Development of marker-free strains of Bacillus subtilis capable of secreting high levels of industrial enzymes

Different strategies have been employed to achieve high-level expression of single-copy genes encoding secreted enzymes in Bacillus subtilis, A model system was developed which utilizes the aprL gene from Bacillus clausii as a reporter gene for monitoring expression levels during stationary phase. A n exceptionally strong promoter was constructed by altering the nuceotide s equence in the -10 and -35 regions of the promoter for the amyQ gene of Bac illus amyloliquefaciens. In addition, two or three tandem copies of this pr omoter were shown to increase expression levels substantially in comparison to the monomer promoter alone. Finally, the promoter and mRNA stabilizatio n sequences derived from the cry3A gene of Bacillus thuringiensis were used in combination with the mutant amyQ promoter to achieve the highest levels of aprL expression, These promoters were shown to be fully functional in a high-expressing Bacillus strain grown under industrial fermentation condit ions. The ability to obtain maximum expression levels from a single copy ge ne now makes it feasible to construct environmentally friendly, marker-free industrial strains of B. subtilis.