Sunday, August 10, 2008
P5

Utilization of a Novel Lysine Racemase Gene as a Selectable Marker for Microorganisms transformation

I-Chieh Chen1, Shih-Kuang Hsu2, and Wen-Hwei Hsu1. (1) Institute of Molecular Biology, National Chung Hsing University, 250, Kuo Kuang Rd., Taichung 402, Taiwan, (2) Department of Dental Laboratory Technology, Central Taiwan University of Science and Technology, No.11, Buzih Lane, Beitun District, Taichung 406, Taiwan

Genetic engineering of microorganisms is highly dependent on the application of antibiotic resistance genes. Transfromants harboring antibiotic resistance genes may cause the risk of spread of antibiotic resistance traits to environmental microbes. In this study, we reported a novel selection marker for transformation in which lysine racemase (lyr)gene instead of drug-resistant gene is used as selectable marker and wild-type strains can be used as host cells for gene transformation.

Lysine racemase gene ( lyr ) was screened from soil metagenomic libraries. The lyr was 1,181 bp long; the molecular mass of the translated protein was estimated to be 43,068Da. The putative lyr gene was overexpressed and purified for characterization. Optimal conditions for the enzyme activity of Lyr protein were 30 °C and pH 8.0. Lyr can catalyze the racemization of L- and D-lysine. The Km and kcat values of the Lyr for the racemization of L-lysine to the D-form and the conversion of D-lysine to the L-form were measured as 16.21 mM and 0.75 min-1 or 23.48 mM and 1.12 min-1, respectively. Plasmid harboring lyr gene was transformed into E. coli BCRC 51734, E. coli BL21 (DE3), Aspergillus oryzae BCRC 30129, Bacillus subtilis, and Bacillus circulans and transformants could grow in the medium containing D-lysine as sole nitrogen source. Our data indicated that the lyr gene can be used as a selectable marker for the transformation of microorganisms.


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