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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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Chicago
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.
APA
De Muynck, Cassandra, 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.
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.
MLA
De Muynck, Cassandra, Jef Van der Borght, Marjan De Mey, 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.
@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},
  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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