{"doi":"10.1101/2022.09.26.509516","title":"Activity-based directed evolution of a membrane editor in mammalian cells","abstract":"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.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2022,"id":301906,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":2,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9541,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2022-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":646799,"name":"Saket R. Bagde","orcid":"0000-0001-9800-9326","position":1,"is_corresponding":false},{"id":277256,"name":"J. Christopher Fromme","orcid":"0000-0002-8837-0473","position":2,"is_corresponding":false},{"id":293068,"name":"Jeremy M. Baskin","orcid":"0000-0003-2939-3138","position":3,"is_corresponding":false},{"id":993390,"name":"Reika Tei","orcid":"0000-0002-9492-9309","position":0,"is_corresponding":true}],"reference_count":63,"raw_metadata":null,"created_at":"2026-07-19T00:32:06.309890Z","pmid":null,"pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}