Metabolic engineering for the production of shikimic acid in an evolved <it>Escherichia coli </it>strain lacking the phosphoenolpyruvate: carbohydrate phosphotransferase system
<p>Abstract</p> <p>Background</p> <p>Shikimic acid (SA) is utilized in the synthesis of oseltamivir-phosphate, an anti-influenza drug. In this work, metabolic engineering approaches were employed to produce SA in <it>Escherichia coli </it>strains derived fro...
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| Format: | Article |
| Language: | English |
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BMC
2010-04-01
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| Series: | Microbial Cell Factories |
| Online Access: | http://www.microbialcellfactories.com/content/9/1/21 |
| _version_ | 1811243963028340736 |
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| author | Bolívar Francisco Gosset Guillermo Ramírez Octavio T Hernández Georgina de Anda Ramón Valdivia Araceli Calderón Rocío Escalante Adelfo |
| author_facet | Bolívar Francisco Gosset Guillermo Ramírez Octavio T Hernández Georgina de Anda Ramón Valdivia Araceli Calderón Rocío Escalante Adelfo |
| author_sort | Bolívar Francisco |
| collection | DOAJ |
| description | <p>Abstract</p> <p>Background</p> <p>Shikimic acid (SA) is utilized in the synthesis of oseltamivir-phosphate, an anti-influenza drug. In this work, metabolic engineering approaches were employed to produce SA in <it>Escherichia coli </it>strains derived from an evolved strain (PB12) lacking the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS<sup>-</sup>) but with capacity to grow on glucose. Derivatives of PB12 strain were constructed to determine the effects of inactivating <it>aroK</it>, <it>aroL</it>, <it>pykF or pykA </it>and the expression of plasmid-coded genes <it>aroG</it><sup>fbr</sup>, <it>tktA, aroB </it>and <it>aroE</it>, on SA synthesis.</p> <p>Results</p> <p>Batch cultures were performed to evaluate the effects of genetic modifications on growth, glucose consumption, and aromatic intermediate production. All derivatives showed a two-phase growth behavior with initial high specific growth rate (<it>μ</it>) and specific glucose consumption rate (<it>qs</it>), but low level production of aromatic intermediates. During the second growth phase the <it>μ </it>decreased, whereas aromatic intermediate production reached its maximum. The double <it>aroK</it><sup>- </sup><it>aroL</it><sup>- </sup>mutant expressing plasmid-coded genes (strain PB12.SA22) accumulated SA up to 7 g/L with a yield of SA on glucose of 0.29 mol/mol and a total aromatic compound yield (TACY) of 0.38 mol/mol. Single inactivation of <it>pykF or pykA </it>was performed in PB12.SA22 strain. Inactivation of <it>pykF </it>caused a decrease in <it>μ</it>, <it>qs</it>, SA production, and yield; whereas TACY increased by 33% (0.5 mol/mol).</p> <p>Conclusions</p> <p>The effect of increased availability of carbon metabolites, their channeling into the synthesis of aromatic intermediates, and disruption of the SA pathway on SA production was studied. Inactivation of both <it>aroK </it>and <it>aroL</it>, and transformation with plasmid-coded genes resulted in the accumulation of SA up to 7 g/L with a yield on glucose of 0.29 mol/mol PB12.SA22, which represents the highest reported yield. The <it>pykF </it>and <it>pykA </it>genes were inactivated in strain PB12.SA22 to increase the production of aromatic compounds in the PTS<sup>- </sup>background. Results indicate differential roles of Pyk isoenzymes on growth and aromatic compound production. This study demonstrated for the first time the simultaneous inactivation of PTS and <it>pykF </it>as part of a strategy to improve SA production and its aromatic precursors in <it>E. coli</it>, with a resulting high yield of aromatic compounds on glucose of 0.5 mol/mol.</p> |
| first_indexed | 2024-04-12T14:16:33Z |
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| institution | Directory Open Access Journal |
| issn | 1475-2859 |
| language | English |
| last_indexed | 2024-04-12T14:16:33Z |
| publishDate | 2010-04-01 |
| publisher | BMC |
| record_format | Article |
| series | Microbial Cell Factories |
| spelling | doaj.art-66bee72d36d548e3919a0ff991d11efd2022-12-22T03:29:42ZengBMCMicrobial Cell Factories1475-28592010-04-01912110.1186/1475-2859-9-21Metabolic engineering for the production of shikimic acid in an evolved <it>Escherichia coli </it>strain lacking the phosphoenolpyruvate: carbohydrate phosphotransferase systemBolívar FranciscoGosset GuillermoRamírez Octavio THernández Georginade Anda RamónValdivia AraceliCalderón RocíoEscalante Adelfo<p>Abstract</p> <p>Background</p> <p>Shikimic acid (SA) is utilized in the synthesis of oseltamivir-phosphate, an anti-influenza drug. In this work, metabolic engineering approaches were employed to produce SA in <it>Escherichia coli </it>strains derived from an evolved strain (PB12) lacking the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS<sup>-</sup>) but with capacity to grow on glucose. Derivatives of PB12 strain were constructed to determine the effects of inactivating <it>aroK</it>, <it>aroL</it>, <it>pykF or pykA </it>and the expression of plasmid-coded genes <it>aroG</it><sup>fbr</sup>, <it>tktA, aroB </it>and <it>aroE</it>, on SA synthesis.</p> <p>Results</p> <p>Batch cultures were performed to evaluate the effects of genetic modifications on growth, glucose consumption, and aromatic intermediate production. All derivatives showed a two-phase growth behavior with initial high specific growth rate (<it>μ</it>) and specific glucose consumption rate (<it>qs</it>), but low level production of aromatic intermediates. During the second growth phase the <it>μ </it>decreased, whereas aromatic intermediate production reached its maximum. The double <it>aroK</it><sup>- </sup><it>aroL</it><sup>- </sup>mutant expressing plasmid-coded genes (strain PB12.SA22) accumulated SA up to 7 g/L with a yield of SA on glucose of 0.29 mol/mol and a total aromatic compound yield (TACY) of 0.38 mol/mol. Single inactivation of <it>pykF or pykA </it>was performed in PB12.SA22 strain. Inactivation of <it>pykF </it>caused a decrease in <it>μ</it>, <it>qs</it>, SA production, and yield; whereas TACY increased by 33% (0.5 mol/mol).</p> <p>Conclusions</p> <p>The effect of increased availability of carbon metabolites, their channeling into the synthesis of aromatic intermediates, and disruption of the SA pathway on SA production was studied. Inactivation of both <it>aroK </it>and <it>aroL</it>, and transformation with plasmid-coded genes resulted in the accumulation of SA up to 7 g/L with a yield on glucose of 0.29 mol/mol PB12.SA22, which represents the highest reported yield. The <it>pykF </it>and <it>pykA </it>genes were inactivated in strain PB12.SA22 to increase the production of aromatic compounds in the PTS<sup>- </sup>background. Results indicate differential roles of Pyk isoenzymes on growth and aromatic compound production. This study demonstrated for the first time the simultaneous inactivation of PTS and <it>pykF </it>as part of a strategy to improve SA production and its aromatic precursors in <it>E. coli</it>, with a resulting high yield of aromatic compounds on glucose of 0.5 mol/mol.</p>http://www.microbialcellfactories.com/content/9/1/21 |
| spellingShingle | Bolívar Francisco Gosset Guillermo Ramírez Octavio T Hernández Georgina de Anda Ramón Valdivia Araceli Calderón Rocío Escalante Adelfo Metabolic engineering for the production of shikimic acid in an evolved <it>Escherichia coli </it>strain lacking the phosphoenolpyruvate: carbohydrate phosphotransferase system Microbial Cell Factories |
| title | Metabolic engineering for the production of shikimic acid in an evolved <it>Escherichia coli </it>strain lacking the phosphoenolpyruvate: carbohydrate phosphotransferase system |
| title_full | Metabolic engineering for the production of shikimic acid in an evolved <it>Escherichia coli </it>strain lacking the phosphoenolpyruvate: carbohydrate phosphotransferase system |
| title_fullStr | Metabolic engineering for the production of shikimic acid in an evolved <it>Escherichia coli </it>strain lacking the phosphoenolpyruvate: carbohydrate phosphotransferase system |
| title_full_unstemmed | Metabolic engineering for the production of shikimic acid in an evolved <it>Escherichia coli </it>strain lacking the phosphoenolpyruvate: carbohydrate phosphotransferase system |
| title_short | Metabolic engineering for the production of shikimic acid in an evolved <it>Escherichia coli </it>strain lacking the phosphoenolpyruvate: carbohydrate phosphotransferase system |
| title_sort | metabolic engineering for the production of shikimic acid in an evolved it escherichia coli it strain lacking the phosphoenolpyruvate carbohydrate phosphotransferase system |
| url | http://www.microbialcellfactories.com/content/9/1/21 |
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