Development of a selection system for the detection of L-ribose isomerase expressing mutants of <Emphasis Type="Italic">Escherichia coli</Emphasis> |
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Authors: | Cassandra De Muynck Jef Van der Borght Marjan De Mey Sofie L De Maeseneire Inge N A Van Bogaert Joeri Beauprez Wim Soetaert Erick Vandamme |
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Institution: | (1) Laboratory of Industrial Microbiology and Biocatalysis, Department of Biochemical and Microbial Technology,Faculty of Bioscience Engineering, Ghent University, Coupure links 653, B-9000 Ghent, Belgium;(2) Present address: Pharmadvize, Kleimoer 4, B-9030 Mariakerke, Belgium |
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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. |
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Keywords: | L-arabinose isomerase L-ribose isomerase Directed evolution Screening Gene disruption |
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