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Development of a selection system for the detection of L-ribose isomerase expressing mutants of Escherichia coli

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Abstract
L-Arabinose isomerase (E.C. 5.3.1.14) catalyzes the reversible isomerization between L-arabinose and L-ribulose and is highly selective towards L-arabinose. By using a directed evolution approach, enzyme variants with altered substrate specificity were created and screened in this research. More specifically, the screening was directed towards the identification of isomerase mutants with L-ribose isomerizing activity. Random mutagenesis was performed on the Escherichia coli L-arabinose isomerase gene (araA) by error-prone polymerase chain reaction to construct a mutant library. To enable screening of this library, a selection host was first constructed in which the mutant genes were transformed. In this selection host, the genes encoding for L-ribulokinase and L-ribulose-5-phosphate-4-epimerase were brought to constitutive expression and the gene encoding for the native L-arabinose isomerase was knocked out. L-Ribulokinase and L-ribulose-5-phosphate-4-epimerase are necessary to ensure the channeling of the formed product, L-ribulose, to the pentose phosphate pathway. Hence, the mutant clones could be screened on a minimal medium with L-ribose as the sole carbon source. Through the screening, two first-generation mutants were isolated, which expressed a small amount of L-ribose isomerase activity.
Keywords
Screening, Gene disruption, Directed evolution, L-arabinose isomerase, L-ribose isomerase, RIBITOL, OPERON, GENES, K-12, L-ARABINOSE ISOMERASE

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Citation

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MLA
De Muynck, Cassandra et al. “Development of a Selection System for the Detection of L-ribose Isomerase Expressing Mutants of Escherichia Coli.” APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 76.5 (2007): 1051–1057. Print.
APA
De Muynck, C., Van der Borght, J., De Mey, M., De Maeseneire, S., Van Bogaert, I., Beauprez, J., Soetaert, W., et al. (2007). Development of a selection system for the detection of L-ribose isomerase expressing mutants of Escherichia coli. APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 76(5), 1051–1057.
Chicago author-date
De Muynck, Cassandra, Jef Van der Borght, Marjan De Mey, Sofie De Maeseneire, Inge Van Bogaert, Joeri Beauprez, Wim Soetaert, and Erick Vandamme. 2007. “Development of a Selection System for the Detection of L-ribose Isomerase Expressing Mutants of Escherichia Coli.” Applied Microbiology and Biotechnology 76 (5): 1051–1057.
Chicago author-date (all authors)
De Muynck, Cassandra, Jef Van der Borght, Marjan De Mey, Sofie De Maeseneire, Inge Van Bogaert, Joeri Beauprez, Wim Soetaert, and Erick Vandamme. 2007. “Development of a Selection System for the Detection of L-ribose Isomerase Expressing Mutants of Escherichia Coli.” Applied Microbiology and Biotechnology 76 (5): 1051–1057.
Vancouver
1.
De Muynck C, Van der Borght J, De Mey M, De Maeseneire S, Van Bogaert I, Beauprez J, et al. Development of a selection system for the detection of L-ribose isomerase expressing mutants of Escherichia coli. APPLIED MICROBIOLOGY AND BIOTECHNOLOGY. 2007;76(5):1051–7.
IEEE
[1]
C. De Muynck et al., “Development of a selection system for the detection of L-ribose isomerase expressing mutants of Escherichia coli,” APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, vol. 76, no. 5, pp. 1051–1057, 2007.
@article{387816,
  abstract     = {L-Arabinose isomerase (E.C. 5.3.1.14) catalyzes the reversible isomerization between L-arabinose and L-ribulose and is highly selective towards L-arabinose. By using a directed evolution approach, enzyme variants with altered substrate specificity were created and screened in this research. More specifically, the screening was directed towards the identification of isomerase mutants with L-ribose isomerizing activity. Random mutagenesis was performed on the Escherichia coli L-arabinose isomerase gene (araA) by error-prone polymerase chain reaction to construct a mutant library. To enable screening of this library, a selection host was first constructed in which the mutant genes were transformed. In this selection host, the genes encoding for L-ribulokinase and L-ribulose-5-phosphate-4-epimerase were brought to constitutive expression and the gene encoding for the native L-arabinose isomerase was knocked out. L-Ribulokinase and L-ribulose-5-phosphate-4-epimerase are necessary to ensure the channeling of the formed product, L-ribulose, to the pentose phosphate pathway. Hence, the mutant clones could be screened on a minimal medium with L-ribose as the sole carbon source. Through the screening, two first-generation mutants were isolated, which expressed a small amount of L-ribose isomerase activity.},
  author       = {De Muynck, Cassandra and Van der Borght, Jef and De Mey, Marjan and De Maeseneire, Sofie and Van Bogaert, Inge and Beauprez, Joeri and Soetaert, Wim and Vandamme, Erick},
  issn         = {0175-7598},
  journal      = {APPLIED MICROBIOLOGY AND BIOTECHNOLOGY},
  keywords     = {Screening,Gene disruption,Directed evolution,L-arabinose isomerase,L-ribose isomerase,RIBITOL,OPERON,GENES,K-12,L-ARABINOSE ISOMERASE},
  language     = {eng},
  number       = {5},
  pages        = {1051--1057},
  title        = {Development of a selection system for the detection of L-ribose isomerase expressing mutants of Escherichia coli},
  url          = {http://dx.doi.org/10.1007/s00253-007-1084-8},
  volume       = {76},
  year         = {2007},
}

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