An acetoin-regulated expression system of Bacillus subtilis.
An expression system, which is based on the promoter of the acoABCL operon of Bacillus subtilis was developed and characterized. The acoABCL operon codes for the acetoin dehydrogenase complex, which is the major enzyme system responsible for the catabolism of acetoin in B. subtilis. Besides weak organic acids, the neutral overflow metabolite acetoin is metabolized by the cells in the early stationary phase. Transcription of reporter gene fusions with the acoA promoter of this operon is strongly repressed by glucose but induced by acetoin as soon as the preferred carbon source glucose is exhausted. The co-expression of an additional copy of the regulator gene acoR led to more than twofold higher activity of the acoA promoter. It is demonstrated that the induction of this promoter in growing cells with acetoin is possible with non-phosphotransferase system sugars as carbon and energy source and in a ccpA mutant background. Moreover, it could be shown that the activity of the acoA-directed expression system correlates with the level of acetoin in the medium. During glucose limitation, the utilization of the alternative energy source acetoin keeps the protein synthesis machinery of B. subtilis cells active and thus allows for a long lasting acoA-controlled expression of recombinant genes.
Silbersack
Jörg
J
Department of Pharmaceutical Biotechnology, Institute of Pharmacy, Ernst-Moritz-Arndt University, 17487 Greifswald, Germany. schweder@uni-greifswald.de
Jürgen
Britta
B
Hecker
Michael
M
Schneidinger
Bernd
B
Schmuck
Rainer
R
Schweder
Thomas
T
D000093:Acetoin / Q000378:metabolism*; D000681:Amylases / Q000032:analysis / Q000235:genetics; D019677:Artificial Gene Fusion; D001412:Bacillus subtilis / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics; D004274:DNA, Recombinant; D004268:DNA-Binding Proteins / Q000235:genetics; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial; D017930:Genes, Reporter; D005947:Glucose / Q000378:metabolism; D009876:Operon / Q000235:genetics; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000235:genetics; D013223:Statistics as Topic; D014158:Transcription, Genetic; D001616:beta-Galactosidase / Q000032:analysis / Q000235:genetics
D001426:Bacterial Proteins; D004274:DNA, Recombinant; D004268:DNA-Binding Proteins; D012097:Repressor Proteins; C069286:catabolite control proteins, bacteria; D000093:Acetoin; D000681:Amylases; D001616:beta-Galactosidase; D005947:Glucose