diterpenoid precursor. Thus for the metabolic engineering of an effective heterologous natural product biosynthetic pathway a sufficient supportive primary metabolism has to be established. By genetic engineering we generated a S. cerevisiae strain producing high levels of geranylgeranyl diphosphate for supporting effective taxane biosynthesis. Thus building the foundation for the efficient later production of Taxol or other taxoids in yeast. doi:10.1016/j.nbt.2009.06.805
4.1.24 Genome-scale reconstruction of the metabolic network in Chromohalobacter salexigens C. Vargas 1,∗ , A. García-Yoldi 1 , J.M. Rodríguez 2 , M. Argando˜na 1 , M. Cánovas 3 , J.M.P. Hernández 3 , J.J. Nieto 1 1
University of Seville, Spain National Bioinformatics Institute, Spain 3 University of Murcia, Spain 2
Chromohalobacter salexigens is a halophilic bacterium with biotechnological potential as a source of ectoine and hydroxyectoine, two compatible solutes with biostabilizing properties on molecules, whole cells and tissues. Currently, ectoines are commercialized in products for cosmetics and dermopharmacy, as well as components of laboratory kits for molecular biology, where they function as stabilizers of enzymes and antibodies. In addition, they have a
ABSTRACTS
promising potential for use in cryopreservation and neuroprotection. C. salexigens genome has been recently sequenced and an automatically generated annotation is available. This opens the way to the development of a Systems Biology approach, aimed to genetically engineering this microorganism for optimizing production of ectoines. The aim of this study is to generate a genome-based reconstruction of C. salexigens metabolism, and to complement it with data from in vitro metabolic and genetic analyses performed in our laboratory, to produce an in silico model of C. salexigens metabolism that will be used to optimize ectoines production. Currently, bioinformatic tools are being developed to re-annotate the whole genome and designate individual protein functions. Integration of these functions in metabolic pathways will be presented. doi:10.1016/j.nbt.2009.06.806
4.1.25 Enhancement of anthraquinone production in Morinda citrifolia cell suspension cultures after treatment with azetidine-2-carboxylic acid and thiazolidine-4carboxylic acid C.V. Quevedo ∗ , M. Perassolo, A.M. Giulietti, J. Rodríguez Talou Facultad de Farmacia y Biquimica, UBA, Buenos Aires, Argentina
Plant cell suspension cultures are attractive alternatives for large scale production of plant-derived natural products, in particular of secondary metabolites. Morinda citrifolia is a member of Rubiaceae family that produces anthraquinones (AQs), anthracene derivatives which exhibit biological interesting properties. The basal compound, anthraquinone (9,10-dioxoanthracene), can be substituted in various ways, resulting in a great diversity of structures. Different metabolic routes are involved in AQs synthesis: shikimate pathway produces chorismic acid, which is then converted into isochorismic acid by the enzyme isochorismate synthase. The reaction of this compound with ␣-ketoglutaric acid generates osuccinylbenzoic acid, the precursor of A and B rings of the AQs structure. C ring is derived from an isoprene unit produced by the 2-C-methyl-D-erythritol-4-phosphate pathway. The proline cycle was proposed to be linked to the pentose phosphate pathway (PPP), as the first one generates two NADP+ which are cofactors of the two first enzymes in the PPP. The PPP produces erithrose-4phosphate, which is substrate of the shikimate pathway. The aim of this work was to study a possible link between proline cycle and AQs production and to evaluate this link as a possible strategy for AQs accumulation. M. citrifolia cell suspension cultures were treated with azetidine-2-carboxylic acid (A2C; 25 and 50 M) and thiazolidine-4-carboxylic acid (T4C; 100 and 200 M), two proline analogs. All treatments except from A2C 25 M showed a higher AQs content (P < 0.05) compared to the control line, while only T4C 200 M treatment increased total phenolics (TP) content after a six-day culture. After ten days of culture, both T4C treatments enhanced AQs and TP production compared to control. Accumulation of proline was significantly increased by all treatments after six days of culture, and by A2C 50 M and both T4C treatments after www.elsevier.com/locate/nbt S333
from their natural source or too complex to be produced by chemical synthesis. Taxol (paclitaxel), a complex polyoxygenated diterpenoid natural product of the pacific yew tree (Taxus brevifolia Nutt.), is used extensively for the treatment of a variety of human cancers. Today Taxol is derived by semisynthesis from other advanced taxoids (obtained from various Taxus sp.) or extracted from plant cell culture material. The various Taxus species produce arrays of chemically similar taxoid structures, and levels of production vary extensively due to genetic, environmental and epigenetic factors. The extraction of specific taxoids from plant material can be hampered by low yields of the desired target compound and the complex background of chemically similar (co-purifying) taxoid structures. The biosynthesis of Taxol involves at least 19 enzyme-catalysed steps starting form geranylgeranyl diphosphate. Several of these steps are now characterized and the underlying biosynthetic genes have been cloned. Thus, the biotechnological production of Taxol or of other advanced taxoids (polyoxygenated taxanes) is now becoming possible, and would offer an economic alternative to current means of production. Furthermore the taxoid synthesis in Taxus sp. represents an anastamosing pathway, therefore metabolic engineering of a heterologous microbial host like yeast would offer the possibility to establish a linear pathway to defined products. However, the host primary metabolism must be able to support the heterologous introduced natural product pathway. For example Saccharomyces cerevisiae does not possess an extensive diterpenoid metabolism, and therefore can offer only very limited amounts of geranylgeranyl diphosphate, the universal diterpenoid precursor. Thus for the metabolic engineering of an effective heterologous natural product biosynthetic pathway a sufficient supportive primary metabolism has to be established. By genetic engineering we generated a S. cerevisiae strain producing high levels of geranylgeranyl diphosphate for supporting effective taxane biosynthesis. Thus building the foundation for the efficient later production of Taxol or other taxoids in yeast. doi:10.1016/j.nbt.2009.06.805
4.1.24 Genome-scale reconstruction of the metabolic network in Chromohalobacter salexigens C. Vargas 1,∗ , A. García-Yoldi 1 , J.M. Rodríguez 2 , M. Argando˜na 1 , M. Cánovas 3 , J.M.P. Hernández 3 , J.J. Nieto 1 1
University of Seville, Spain National Bioinformatics Institute, Spain 3 University of Murcia, Spain 2
Chromohalobacter salexigens is a halophilic bacterium with biotechnological potential as a source of ectoine and hydroxyectoine, two compatible solutes with biostabilizing properties on molecules, whole cells and tissues. Currently, ectoines are commercialized in products for cosmetics and dermopharmacy, as well as components of laboratory kits for molecular biology, where they function as stabilizers of enzymes and antibodies. In addition, they have a
ABSTRACTS
promising potential for use in cryopreservation and neuroprotection. C. salexigens genome has been recently sequenced and an automatically generated annotation is available. This opens the way to the development of a Systems Biology approach, aimed to genetically engineering this microorganism for optimizing production of ectoines. The aim of this study is to generate a genome-based reconstruction of C. salexigens metabolism, and to complement it with data from in vitro metabolic and genetic analyses performed in our laboratory, to produce an in silico model of C. salexigens metabolism that will be used to optimize ectoines production. Currently, bioinformatic tools are being developed to re-annotate the whole genome and designate individual protein functions. Integration of these functions in metabolic pathways will be presented. doi:10.1016/j.nbt.2009.06.806
4.1.25 Enhancement of anthraquinone production in Morinda citrifolia cell suspension cultures after treatment with azetidine-2-carboxylic acid and thiazolidine-4carboxylic acid C.V. Quevedo ∗ , M. Perassolo, A.M. Giulietti, J. Rodríguez Talou Facultad de Farmacia y Biquimica, UBA, Buenos Aires, Argentina
Plant cell suspension cultures are attractive alternatives for large scale production of plant-derived natural products, in particular of secondary metabolites. Morinda citrifolia is a member of Rubiaceae family that produces anthraquinones (AQs), anthracene derivatives which exhibit biological interesting properties. The basal compound, anthraquinone (9,10-dioxoanthracene), can be substituted in various ways, resulting in a great diversity of structures. Different metabolic routes are involved in AQs synthesis: shikimate pathway produces chorismic acid, which is then converted into isochorismic acid by the enzyme isochorismate synthase. The reaction of this compound with ␣-ketoglutaric acid generates osuccinylbenzoic acid, the precursor of A and B rings of the AQs structure. C ring is derived from an isoprene unit produced by the 2-C-methyl-D-erythritol-4-phosphate pathway. The proline cycle was proposed to be linked to the pentose phosphate pathway (PPP), as the first one generates two NADP+ which are cofactors of the two first enzymes in the PPP. The PPP produces erithrose-4phosphate, which is substrate of the shikimate pathway. The aim of this work was to study a possible link between proline cycle and AQs production and to evaluate this link as a possible strategy for AQs accumulation. M. citrifolia cell suspension cultures were treated with azetidine-2-carboxylic acid (A2C; 25 and 50 M) and thiazolidine-4-carboxylic acid (T4C; 100 and 200 M), two proline analogs. All treatments except from A2C 25 M showed a higher AQs content (P < 0.05) compared to the control line, while only T4C 200 M treatment increased total phenolics (TP) content after a six-day culture. After ten days of culture, both T4C treatments enhanced AQs and TP production compared to control. Accumulation of proline was significantly increased by all treatments after six days of culture, and by A2C 50 M and both T4C treatments after www.elsevier.com/locate/nbt S333