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

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 dodecyl sulfate.

Graphical Abstract

Fluorescence micrographs of B. subtilis reporter strains carrying each egfp fusion and strain 168 (control), which had been cultivated in the presence of glucose or rhamnose.

KEYWORDS:

• Bacillus subtilis
• protein expression
• glucose
• rhamnose

Acknowledgements

We are grateful to Dr. Jun Kamishikiryo for assistance with confocal fluorescence microscopy. We also thank Saki Shioda, Rikiya Yanai, and Mizuki Handa for their help with the experiments.

Disclosure statement

No potential conflict of interest was reported by the authors.

Author contributions

AT conducted most of the experiments and analyzed the results under the supervision of KH. KH drafted the research plan and wrote the manuscript with AT.