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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.


Documento: Artículo
Título:Metabolic selective pressure stabilizes plasmids carrying biosynthetic genes for reduced biochemicals in Escherichia coli redox mutants
Autor:Nikel, P.I.; Pettinari, M.J.; Galvagno, M.A.; Méndez, B.S.
Filiación:Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Av. Intendente Güiraldes 2160, C1428EGA Ciudad Autónoma de Buenos Aires, Argentina
Instituto de Investigaciones Biotecnológicas, Universidad Nacional de San Martín, Av. General Paz 5445, San Martín, Buenos Aires B1650KNA, Argentina
Departamento de Ingeniería Química, Facultad de Ingeniería, Universidad de Buenos Aires, Av. Intendente Güiraldes 2160, C1428EGA Ciudad Autónoma de Buenos Aires, Argentina
Palabras clave: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
Página de inicio:563
Página de fin:573
Título revista:Applied Microbiology and Biotechnology
Título revista abreviado:Appl. Microbiol. Biotechnol.
CAS:alcohol, 64-17-5; aldehyde dehydrogenase, 37353-37-0, 9028-86-8; glycerol, 56-81-5; nicotinamide adenine dinucleotide, 53-84-9; reduced nicotinamide adenine dinucleotide, 58-68-4; Alcohol Dehydrogenase,; Aldehyde Oxidoreductases, 1.2.-; acetaldehyde dehydrogenase,


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---------- APA ----------
Nikel, P.I., Pettinari, M.J., Galvagno, M.A. & Méndez, B.S. (2010) . Metabolic selective pressure stabilizes plasmids carrying biosynthetic genes for reduced biochemicals in Escherichia coli redox mutants. Applied Microbiology and Biotechnology, 88(2), 563-573.
---------- CHICAGO ----------
Nikel, P.I., Pettinari, M.J., Galvagno, M.A., Méndez, B.S. "Metabolic selective pressure stabilizes plasmids carrying biosynthetic genes for reduced biochemicals in Escherichia coli redox mutants" . Applied Microbiology and Biotechnology 88, no. 2 (2010) : 563-573.
---------- MLA ----------
Nikel, P.I., Pettinari, M.J., Galvagno, M.A., Méndez, B.S. "Metabolic selective pressure stabilizes plasmids carrying biosynthetic genes for reduced biochemicals in Escherichia coli redox mutants" . Applied Microbiology and Biotechnology, vol. 88, no. 2, 2010, pp. 563-573.
---------- VANCOUVER ----------
Nikel, P.I., Pettinari, M.J., Galvagno, M.A., Méndez, B.S. Metabolic selective pressure stabilizes plasmids carrying biosynthetic genes for reduced biochemicals in Escherichia coli redox mutants. Appl. Microbiol. Biotechnol. 2010;88(2):563-573.