{"doi":"10.1101/2025.06.26.661824","title":"Directed evolution of L-DOPA sensing and production enables efficient incorporation of an expanded genetic code into proteins","abstract":"Abstract 3,4-dihydroxyphenylalanine (L-DOPA) is a catechol-containing amino acid derived from L-tyrosine that can be introduced into proteins primarily as a post-translational modification. It can also be introduced in a site-specific manner into proteins via orthogonal tRNA synthetase:tRNA pairs. However, despite the interest in expanded genetic codes containing L-DOPA, efforts to improve metabolism relating to its production have been limited. Here, we report and engineer a LysR-family transcription factor, PP_2251, that can serve as a biosensor for L-DOPA, thereby allowing the optimization of genetic circuits for vastly improved L-DOPA production via compartmentalized partnered replication (CPR), an emulsion PCR-based directed evolution scheme. Using this biosensor, a promiscuous E. coli flavin-dependent monooxygenase (HpaB) was optimized via CPR for enhanced L-DOPA biosynthesis. Engineered HpaB variants could support efficient translational incorporation of L-DOPA into proteins with 80% incorporation efficiency and yields in excess of 250 mg.L −1 . Improvements in L-DOPA production are not only of great interest for protein engineering and genetic code expansion but should impact the production of DOPA-derived natural products including catecholamine neurotransmitters and benzylisoquinoline alkaloids (BIAs), molecules of intense pharmaceutical interest.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2025,"id":569226,"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":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9461,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1474175,"name":"Qiyao Wei","orcid":"0000-0002-1147-6405","position":1,"is_corresponding":false},{"id":1305100,"name":"Chad Wang","orcid":"0000-0002-8431-3199","position":2,"is_corresponding":false},{"id":1474176,"name":"Jessica Hellinger","orcid":"0000-0002-3664-7856","position":3,"is_corresponding":false},{"id":1474483,"name":"Vidya Pandarinath","orcid":null,"position":4,"is_corresponding":false},{"id":813521,"name":"Simon d’Oelsnitz","orcid":"0000-0001-7512-9157","position":5,"is_corresponding":false},{"id":1474484,"name":"E. O. Powell","orcid":null,"position":6,"is_corresponding":false},{"id":215664,"name":"Jennifer S. Brodbelt","orcid":"0000-0003-3207-0217","position":7,"is_corresponding":false},{"id":392860,"name":"Andrew D. Ellington","orcid":"0000-0001-6246-5338","position":8,"is_corresponding":false},{"id":813523,"name":"Ross Thyer","orcid":"0000-0002-0356-5790","position":9,"is_corresponding":false},{"id":1474174,"name":"Andrew R. Gilmour","orcid":"0000-0001-8422-2863","position":0,"is_corresponding":true}],"reference_count":56,"raw_metadata":null,"created_at":"2026-07-19T02:56:59.652443Z","pmid":"40667249","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":[]}