Metabolic selective pressure stabilizes plasmids carrying biosynthetic genes for reduced biochemicals in Escherichia coli redox mutants
Several biotechnological processes rely on the utilization of high-copy-number plasmids for heterologous gene expression, and understanding the interactions between plasmid DNA and bacterial hosts is highly relevant for bioprocess optimization. We assessed metabolic modifications and physiological c...
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2010
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| Acceso en línea: | https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_01757598_v88_n2_p563_Nikel http://hdl.handle.net/20.500.12110/paper_01757598_v88_n2_p563_Nikel |
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paper:paper_01757598_v88_n2_p563_Nikel2023-06-08T15:19:00Z Metabolic selective pressure stabilizes plasmids carrying biosynthetic genes for reduced biochemicals in Escherichia coli redox mutants Pettinari, María Julia Galvagno, Miguel Angel Méndez, Beatriz Silvia ArcAB CreBC Escherichia coli Microaerobiosis Plasmid copy number Redox mutants ArcAB Copy number CreBC Microaerobiosis Redox mutants Aldehydes Bearings (structural) Bioconversion Bioreactors Cell culture Chromosomes Escherichia coli Ethanol Gene expression Glycerol Maintenance Metabolism Organic polymers Microbiology alcohol alcohol acetaldehyde dehydrogenase aldehyde dehydrogenase glycerol nicotinamide adenine dinucleotide reduced nicotinamide adenine dinucleotide unclassified drug biochemistry bioreactor chromosome coliform bacterium DNA enzyme ethanol gene expression metabolism mutagenicity plasmid article bacterial chromosome bacterial gene bacterium culture bioreactor biosynthesis controlled study Escherichia coli Leuconostoc mesenteroides mutant nonhuman oxidation oxidation reduction reaction physiology plasmid Alcohol Dehydrogenase Aldehyde Oxidoreductases Escherichia coli Leuconostoc Mutation Oxidation-Reduction Plasmids Selection, Genetic Arca Bacteria (microorganisms) Escherichia coli Leuconostoc mesenteroides Several biotechnological processes rely on the utilization of high-copy-number plasmids for heterologous gene expression, and understanding the interactions between plasmid DNA and bacterial hosts is highly relevant for bioprocess optimization. We assessed metabolic modifications and physiological changes exerted by expression of a plasmid-encoded alcohol-acetaldehyde dehydrogenase from Leuconostoc mesenteroides (adhE Lm ) in Escherichia coli redox mutants. Plasmid pET Lm, a pBluescript II KS(-)-derivative carrying adhE Lm, was introduced in E. coli CT1061 [arcA creC(Con)]. This recombinant was able to attain a higher ethanol concentration in glycerol cultures compared to the parental strain. pBluescript II KS(-) was rapidly lost in 72-h bioreactor cultures (7.8±1.2% of plasmid-bearing cells), while pET Lm was present in 92.4±7.2% of the cells. In E. coli CT1061 carrying pBluescript II KS(-) the plasmid copy number steadily diminished in bioreactor cultures to reach 334±45 copies per chromosome at 72 h, while pET Lm was stably maintained, reaching 498±18 copies per chromosome at the end of the cultivation. Plasmid pETΩ Lm, bearing a defective copy of adhE Lm interrupted by cat, reached 293±62 copies per chromosome, implying a functional role of adhE Lm on plasmid maintenance. The intracellular NADH/NAD+ content suggest that regeneration of oxidized co-factors by the heterologous bioreaction might play a relevant role in plasmid maintenance. © 2010 Springer-Verlag. Fil:Pettinari, M.J. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Galvagno, M.A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Méndez, B.S. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. 2010 https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_01757598_v88_n2_p563_Nikel http://hdl.handle.net/20.500.12110/paper_01757598_v88_n2_p563_Nikel |
| institution |
Universidad de Buenos Aires |
| institution_str |
I-28 |
| repository_str |
R-134 |
| collection |
Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) |
| topic |
ArcAB CreBC Escherichia coli Microaerobiosis Plasmid copy number Redox mutants ArcAB Copy number CreBC Microaerobiosis Redox mutants Aldehydes Bearings (structural) Bioconversion Bioreactors Cell culture Chromosomes Escherichia coli Ethanol Gene expression Glycerol Maintenance Metabolism Organic polymers Microbiology alcohol alcohol acetaldehyde dehydrogenase aldehyde dehydrogenase glycerol nicotinamide adenine dinucleotide reduced nicotinamide adenine dinucleotide unclassified drug biochemistry bioreactor chromosome coliform bacterium DNA enzyme ethanol gene expression metabolism mutagenicity plasmid article bacterial chromosome bacterial gene bacterium culture bioreactor biosynthesis controlled study Escherichia coli Leuconostoc mesenteroides mutant nonhuman oxidation oxidation reduction reaction physiology plasmid Alcohol Dehydrogenase Aldehyde Oxidoreductases Escherichia coli Leuconostoc Mutation Oxidation-Reduction Plasmids Selection, Genetic Arca Bacteria (microorganisms) Escherichia coli Leuconostoc mesenteroides |
| spellingShingle |
ArcAB CreBC Escherichia coli Microaerobiosis Plasmid copy number Redox mutants ArcAB Copy number CreBC Microaerobiosis Redox mutants Aldehydes Bearings (structural) Bioconversion Bioreactors Cell culture Chromosomes Escherichia coli Ethanol Gene expression Glycerol Maintenance Metabolism Organic polymers Microbiology alcohol alcohol acetaldehyde dehydrogenase aldehyde dehydrogenase glycerol nicotinamide adenine dinucleotide reduced nicotinamide adenine dinucleotide unclassified drug biochemistry bioreactor chromosome coliform bacterium DNA enzyme ethanol gene expression metabolism mutagenicity plasmid article bacterial chromosome bacterial gene bacterium culture bioreactor biosynthesis controlled study Escherichia coli Leuconostoc mesenteroides mutant nonhuman oxidation oxidation reduction reaction physiology plasmid Alcohol Dehydrogenase Aldehyde Oxidoreductases Escherichia coli Leuconostoc Mutation Oxidation-Reduction Plasmids Selection, Genetic Arca Bacteria (microorganisms) Escherichia coli Leuconostoc mesenteroides Pettinari, María Julia Galvagno, Miguel Angel Méndez, Beatriz Silvia Metabolic selective pressure stabilizes plasmids carrying biosynthetic genes for reduced biochemicals in Escherichia coli redox mutants |
| topic_facet |
ArcAB CreBC Escherichia coli Microaerobiosis Plasmid copy number Redox mutants ArcAB Copy number CreBC Microaerobiosis Redox mutants Aldehydes Bearings (structural) Bioconversion Bioreactors Cell culture Chromosomes Escherichia coli Ethanol Gene expression Glycerol Maintenance Metabolism Organic polymers Microbiology alcohol alcohol acetaldehyde dehydrogenase aldehyde dehydrogenase glycerol nicotinamide adenine dinucleotide reduced nicotinamide adenine dinucleotide unclassified drug biochemistry bioreactor chromosome coliform bacterium DNA enzyme ethanol gene expression metabolism mutagenicity plasmid article bacterial chromosome bacterial gene bacterium culture bioreactor biosynthesis controlled study Escherichia coli Leuconostoc mesenteroides mutant nonhuman oxidation oxidation reduction reaction physiology plasmid Alcohol Dehydrogenase Aldehyde Oxidoreductases Escherichia coli Leuconostoc Mutation Oxidation-Reduction Plasmids Selection, Genetic Arca Bacteria (microorganisms) Escherichia coli Leuconostoc mesenteroides |
| description |
Several biotechnological processes rely on the utilization of high-copy-number plasmids for heterologous gene expression, and understanding the interactions between plasmid DNA and bacterial hosts is highly relevant for bioprocess optimization. We assessed metabolic modifications and physiological changes exerted by expression of a plasmid-encoded alcohol-acetaldehyde dehydrogenase from Leuconostoc mesenteroides (adhE Lm ) in Escherichia coli redox mutants. Plasmid pET Lm, a pBluescript II KS(-)-derivative carrying adhE Lm, was introduced in E. coli CT1061 [arcA creC(Con)]. This recombinant was able to attain a higher ethanol concentration in glycerol cultures compared to the parental strain. pBluescript II KS(-) was rapidly lost in 72-h bioreactor cultures (7.8±1.2% of plasmid-bearing cells), while pET Lm was present in 92.4±7.2% of the cells. In E. coli CT1061 carrying pBluescript II KS(-) the plasmid copy number steadily diminished in bioreactor cultures to reach 334±45 copies per chromosome at 72 h, while pET Lm was stably maintained, reaching 498±18 copies per chromosome at the end of the cultivation. Plasmid pETΩ Lm, bearing a defective copy of adhE Lm interrupted by cat, reached 293±62 copies per chromosome, implying a functional role of adhE Lm on plasmid maintenance. The intracellular NADH/NAD+ content suggest that regeneration of oxidized co-factors by the heterologous bioreaction might play a relevant role in plasmid maintenance. © 2010 Springer-Verlag. |
| author |
Pettinari, María Julia Galvagno, Miguel Angel Méndez, Beatriz Silvia |
| author_facet |
Pettinari, María Julia Galvagno, Miguel Angel Méndez, Beatriz Silvia |
| author_sort |
Pettinari, María Julia |
| title |
Metabolic selective pressure stabilizes plasmids carrying biosynthetic genes for reduced biochemicals in Escherichia coli redox mutants |
| title_short |
Metabolic selective pressure stabilizes plasmids carrying biosynthetic genes for reduced biochemicals in Escherichia coli redox mutants |
| title_full |
Metabolic selective pressure stabilizes plasmids carrying biosynthetic genes for reduced biochemicals in Escherichia coli redox mutants |
| title_fullStr |
Metabolic selective pressure stabilizes plasmids carrying biosynthetic genes for reduced biochemicals in Escherichia coli redox mutants |
| title_full_unstemmed |
Metabolic selective pressure stabilizes plasmids carrying biosynthetic genes for reduced biochemicals in Escherichia coli redox mutants |
| title_sort |
metabolic selective pressure stabilizes plasmids carrying biosynthetic genes for reduced biochemicals in escherichia coli redox mutants |
| publishDate |
2010 |
| url |
https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_01757598_v88_n2_p563_Nikel http://hdl.handle.net/20.500.12110/paper_01757598_v88_n2_p563_Nikel |
| work_keys_str_mv |
AT pettinarimariajulia metabolicselectivepressurestabilizesplasmidscarryingbiosyntheticgenesforreducedbiochemicalsinescherichiacoliredoxmutants AT galvagnomiguelangel metabolicselectivepressurestabilizesplasmidscarryingbiosyntheticgenesforreducedbiochemicalsinescherichiacoliredoxmutants AT mendezbeatrizsilvia metabolicselectivepressurestabilizesplasmidscarryingbiosyntheticgenesforreducedbiochemicalsinescherichiacoliredoxmutants |
| _version_ |
1768546440453292032 |