Development of a selection system for the detection of L-ribose isomerase expressing mutants of Escherichia coli.
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.
De Muynck
Cassandra
C
Pharmadvize, Kleimoer 4, B-9030, Mariakerke, Belgium. cdmuynck@hotmail.com
Van der Borght
Jef
J
De Mey
Marjan
M
De Maeseneire
Sofie L
SL
Van Bogaert
Inge N A
IN
Beauprez
Joeri
J
Soetaert
Wim
W
Vandamme
Erick
E
D019747:Aldose-Ketose Isomerases / Q000235:genetics / Q000378:metabolism*; D001089:Arabinose / Q000378:metabolism; D001709:Biotechnology / Q000379:methods; D003001:Cloning, Molecular; D003470:Culture Media; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000145:classification / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D007536:Isomerism; D009154:Mutation; D012641:Selection, Genetic
D003470:Culture Media; D001089:Arabinose; D019747:Aldose-Ketose Isomerases; C022546:ribose isomerase