Production of recombinant α-galactosidases in Thermus thermophilus

TLDR: A Thermus thermophilus selector strain for production of thermostable and thermoactive α-galactosidase was constructed by applying integration mutagenesis in combination with phenotypic selection and growth on minimal agar medium containing melibiose was achieved.

Abstract: α-Galactosidases catalyze the hydrolysis of α-1,6-linked α-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 α-galactosidases. We have been studying α-galactosidases from various bacteria with regard to their application as oligosaccharide-hydrolyzing enzymes (9, 11, 14). Our intention was to subject α-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 (α-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 application of autonomously replicating plasmids, as well as integration vectors or vectors containing cassettes, for chromosomal integration have been established (13, 18, 22, 23, 35, 40). So far, the only antibiotic selection markers described for Thermus bacteria are thermostabilized variants of the kanamycin nucleotidyltransferase gene derived from a thermophilic Bacillus gene (28) or from the kan gene of Staphylococcus aureus (24). Expression of heterologous genes requires the inactivation of analogous genes in the host strain. In our previous work (10), we cloned the α-galactosidase gene (agaT) from T. thermophilus TH125 into Escherichia coli and subsequently disrupted the gene by site-specific integration of the kanamycin resistance marker into the agaT locus in the T. thermophilus chromosome. Sequence analysis of agaT along with flanking sequences revealed an open reading frame (ORF) downstream of and overlapping the agaT gene. The predicted translation product displayed similarity to galactose-1-phosphate uridylyltransferases (GalT) of E. coli (2) and Streptomyces lividans (1). The 3′ region of agaT and the 5′ region of galT were left intact in the site-specific integration due to the overlapping coding regions. However, characterization of the integration mutants revealed their inability to use melibiose as well as galactose. This indicated a polar transcriptional effect on the downstream galT gene. The polar effect was considered an obstruction for our purpose due to a potential growth inhibition effect of the accumulated galactose (or its metabolite derivatives) produced from melibiose hydrolysis in Gal− strains harboring recombinant α-galactosidases. Interference with growth by galactose has been described for Gal− mutants of, e.g., E. coli (30) and Bacillus subtilis (20). In this paper, we describe the establishment of a Thermus strain suitable for production of heterologous α-galactosidases. Thereby, two problems were circumvented which restricted the use of the previously constructed agaT deletion strains (10): the galactose-negative phenotype and their kanamycin resistance, which otherwise prevented plasmid selection with the kanamycin marker. Further, we demonstrate the practical value of the strain established in this work for the production of recombinant α-galactosidases and as a potential selection system for α-galactosidases active at high temperatures.