Mutant aprE promoter
The present invention provides a mutant aprE promoter and methods for the production of a desired protein in a Bacillus host cell, which comprises the mutant aprE promoter. The present invention provides the sequence of a preferred aprE mutant promoter, which provided for a 100-fold increase in the production of a protein from Bacillus subtilis.
1. A method for producing a desired protein in a Bacillus comprising the steps of culturing a Bacillus comprising nucleic acid encoding the desired protein, wherein said Bacillus further comprises an isolated mutant aprE promoter, wherein said mutant aprE promoter comprises a modification in any of the nucleotides of the −35 consensus box of the wild-type promoter as illustrated in FIG. 1 , and further wherein said mutant aprE promoter is operatively linked to said nucleic acid encoding said desired protein, and optionally recovering the protein.
2. The method of claim 1 wherein said Bacillus is selected from the group consisting of B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, amyloliquefaciens, B. coagulans, B. circulans, B. lautus , and B. thuringiensis.
3. The method of claim 1 wherein said desired protein is selected from the group consisting of proteases, cellulases, amylases, carbohydrases, lipases, isomerases, racemases, epimerases, tautomerases, mutases, transferases, kinases, and phosphatases.
4. The method of claim 2 wherein said nucleic acid is integrated into the genome of said Bacillus.
5. The method of claim 2 wherein said nucleic acid is on a replicating plasmid.
6. The method of claim 1 wherein said Bacillus is B. subtilis and said desired protein is a protease.
7. The method of claim 6 wherein said protease is subtilisin.
8. The method of claim 1 wherein said isolated mutant aprE promoter comprises the sequence set forth in SEQ ID NO:1.