Production of Recombinant α-Galactosidases in Thermus thermophilus
Open Access
- 1 September 2001
- journal article
- Published by American Society for Microbiology in Applied and Environmental Microbiology
- Vol. 67 (9) , 4192-4198
- https://doi.org/10.1128/aem.67.9.4192-4198.2001
Abstract
A Thermus thermophilus selector strain for production of thermostable and thermoactive α-galactosidase was constructed. For this purpose, the native α-galactosidase gene ( agaT ) of T. thermophilus TH125 was inactivated to prevent background activity. In our first attempt, insertional mutagenesis of agaT by using a cassette carrying a kanamycin resistance gene led to bacterial inability to utilize melibiose (α-galactoside) and galactose as sole carbohydrate sources due to a polar effect of the insertional inactivation. A Gal + phenotype was assumed to be essential for growth on melibiose. In a Gal − background, accumulation of galactose or its metabolite derivatives produced from melibiose hydrolysis could interfere with the growth of the host strain harboring recombinant α-galactosidase. Moreover, the AgaT − strain had to be Km s for establishment of the plasmids containing α-galactosidase genes and the kanamycin resistance marker. Therefore, a suitable selector strain (AgaT − Gal + Km s ) was generated by applying integration mutagenesis in combination with phenotypic selection. To produce heterologous α-galactosidase in T. thermophilus , the isogenes agaA and agaB of Bacillus stearothermophilus KVE36 were cloned into an Escherichia coli-Thermus shuttle vector. The region containing the E. coli plasmid sequence (pUC-derived vector) was deleted before transformation of T. thermophilus with the recombinant plasmids. As a result, transformation efficiency and plasmid stability were improved. However, growth on minimal agar medium containing melibiose was achieved only following random selection of the clones carrying a plasmid-based mutation that had promoted a higher copy number and greater stability of the plasmid.Keywords
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