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A simple selection strategy for evolving highly efficient enzymes

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Abstract

Combining tunable transcription with an enzyme-degradation tag affords an effective means to reduce intracellular enzyme concentrations from high to very low levels. Such fine-tuned control allows selection pressure to be systematically increased in directed-evolution experiments. This facilitates identification of mutants with wild-type activity, as shown here for an engineered chorismate mutase. Numerous selection formats and cell-based screening methodologies may benefit from the large dynamic range afforded by this easily implemented strategy.

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Figure 1: Selection plasmid design.
Figure 2: Tetracycline-dependent growth in selective M9c medium and influence of tetracycline concentration on the selection process.

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Acknowledgements

This work was generously supported by the Schweizerischer Nationalfonds and the ETH Zurich.

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Authors and Affiliations

  1. Laboratory of Organic Chemistry, ETH Zurich, Hönggerberg HCI F 339, Zurich, CH-8093, Switzerland

    Martin Neuenschwander, Maren Butz, Caroline Heintz, Peter Kast & Donald Hilvert

Authors
  1. Martin Neuenschwander
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  2. Maren Butz
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  3. Caroline Heintz
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  4. Peter Kast
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  5. Donald Hilvert
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Contributions

M.N., P.K. and D.H. designed research; M.N., M.B. and C.H. performed the experiments; M.N., M.B., P.K. and D.H. analyzed data; M.N., P.K. and D.H. wrote the paper.

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Correspondence to Donald Hilvert.

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Supplementary Text and Figures

Supplementary Figs. 1–4; Supplementary Table 1; Supplementary Methods (PDF 226 kb)

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Neuenschwander, M., Butz, M., Heintz, C. et al. A simple selection strategy for evolving highly efficient enzymes. Nat Biotechnol 25, 1145–1147 (2007). https://doi.org/10.1038/nbt1341

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  • DOI: https://doi.org/10.1038/nbt1341

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