A cell-penetrating artificial metalloenzyme regulates a gene switch in a designer mammalian cell

Yasunori Okamoto,Ryosuke Kojima,Martin Fussenegger,Fabian Schwizer,Eline Bartolami,Thomas R Ward,Stefan Matile,Tillmann Heinisch

doi:10.1038/s41467-018-04440-0

Yasunori Okamoto, Ryosuke Kojima + Show 6 more

Open Access

https://doi.org/10.1038/s41467-018-04440-0

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Abstract

Complementing enzymes in their native environment with either homogeneous or heterogeneous catalysts is challenging due to the sea of functionalities present within a cell. To supplement these efforts, artificial metalloenzymes are drawing attention as they combine attractive features of both homogeneous catalysts and enzymes. Herein we show that such hybrid catalysts consisting of a metal cofactor, a cell-penetrating module, and a protein scaffold are taken up into HEK-293T cells where they catalyze the uncaging of a hormone. This bioorthogonal reaction causes the upregulation of a gene circuit, which in turn leads to the expression of a nanoluc-luciferase. Relying on the biotin–streptavidin technology, variation of the biotinylated ruthenium complex: the biotinylated cell-penetrating poly(disulfide) ratio can be combined with point mutations on streptavidin to optimize the catalytic uncaging of an allyl-carbamate-protected thyroid hormone triiodothyronine. These results demonstrate that artificial metalloenzymes offer highly modular tools to perform bioorthogonal catalysis in live HEK cells.

Highlights

Complementing enzymes in their native environment with either homogeneous or heterogeneous catalysts is challenging due to the sea of functionalities present within a cell
The assembly of gene switches allows for the construction of engineered mammalian cells that are capable of sensing the extracellular environment and producing output molecules on demand (Fig. 1b)[29,30,31]
To achieve efficient delivery of an organometallic catalyst into cells, we capitalize on the homotetrameric nature of a streptavidin scaffold to combine an abiotic biotinylated catalyst with a biotinylated cell-penetrating moiety

Summary

Introduction

Complementing enzymes in their native environment with either homogeneous or heterogeneous catalysts is challenging due to the sea of functionalities present within a cell. Relying on the biotin–streptavidin technology, variation of the biotinylated ruthenium complex: the biotinylated cell-penetrating poly(disulfide) ratio can be combined with point mutations on streptavidin to optimize the catalytic uncaging of an allyl-carbamate-protected thyroid hormone triiodothyronine These results demonstrate that artificial metalloenzymes offer highly modular tools to perform bioorthogonal catalysis in live HEK cells. In addition to these efforts, metabolic engineering in E. coli, yeast, and higher organisms has received increasing attention thanks to its enormous potential to produce either high-added value products and biofuels or to cure diseases[28]. This cell-penetrating ArM is taken up into mammalian cells and catalyzes an abiotic reaction, leading to the upregulation of a designed gene circuit

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