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Engineering prokaryotic transcriptional activators as metabolite biosensors in yeast

Nature Chemical Biology volume 12, pages 951–958 (2016)Cite this article

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Abstract

Whole-cell biocatalysts have proven a tractable path toward sustainable production of bulk and fine chemicals. Yet the screening of libraries of cellular designs to identify best-performing biocatalysts is most often a low-throughput endeavor. For this reason, the development of biosensors enabling real-time monitoring of production has attracted attention. Here we applied systematic engineering of multiple parameters to search for a general biosensor design in the budding yeast Saccharomyces cerevisiae based on small-molecule binding transcriptional activators from the prokaryote superfamily of LysR-type transcriptional regulators (LTTRs). We identified a design supporting LTTR-dependent activation of reporter gene expression in the presence of cognate small-molecule inducers. As proof of principle, we applied the biosensors for in vivo screening of cells producing naringenin or cis,cis-muconic acid at different levels, and found that reporter gene output correlated with production. The transplantation of prokaryotic transcriptional activators into the eukaryotic chassis illustrates the potential of a hitherto untapped biosensor resource useful for biotechnological applications.

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Figure 1: Engineering the CCM-responsive prokaryotic transcriptional activator BenM in yeast.
Figure 2: High-throughput engineering and screening of BenM variants with improved CCM inducibility.
Figure 3: Biosensor specificity and transcriptional orthogonality.
Figure 4: Application of transcriptional activators from the LTTR family as biosensors in yeast.
Figure 5: Biosensor sensitivity and operational range.
Figure 6: In vivo application of CCM and naringenin biosensors in yeast.

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Acknowledgements

This work was supported by the Novo Nordisk Foundation and by the European Union Seventh Framework Programme (FP7-KBBE-2013-7-single-stage) under grant agreement no. 613745, Promys (M.E. & S.S.). We acknowledge A. Koza and E. Özdemir for technical assistance.

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Author notes

  1. Mette L Skjoedt and Tim Snoek: These authors contributed equally to this work.

Authors and Affiliations

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

    Mette L Skjoedt, Tim Snoek, Kanchana R Kildegaard, Dushica Arsovska, Tobias J Goedecke, Arun S Rajkumar, Jie Zhang, Mette Kristensen, Solvej Siedler, Irina Borodina, Michael K Jensen & Jay D Keasling

  2. Evolva SA, Reinach, Switzerland

    Michael Eichenberger

  3. Department of Biology, Technical University Darmstadt, Darmstadt, Germany

    Michael Eichenberger

  4. Evolva Biotech A/S, Copenhagen, Denmark

    Beata J Lehka

  5. Department of Science and Environment, Roskilde University, Roskilde, Denmark

    Beata J Lehka

  6. Joint BioEnergy Institute, Emeryville, California, USA

    Jay D Keasling

  7. Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA

    Jay D Keasling

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

    Jay D Keasling

  9. Department of Bioengineering, University of California, Berkeley, Berkeley, California, USA

    Jay D Keasling

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  1. Mette L Skjoedt
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Contributions

M.L.S., T.S., J.D.K. and M.K.J. conceived this project. M.L.S., T.S. and M.K.J. designed all of the experiments. M.L.S., T.S. and D.A. performed all flow cytometry analyses. M.L.S., T.S., D.A., B.J.L., J.Z., K.R.K., S.S., T.J.G. and M.E. constructed all strains and plasmids. M.K. and K.R.K. performed all analytical measurements, and M.K.J. performed the RNA-seq experiment. M.L.S., T.S., M.K.J., I.B., A.S.R. and K.R.K. analyzed the data. M.K.J. wrote the paper.

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Correspondence to Michael K Jensen.

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Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Figures 1–7 and Supplementary Tables 1–5. (PDF 2914 kb)

Supplementary Dataset 1

RNA-seq gene list. (XLSX 947 kb)

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Skjoedt, M., Snoek, T., Kildegaard, K. et al. Engineering prokaryotic transcriptional activators as metabolite biosensors in yeast. Nat Chem Biol 12, 951–958 (2016). https://doi.org/10.1038/nchembio.2177

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