title: Improvement of the production of foreign proteins using a heterologous secretion vector system in Bacillus subtilis: effects of resistance to glucose-mediated catabolite repression.
abstract: To improve protein production, a heterologous secretion vector system was constructed with the aid of the amyR2 region. The operator sequence (amyO) of the amyR2 region on the secretion vector was changed through site-directed mutagenesis to eliminate carbon-source-mediated catabolite repression. Three substitutional (AG, G5, G10), one deletional (delta HH), and one insertional (AGHF) mutant promoters were obtained. The expression level and the degree of catabolite repression of amyR2 and the mutant promoters were examined with a single copy system using an integrational promoter probe vector, pDH32. Under glucose-free culture conditions, expression levels from all mutant promoters except HH were 1.4 to 1.5 fold higher than that from amyR2. While the expression of the amyR2 promoter was repressed by 90% in the presence of 2% glucose, expression levels of the mutant promoters were repressed by only 1% to 50%. To evaluate the advantage of the mutant promoters in production of foreign proteins by the heterologous secretion system, beta-lactamase and human pancreatic secretory trypsin inhibitor (hPSTI) were expressed by the mutant promoters. When B. subtilis LKS87 was used as a host strain, the production of the target proteins using the respective mutant promoter was increased by about 1.5 fold under glucose-free culture conditions. Under the high glucose culture conditions, secretion of target proteins produced from the mutant promoters increased 1.5 to 2 fold, whereas those by the amyR2 promoter were reduced to between 50% and 60%. The additive effect of degUh mutation on protein production was not observed under high glucose culture conditions. In addition, such culture conditions inhibited proteolytic degradation of secreted target proteins after the stationary growth phase even in B. subtilis LKS88 (degUh mutant). Thus, our results indicated that the mutant promoters, which are resistant to glucose-mediated catabolite repression, are very useful for over-production of foreign proteins under the high glucose culture conditions using the heterologous expression-secretion system in B. subtilis.
authors.affiliation: Graduate School of Biotechnology, Korea University, Seoul, Korea.
authors.forename: S I
authors.initials: SI
authors.lastname: Kim
authors.forename: Y S
authors.initials: YS
authors.lastname: Nam
authors.forename: S Y
authors.initials: SY
authors.lastname: Lee
mesh_terms: D001412:Bacillus subtilis / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics*; D005798:Genes, Bacterial / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D005947:Glucose / Q000494:pharmacology*; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification*; D014170:Transformation, Genetic / Q000235:genetics; D014361:Trypsin Inhibitors / Q000235:genetics / Q000302:isolation & purification; D001616:beta-Galactosidase / Q000378:metabolism; D001618:beta-Lactamases / Q000235:genetics / Q000302:isolation & purification
chemical_list: D011994:Recombinant Proteins; D014361:Trypsin Inhibitors; D001616:beta-Galactosidase; D001618:beta-Lactamases; D005947:Glucose