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Construction of a butyrate-producing E. coli strain without the use of heterologous genes

Construction of a butyrate-producing E. coli strain without the use of heterologous genes Multistage construction of an E. coli strain containing no foreign genes which is capable of producing butyrate has been carried out. At the first stage, deletions in gene fadR encoding a protein repressor of an operon for fatty acid degradation and gene aceF responsible for the synthesis of pyruvate dehydrogenase were introduced in the strain MG1655 genome. Then, a mutant obtained from the above strain by induced mutagenesis and capable of growth on ethanol as a sole carbon source under aerobic conditions was selected. It was shown that growth of the mutant on ethanol is provided by two mutations. One of them (a substitution: 257G → A) is located in the regulatory region of gene adhE that controls the synthesis of alcohol-dehydrogenase; the other, containing a substitution Glu568 → Lys, affects the structural portion of the gene. As a result of the consequent mutagenesis of the obtained strain and selection on indicating media, variants capable of growing on butyrate and butanol as sole carbon sources and putatively bearing mutations in gene atoC (encoding transcriptional activator of atoDAB operon) were selected. At the last stage of the work, gene atoB, encoding the synthesis of the thiolase II enzyme, was placed under the control of a constitutive promoter P tet , and the functional allele of gene aceF was introduced. The resulting E. coli strain (ΔfadR, adhE, atoC, P tet -atoB) accumulates 800 mg/l of butyrate upon growth on glucose-containing medium under semi-anaerobic (oxygen limited) conditions. Introduction of an additional deletion in gene ldhA encoding lactate dehydrogenase in the strain genome leads to a further growth of a butyrate production up to 1.3 g/l. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Applied Biochemistry and Microbiology Springer Journals

Construction of a butyrate-producing E. coli strain without the use of heterologous genes

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References (26)

Publisher
Springer Journals
Copyright
Copyright © 2010 by Pleiades Publishing, Ltd.
Subject
Life Sciences; Medical Microbiology ; Microbiology ; Biochemistry, general
ISSN
0003-6838
eISSN
1608-3024
DOI
10.1134/S000368381008003X
Publisher site
See Article on Publisher Site

Abstract

Multistage construction of an E. coli strain containing no foreign genes which is capable of producing butyrate has been carried out. At the first stage, deletions in gene fadR encoding a protein repressor of an operon for fatty acid degradation and gene aceF responsible for the synthesis of pyruvate dehydrogenase were introduced in the strain MG1655 genome. Then, a mutant obtained from the above strain by induced mutagenesis and capable of growth on ethanol as a sole carbon source under aerobic conditions was selected. It was shown that growth of the mutant on ethanol is provided by two mutations. One of them (a substitution: 257G → A) is located in the regulatory region of gene adhE that controls the synthesis of alcohol-dehydrogenase; the other, containing a substitution Glu568 → Lys, affects the structural portion of the gene. As a result of the consequent mutagenesis of the obtained strain and selection on indicating media, variants capable of growing on butyrate and butanol as sole carbon sources and putatively bearing mutations in gene atoC (encoding transcriptional activator of atoDAB operon) were selected. At the last stage of the work, gene atoB, encoding the synthesis of the thiolase II enzyme, was placed under the control of a constitutive promoter P tet , and the functional allele of gene aceF was introduced. The resulting E. coli strain (ΔfadR, adhE, atoC, P tet -atoB) accumulates 800 mg/l of butyrate upon growth on glucose-containing medium under semi-anaerobic (oxygen limited) conditions. Introduction of an additional deletion in gene ldhA encoding lactate dehydrogenase in the strain genome leads to a further growth of a butyrate production up to 1.3 g/l.

Journal

Applied Biochemistry and Microbiology – Springer Journals

Published: Nov 14, 2010

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