Artificial signaling in mammalian cells enabled by prokaryotic two-component system


Augmenting live cells with new signal transduction capabilities is a key objective in genetic engineering and synthetic biology. We showed earlier that two-component signaling pathways could function in mammalian cells, albeit while losing their ligand sensitivity. Here, we show how to transduce small-molecule ligands in a dose-dependent fashion into gene expression in mammalian cells using two-component signaling machinery. First, we engineer mutually complementing truncated mutants of a histidine kinase unable to dimerize and phosphorylate the response regulator. Next, we fuse these mutants to protein domains capable of ligand-induced dimerization, which restores the phosphoryl transfer in a ligand-dependent manner. Cytoplasmic ligands are transduced by facilitating mutant dimerization in the cytoplasm, while extracellular ligands trigger dimerization at the inner side of a plasma membrane. These findings point to the potential of two-component regulatory systems as enabling tools for orthogonal signaling pathways in mammalian cells.

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Fig. 1: Identification of HK domains with reduced intrinsic signaling.
Fig. 2: Restoration of two-component signaling via forced dimerization.
Fig. 3: Transduction of cytoplasmic ligand to gene expression.
Fig. 4: Rewiring the GPCR activity to the expression of a reporter gene.
Fig. 5: Multiple GPCRs are rewired to induce gene expression.
Fig. 6: Dynamic characterization of the various signaling approaches.

Data availability

All plasmid sequences and data supporting the findings are available from the corresponding author upon request.

Code availability

The code used to process the time-lapse imaging data is available from the corresponding author upon request.


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The study was funded by ETH Zurich, NCCR Molecular Systems Engineering (grant no. 51NF40-182895) and Swiss National Science foundation (grant no. 31003A_149802). We thank B. Haefliger, R. Altamura, J. Hansen, T. Littmann, G. Bernhardt and C. Stelzer for plasmids, Benenson lab members for discussions, H.M. Kaltenbach for help with statistical analysis and the members of the Single Cell Unit for their help with imaging and flow cytometry.

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A.M. conceived research, performed the experiments, analyzed data and wrote the paper. Y.B. conceived research, analyzed data and wrote the paper.

Correspondence to Yaakov Benenson.

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Competing interests

A patent application has been filed describing the result in this study, with A.M. and Y.B. listed as coinventors. Y.B. is a coinventor of a background patent to this filing.

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Supplementary Information

Supplementary Tables 1–27, Figs. 1–13 and Notes 1 and 2.

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Mazé, A., Benenson, Y. Artificial signaling in mammalian cells enabled by prokaryotic two-component system. Nat Chem Biol 16, 179–187 (2020).

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