Production of Recombinant α-Galactosidases in Thermus thermophilus

Abstract

A Thermus thermophilus selector strain for production of thermostable and thermoactive ␣-galactosidase was constructed. For this purpose, the native ␣-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 (␣-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 ␣-galactosidase. Moreover, the AgaT ؊ strain had to be Km s for establishment of the plasmids containing ␣-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 ␣-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.␣-Galactosidases catalyze the hydrolysis of ␣-1,6-linked ␣-galactose residues from oligosaccharides and polymeric galactomannans (9,25,26,42). They have considerable potential in various industrial applications, e.g., in the sugar industry for the elimination of D-raffinose from sugar beet syrup. Due to the elevated temperatures used during the sugar manufacturing process, as well as in other industrial applications, stability and activity at high temperatures are important properties of ␣-galactosidases.We have been studying ␣-galactosidases from various bacteria with regard to their application as oligosaccharidehydrolyzing enzymes (9,11,14). Our intention was to subject ␣-galactosidase to thermoadaptation by introducing genes encoding enzymes inactive at high temperatures into a thermophilic bacterium for subsequent selection of enzyme variants active at high temperatures. We chose Thermus thermophilus as a host due to its high transformation ability (17) and ability to use melibiose (␣-galactoside) as a sole carbohydrate source (10). Thermus species have been used for expression of heterologous genes and selection of thermostable enzyme variants (16,19,34,36). They possess a natural transformation system (17) and are competent regardless of their growth phase (12). Genetic systems based on the applic...

Thermoadaptation of α-Galactosidase AgaB1 in Thermus thermophilus

“…The resulting chimeric gene encoded an enzyme with the characteristic phenotypes of AgaA. The construction of pOF10726 is described elsewhere (8). The plasmid contains a stable plasmid mutant of pOF5714 (8) in a pUC19 derivative.…”

“…Growth was carried out at 65°C under nonselective conditions and at 60°C when cultures contained kanamycin (20 g ml Ϫ1 ) for plasmid selection. Growth on a single carbon source (melibiose, 0.1, 0.2, and 0.4%) was tested on agar plates containing the minimal medium 162 (4) with a slight modification as previously described (8). The method of Koyama et al (14) was used for the transformation of T. thermophilus, with a slight modification as previously described (7).…”

“…Hybrid genes, based on agaA and agaB, of B. stearothermophilus KVE39 were cloned into a Thermus autonomously replicating plasmid, downstream of the E. coli tac promoter (1) and a ribosome binding site from T. thermophilus (8). The construction of plasmid pOF2811, carrying an AgaA type encoding gene, agaA1, which served as a positive control, was as follows.…”

“…It was constructed by replacing the kan gene of pOF1155 (8), which contains the marker between flanking sequences of the native ␣-galactosidase gene agaT (7), with agaA1. Due to an NdeI restriction site in agaA, a three-fragment ligation was performed with the following: the agaA gene following amplification (8) and NdeI-HindIII digestion; agaB, following amplification (8) and HindIII-BglII digestion; and the pOF1155 following NdeI-BamHI digestion. The resulting chimeric gene encoded an enzyme with the characteristic phenotypes of AgaA.…”

“…A suitable host strain for a selection of thermostable ␣-galactosidases active at elevated temperatures was constructed by applying integration mutagenesis in combination with phenotypic selection and designated T. thermophilus OF1053GD, as previously described (8).…”