Bacillus subtilis highly efficient protein expression systems that are chromosomally integrated and controllable by glucose and rhamnose

Bioscience, Biotechnology, and Biochemistry
Kazutake Hirooka, Ayaka Tamano

Abstract

To achieve rhamnose-inducible efficient protein expression in Bacillus subtilis, we assembled the strong promoters of B. subtilis cdd and ylbP genes and the regulatory region (PrhaEW) of B. subtilis rhaEWRBMA operon, whose transcription is induced by rhamnose and repressed by glucose, to produce various hybrid constructs. These constructs were evaluated using B. subtilis strains carrying a fusion of each construct to the gene encoding a mutated green fluorescent protein in the chromosome. When these strains were cultivated in the presence of glucose or rhamnose, the strain carrying a fusion of a partial PrhaEW region, lacking the intrinsic Shine-Dalgarno (SD) sequence, and the ylbP SD sequence most strictly controlled the promoter activity depending on sugar species. Moreover, the strain carrying a fusion of the cdd core promoter and the ylbP SD sequence showed the highest promoter activity when it was cultivated in the presence of glucose until the late stationary phase. Abbreviations: RNAP: RNA polymerase; cre: catabolite-responsive element; SD: Shine-Dalgarno; PAGE: polyacrylamide gel electrophoresis; GFP: green fluorescent protein; OD600: optical density at 600 nm; LB: Luria-Bertani; a.u.: arbitrary unit; SDS: sodium dodecy...Continue Reading

References

Oct 6, 1998·Annual Review of Biochemistry·R Y Tsien
Sep 6, 2001·Microbiology·H JarmerS Knudsen
Sep 18, 2007·Advances in Applied Microbiology·Wolfgang Schumann
Feb 10, 2009·Bioscience, Biotechnology, and Biochemistry·Yasutaro Fujita
Jun 3, 2014·Microbial Cell Factories·Huina Dong, Dawei Zhang
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Sep 24, 2015·Microbial Cell Factories·Chengran GuanZhemin Zhou
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Methods Mentioned

BETA
phosphotransferase
footprinting
PCR
electrophoresis
protein assay
fluorescence measurement
four hybrid
fluorescence microscopy

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