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 ‘superPLDs’ 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.

细胞膜含有多种脂质种类，由于缺乏原位控制调节膜成分的方法，理解单个脂质的生物学功能的努力受到阻碍。在这里，我们提出了一种编辑磷脂（生物膜中最丰富的脂质）的策略。我们的膜编辑器基于细菌磷脂酶 D (PLD)，它通过用水或外源醇水解或磷脂酰胆碱转磷脂酰化来交换磷脂头基。利用哺乳动物细胞中活性依赖性定向酶进化，我们开发了一系列“superPLD”，并对其结构进行了表征，其细胞内活性增强了 100 倍。我们展示了 superPLD 在活细胞特定细胞器膜内的光遗传学编辑磷脂以及体外天然和非天然设计磷脂的生物催化合成中的实用性。除了 superPLD 之外，哺乳动物细胞中基于活性的定向酶进化是设计其他化学酶生物分子编辑器的通用方法。