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Design, Engineering, and Characterization of Prokaryotic Ligand-Binding Transcriptional Activators as Biosensors in Yeast

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Synthetic Metabolic Pathways

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1671))

Abstract

In cell factory development, screening procedures, often relying on low-throughput analytical methods, are lagging far behind diversity generation methods. This renders the identification and selection of the best cell factory designs tiresome and costly, conclusively hindering the manufacturing process. In the yeast Saccharomyces cerevisiae, implementation of allosterically regulated transcription factors from prokaryotes as metabolite biosensors has proven a valuable strategy to alleviate this screening bottleneck. Here, we present a protocol to select and incorporate prokaryotic transcriptional activators as metabolite biosensors in S. cerevisiae. As an example, we outline the engineering and characterization of the LysR-type transcriptional regulator (LTTR) family member BenM from Acetinobacter sp. ADP1 for monitoring accumulation of cis,cis-muconic acid, a bioplast precursor, in yeast by means of flow cytometry.

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Author information

Author notes

  1. Francesca Ambri and Tim Snoek contributed equally to this work.

Authors and Affiliations

  1. The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kongens Lyngby, Denmark

    Francesca Ambri, Tim Snoek, Mette L. Skjoedt, Michael K. Jensen & Jay D. Keasling

  2. Joint BioEnergy Institute, Emeryville, CA, USA

    Jay D. Keasling

  3. Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA

    Jay D. Keasling

  4. Department of Chemical and Biomolecular Engineering, University of California, Berkeley, CA, USA

    Jay D. Keasling

  5. Department of Bioengineering, University of California, Berkeley, CA, USA

    Jay D. Keasling

Authors
  1. Francesca Ambri
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  2. Tim Snoek
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  3. Mette L. Skjoedt
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  4. Michael K. Jensen
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  5. Jay D. Keasling
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Corresponding author

Correspondence to Michael K. Jensen .

Editor information

Editors and Affiliations

  1. The Novo Nordisk Foundation, Center for Biosustainability, Technical University of Denmark, Kongens Lyngby, Denmark

    Michael Krogh Jensen

  2. Joint BioEnergy Institute, Emeryville, California, USA

    Jay D. Keasling

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Ambri, F., Snoek, T., Skjoedt, M.L., Jensen, M.K., Keasling, J.D. (2018). Design, Engineering, and Characterization of Prokaryotic Ligand-Binding Transcriptional Activators as Biosensors in Yeast. In: Jensen, M.K., Keasling, J.D. (eds) Synthetic Metabolic Pathways. Methods in Molecular Biology, vol 1671. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7295-1_17

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  • DOI: https://doi.org/10.1007/978-1-4939-7295-1_17

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7294-4

  • Online ISBN: 978-1-4939-7295-1

  • eBook Packages: Springer Protocols

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