Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY, USA.
Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY, USA.
Nat Chem. 2023 Jul;15(7):1030-1039. doi: 10.1038/s41557-023-01214-0. Epub 2023 May 22.
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.
细胞膜含有许多脂质种类，由于缺乏原位控制调节膜组成的方法，因此人们对单个脂质的生物学功能的研究一直受到阻碍。在这里，我们提出了一种编辑磷脂的策略，磷脂是生物膜中最丰富的脂质。我们的膜编辑器基于一种细菌磷脂酶 D（PLD），它通过水解或通过与水或外源性醇的转磷酸化作用将磷脂酰胆碱的磷脂头基交换。利用哺乳动物细胞中基于活性的定向酶进化，我们开发并对具有高达 100 倍细胞内活性增强的“超级 PLD”家族进行了结构表征。我们证明了超级 PLD 可用于在活细胞中对特定细胞器膜内的磷脂进行光遗传学编辑，以及在体外进行天然和非天然设计磷脂的生物催化合成。除了超级 PLD 之外，哺乳动物细胞中的基于活性的定向酶进化是一种可推广的方法，可以用于设计其他化学酶生物分子编辑器。
