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

Publication Type:

Journal Article


Nat Chem, Volume 15, Issue 7, p.1030-1039 (2023)


Animals, Cell Membrane, Hydrolysis, Mammals, Phosphatidylcholines, Phospholipase D, Phospholipids


<p>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 on 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 have developed and structurally characterized a family of &#39;superPLDs&#39; with up to a 100-fold enhancement in intracellular activity. We demonstrate 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.</p>

uperPLD (2–48) and superPLD (2–23) have been deposited in the RCSB PDB under accession nos. 8CTQ and 8CTP