{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,24]],"date-time":"2026-10-24T00:30:54Z","timestamp":1792801854578,"version":"4.5.0"},"reference-count":71,"publisher":"American Chemical Society (ACS)","issue":"8","license":[{"start":{"date-parts":[[2026,8,4]],"date-time":"2026-08-04T00:00:00Z","timestamp":1785801600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001665","name":"Agence Nationale de la Recherche","doi-asserted-by":"publisher","award":["ANR-22-CPJ2-0091-01"],"award-info":[{"award-number":["ANR-22-CPJ2-0091-01"]}],"id":[{"id":"10.13039\/501100001665","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100003245","name":"Ministerie van Onderwijs, Cultuur en Wetenschap","doi-asserted-by":"publisher","award":["024.003.019"],"award-info":[{"award-number":["024.003.019"]}],"id":[{"id":"10.13039\/501100003245","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100003246","name":"Nederlandse Organisatie voor Wetenschappelijk Onderzoek","doi-asserted-by":"publisher","award":["SUMMIT.1.004"],"award-info":[{"award-number":["SUMMIT.1.004"]}],"id":[{"id":"10.13039\/501100003246","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,8,21]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>In vitro reconstitution of protein systems\u2500e.g., metabolic pathways, genetic circuits, or biosensors\u2500often requires optimization to enhance their activity. Combinatorial DNA libraries that simultaneously target multiple genes allow for a holistic optimization strategy by studying the interplay between the systems\u2019 components, which may reveal DNA variants that would be hidden when testing each element in isolation. Here, we screen large populations of synthetic vesicles that express combinatorial DNA variants of a DNA self-replicator or a phospholipid synthesis pathway. We simultaneously vary the strengths of multiple RBSs or synonymously mutate the first codons of multiple genes to explore the effects of the protein translation rates directly on the functionality of the two core synthetic cell modules. We isolated high performers through DNA self-selection or functional screening by fluorescence-activated cell sorting. Long-read sequencing of the fittest variants revealed the optimal RBS strengths and base substitutions in the first codons and indicated which genes were most impactful in regulating the functionality of the protein systems. Single-mutation data were used to predict the fitness of combinatorial variants, which was compared with the experimental fitness observed. The theoretical fitness of combinatorial variants was extremely predictive for the two-gene library of the DNA replicator but less for the larger pathway library. Altogether, our approach exemplifies how combinatorial testing can be expanded from single proteins to multiprotein systems, which can in the future be extended to the evolutionary engineering of even larger genetic and metabolic networks, and eventually an entire artificial cell.<\/jats:p>","DOI":"10.1021\/acssynbio.6c00166","type":"journal-article","created":{"date-parts":[[2026,8,4]],"date-time":"2026-08-04T15:04:12Z","timestamp":1785855852000},"page":"3285-3299","source":"Crossref","is-referenced-by-count":1,"title":["Combinatorial\nOptimization of Protein Systems in Synthetic\nCells"],"prefix":"10.1021","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0009-0006-9286-0746","authenticated-orcid":true,"given":"Marijn","family":"van den Brink","sequence":"first","affiliation":[{"name":"Kavli Institute of Nanoscience, Delft University of Technology , , ,","place":["Delft, The Netherlands, 2629 HZ"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1593-0377","authenticated-orcid":true,"given":"Nico J.","family":"Claassens","sequence":"additional","affiliation":[{"name":"Wageningen University , , Stippeneng 4 , ,","place":["Wageningen, The Netherlands, 6708 WE"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0961-6640","authenticated-orcid":true,"given":"Christophe","family":"Danelon","sequence":"additional","affiliation":[{"name":"Kavli Institute of Nanoscience, Delft University of Technology , , ,","place":["Delft, The Netherlands, 2629 HZ"]},{"name":"Universite\u0301 de Toulouse, CNRS, INRAE, INSA , , ,","place":["Toulouse, France, 31077"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,8,4]]},"reference":[{"key":"2026082212130128500_cit1","doi-asserted-by":"publisher","first-page":"91","DOI":"10.1021\/acs.chemrev.4c00116","article-title":"Cell-free gene expression:\nmethods and applications","volume":"125","author":"Hunt","year":"2025","journal-title":"Chem. Rev."},{"key":"2026082212130128500_cit2","doi-asserted-by":"publisher","first-page":"1221","DOI":"10.1021\/acs.biochem.3c00023","article-title":"The long road to a synthetic self-replicating\ncentral dogma","volume":"62","author":"De Capitani","year":"2023","journal-title":"Biochemistry"},{"key":"2026082212130128500_cit3","doi-asserted-by":"publisher","first-page":"193","DOI":"10.1016\/j.molcel.2020.09.014","article-title":"The ongoing\nquest to crack the genetic code for protein production","volume":"80","author":"Nieuwkoop","year":"2020","journal-title":"Mol. Cell"},{"key":"2026082212130128500_cit4","doi-asserted-by":"publisher","first-page":"151","DOI":"10.1016\/j.ab.2003.10.005","article-title":"In vitro compartmentalization\nby double emulsions: sorting and gene enrichment by fluorescence activated\ncell sorting","volume":"325","author":"Bernath","year":"2004","journal-title":"Anal. Biochem."},{"key":"2026082212130128500_cit5","doi-asserted-by":"publisher","first-page":"5017","DOI":"10.1021\/ac300678w","article-title":"Construction of a gene screening\nsystem using giant unilamellar liposomes\nand a fluorescence-activated cell sorter","volume":"84","author":"Nishikawa","year":"2012","journal-title":"Anal.\nChem."},{"key":"2026082212130128500_cit6","doi-asserted-by":"publisher","first-page":"16796","DOI":"10.1073\/pnas.1314585110","article-title":"In vitro evolution\nof \u03b1-hemolysin using a liposome display","volume":"110","author":"Fujii","year":"2013","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"2026082212130128500_cit7","doi-asserted-by":"publisher","first-page":"1578","DOI":"10.1038\/nprot.2014.107","article-title":"Liposome display for\nin vitro selection and evolution of membrane proteins","volume":"9","author":"Fujii","year":"2014","journal-title":"Nat. Protoc."},{"key":"2026082212130128500_cit8","doi-asserted-by":"publisher","first-page":"1797","DOI":"10.1002\/cbic.201500174","article-title":"Liposome-based in vitro\nevolution of aminoacyl-tRNA synthetase for enhanced pyrrolysine derivative\nincorporation","volume":"16","author":"Uyeda","year":"2015","journal-title":"ChemBioChem"},{"key":"2026082212130128500_cit9","doi-asserted-by":"publisher","first-page":"12028","DOI":"10.1021\/acs.analchem.6b02308","article-title":"Construction\nof an in vitro gene screening system of the E. coli EmrE transporter using liposome display","volume":"88","author":"Uyeda","year":"2016","journal-title":"Anal.\nChem."},{"key":"2026082212130128500_cit10","doi-asserted-by":"publisher","first-page":"576","DOI":"10.1021\/acssynbio.7b00333","article-title":"In vitro evolution\nof unmodified 16S rRNA for simple ribosome reconstitution","volume":"7","author":"Murase","year":"2018","journal-title":"ACS Synth. Biol."},{"key":"2026082212130128500_cit11","doi-asserted-by":"crossref","first-page":"121","DOI":"10.1007\/10_2023_219","article-title":"Progresses in\nCell-Free In Vitro Evolution","volume-title":"Cell-free\nProduction: System Development","author":"Seo","year":"2023"},{"key":"2026082212130128500_cit12","doi-asserted-by":"publisher","first-page":"2108","DOI":"10.1021\/acssynbio.2c00050","article-title":"High-throughput regulatory\npart prototyping and analysis by cell-free protein synthesis and droplet\nmicrofluidics","volume":"11","author":"Gan","year":"2022","journal-title":"ACS Synth. Biol."},{"key":"2026082212130128500_cit13","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-022-28396-4","article-title":"Droplet-based screening\nof phosphate transfer catalysis reveals how epistasis shapes MAP kinase\ninteractions with substrates","volume":"13","author":"Scheele","year":"2022","journal-title":"Nat. Commun."},{"key":"2026082212130128500_cit14","doi-asserted-by":"publisher","first-page":"142","DOI":"10.1016\/j.copbio.2017.06.014","article-title":"Combinatorial pathway optimization\nfor streamlined metabolic engineering","volume":"47","author":"Jeschek","year":"2017","journal-title":"Curr.\nOpin. Biotechnol."},{"key":"2026082212130128500_cit15","doi-asserted-by":"publisher","DOI":"10.1016\/j.coisb.2023.100499","article-title":"From sequence\nto function\nand back \u2013 High-throughput sequence-function mapping in synthetic\nbiology","volume":"37","author":"Ho\u0308llerer","year":"2024","journal-title":"Curr. Opin. Syst. Biol."},{"key":"2026082212130128500_cit16","doi-asserted-by":"publisher","first-page":"379","DOI":"10.1038\/nrg3927","article-title":"Methods for the\ndirected evolution of proteins","volume":"16","author":"Packer","year":"2015","journal-title":"Nat. Rev. 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Biol."},{"key":"2026082212130128500_cit33","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-020-17863-5","article-title":"Genetically controlled\nmembrane synthesis in liposomes","volume":"11","author":"Blanken","year":"2020","journal-title":"Nat. Commun."},{"key":"2026082212130128500_cit34","doi-asserted-by":"publisher","DOI":"10.1128\/mBio.02703-19","article-title":"Posttranslational control\nof PlsB is sufficient to coordinate membrane synthesis with growth\nin Escherichia coli","volume":"11","author":"Noga","year":"2020","journal-title":"mBio"},{"key":"2026082212130128500_cit35","doi-asserted-by":"publisher","first-page":"111","DOI":"10.1271\/bbb.60.111","article-title":"A regulatory\nmechanism for the balanced synthesis of membrane phospholipid species\nin Escherichia coli","volume":"60","author":"Saha","year":"1996","journal-title":"Biosci. Biotechnol. Biochem."},{"key":"2026082212130128500_cit36","doi-asserted-by":"publisher","DOI":"10.1038\/ncomms11163","article-title":"Rationally reduced libraries for\ncombinatorial pathway optimization minimizing experimental effort","volume":"7","author":"Jeschek","year":"2016","journal-title":"Nat. Commun."},{"key":"2026082212130128500_cit37","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkt151","article-title":"Spanning high-dimensional\nexpression space using ribosome-binding site combinatorics","volume":"41","author":"Zelcbuch","year":"2013","journal-title":"Nucleic Acids Res."},{"key":"2026082212130128500_cit38","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0163058","article-title":"Cell-free phospholipid\nbiosynthesis by gene-encoded enzymes reconstituted in liposomes","volume":"11","author":"Scott","year":"2016","journal-title":"PLoS One"},{"key":"2026082212130128500_cit39","doi-asserted-by":"publisher","first-page":"1252","DOI":"10.1021\/acssynbio.3c00014","article-title":"In vitro transcription\/translation-coupled\nDNA replication\nthrough partial regeneration of 20 aminoacyl-tRNA synthetases","volume":"12","author":"Hagino","year":"2023","journal-title":"ACS Synth. Biol."},{"key":"2026082212130128500_cit40","unstructured":"GeneFrontier Corporation.\n          . Influence of 5\u2032UTR and N-terminal ORF region\non protein synthesis\nefficiency in PUREfrex|Posters|Resources|PUREfrex|, 2023. https:\/\/purefrex.genefrontier.com\/resources\/posters\/P040_CFSC2023.html."},{"key":"2026082212130128500_cit41","doi-asserted-by":"publisher","first-page":"1559","DOI":"10.1039\/C2MB25483B","article-title":"Building synthetic\ngene circuits from combinatorial\nlibraries: screening and selection strategies","volume":"9","author":"Schaerli","year":"2013","journal-title":"Mol. BioSyst."},{"key":"2026082212130128500_cit42","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-022-31245-z","article-title":"A versatile active learning\nworkflow for optimization of genetic and metabolic networks","volume":"13","author":"Pandi","year":"2022","journal-title":"Nat. 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Adv."},{"key":"2026082212130128500_cit49","doi-asserted-by":"publisher","DOI":"10.1186\/s12864-016-2533-5","article-title":"Sort-seq under the hood: implications of design choices\non large-scale characterization of sequence-function relations","volume":"17","author":"Peterman","year":"2016","journal-title":"BMC Genomics"},{"key":"2026082212130128500_cit50","doi-asserted-by":"publisher","first-page":"8852","DOI":"10.1073\/pnas.1901979116","article-title":"Machine\nlearning-assisted directed protein evolution with combinatorial libraries","volume":"116","author":"Wu","year":"2019","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"2026082212130128500_cit51","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-025-55987-8","article-title":"Active learning-assisted\ndirected evolution","volume":"16","author":"Yang","year":"2025","journal-title":"Nat. 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Methods"},{"key":"2026082212130128500_cit55","doi-asserted-by":"publisher","first-page":"14508","DOI":"10.1073\/pnas.1208715109","article-title":"Detection of ultra-rare\nmutations by next-generation sequencing","volume":"109","author":"Schmitt","year":"2012","journal-title":"Proc.\nNatl. Acad. Sci. U.S.A."},{"key":"2026082212130128500_cit56","unstructured":"Nanopore Sequencing Accuracy, Oxford Nanopore\nTechnologieshttps:\/\/nanoporetech.com\/platform\/accuracy."},{"key":"2026082212130128500_cit57","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-020-19687-9","article-title":"UMI-linked\nconsensus sequencing enables phylogenetic\nanalysis of directed evolution","volume":"11","author":"Zurek","year":"2020","journal-title":"Nat. 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Sci."},{"key":"2026082212130128500_cit61","doi-asserted-by":"publisher","DOI":"10.1080\/21690731.2017.1327006","article-title":"Dissecting limiting\nfactors of the Protein synthesis Using Recombinant Elements (PURE)\nsystem","volume":"5","author":"Li","year":"2017","journal-title":"Translation"},{"key":"2026082212130128500_cit62","doi-asserted-by":"publisher","first-page":"14024","DOI":"10.1073\/pnas.1301301110","article-title":"Composability of regulatory\nsequences controlling transcription and translation in Escherichia coli","volume":"110","author":"Kosuri","year":"2013","journal-title":"Proc. Natl.\nAcad. Sci. U.S.A."},{"key":"2026082212130128500_cit63","doi-asserted-by":"publisher","DOI":"10.1016\/j.crmeth.2022.100237","article-title":"Microscopy-based single-cell proteomic\nprofiling reveals heterogeneity in DNA damage response dynamics","volume":"2","author":"Su","year":"2022","journal-title":"Cell Rep. 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