PMID:17619876
| Citation |
De Muynck, C, Van der Borght, J, De Mey, M, De Maeseneire, SL, Van Bogaert, IN, Beauprez, J, Soetaert, W and Vandamme, E (2007) Development of a selection system for the detection of L-ribose isomerase expressing mutants of Escherichia coli. Appl. Microbiol. Biotechnol. 76:1051-7 |
|---|---|
| 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. |
| Links |
PubMed Online version:10.1007/s00253-007-1084-8 |
| Keywords |
Aldose-Ketose Isomerases/genetics; Aldose-Ketose Isomerases/metabolism; Arabinose/metabolism; Biotechnology/methods; Cloning, Molecular; Culture Media; Directed Molecular Evolution; Escherichia coli/classification; Escherichia coli/enzymology; Escherichia coli/genetics; Escherichia coli/growth & development; Isomerism; Mutation; Selection, Genetic |
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