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An acetoin-regulated expression system of Bacillussubtilis

  • Applied Genetics and Molecular Biotechnology
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Abstract

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.

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Abbreviations

E. coli :

Escherichia coli

B. subtilis :

Bacillus subtilis

OD500 :

optical density at 500 nm

RT:

reverse transcription

PCR:

polymerase chain reaction

LB:

Luria Broth medium

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Acknowl edgements

We are indebted to Jörg Stülke for providing the ccpA mutant strain B. subtilis GP300. We thank Torsten Koburger for preparing RNA samples from a glucose-limited B. subtilis culture, which were used for the acoA real-time RT-PCR analysis. This work was financially supported by the Ministry of Education, Science, and Culture of Mecklenburg-Vorpommern (FK 0202120).

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Authors and Affiliations

  1. Department of Pharmaceutical Biotechnology, Institute of Pharmacy, Ernst-Moritz-Arndt University, 17487, Greifswald, Germany

    Jörg Silbersack, Britta Jürgen & Thomas Schweder

  2. Institute of Microbiology, Ernst-Moritz-Arndt University, 17487, Greifswald, Germany

    Michael Hecker

  3. Dept. LR-DE 4, Enzymology/Genetics, Roche Diagnostics GmbH, 82377, Penzberg, Germany

    Bernd Schneidinger & Rainer Schmuck

Authors
  1. Jörg Silbersack
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  2. Britta Jürgen
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  3. Michael Hecker
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  4. Bernd Schneidinger
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  5. Rainer Schmuck
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  6. Thomas Schweder
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Correspondence to Thomas Schweder.

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Silbersack, J., Jürgen, B., Hecker, M. et al. An acetoin-regulated expression system of Bacillussubtilis . Appl Microbiol Biotechnol 73, 895–903 (2006). https://doi.org/10.1007/s00253-006-0549-5

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