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bioRxiv · 10.1101/2022.09.26.509516

Activity-based directed evolution of a membrane editor in mammalian cells

Tei, R.·Bagde, S. R.·Fromme, J. C.·Baskin, J. M.
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Abstract

Cellular membranes contain numerous lipid species, and efforts to understand the biological functions of individual lipids have been stymied by a lack of approaches for controlled modulation of membrane composition in situ. Here, we present a strategy for editing phospholipids, the most abundant lipids in biological membranes. Our membrane editor is based upon a bacterial phospholipase D (PLD), which exchanges phospholipid head groups through hydrolysis or transphosphatidylation of phosphatidylcholine with water or exogenous alcohols. Exploiting activity-dependent directed enzyme evolution in mammalian cells, we developed and structurally characterized a family of "superPLDs" with up to 100-fold higher activity than wildtype PLD. We demonstrated the utility of superPLDs for both optogenetics-enabled editing of phospholipids within specific organelle membranes in live cells and biocatalytic synthesis of natural and unnatural designer phospholipids in vitro. Beyond the superPLDs, activity-based directed enzyme evolution in mammalian cells is a generalizable approach to engineer additional chemoenzymatic biomolecule editors.

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Tei, R., Bagde, S. R., Fromme, J. C., Baskin, J. M.. 2022-09-26. Activity-based directed evolution of a membrane editor in mammalian cells. https://doi.org/10.1101/2022.09.26.509516

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