Expression of bacillar glutamyl endopeptidase genes in Bacillus subtilis by a new mobilizable single-replicon vector pLF

The pLF1311 natural plasmid from Lactobacillus fermentum 1311 was used to c onstruct a single-replicon vector suitable for rapid cloning in a wide rang e of gram-positive hosts and Escherichia coli. The new vector is capable of conjugative mobilization from E. coli to various hosts by conjugal transfe r. The final vector (3.4 kb) showed a high segregational and structural sta bility and a high copy number. Glutamyl endopeptidase genes from Bacillus l icheniformis (gseBL) and B. intermedius (gseBI) were cloned in both pLF9 an d pLF14 vectors and introduced to B. subtilis. The yield of enzymes in the pLF-derived producers was 6- to 30-fold more than in the natural producers and reached 100-150 mg/L of mature protease. (C) 2000 Academic Press.