{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,8]],"date-time":"2026-10-08T07:06:03Z","timestamp":1791443163214,"version":"4.3.3"},"reference-count":58,"publisher":"National Academy of Sciences","issue":"27","license":[{"start":{"date-parts":[[2025,6,30]],"date-time":"2025-06-30T00:00:00Z","timestamp":1751241600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000001","name":"NSF","doi-asserted-by":"publisher","award":["2244770"],"award-info":[{"award-number":["2244770"]}],"id":[{"id":"10.13039\/100000001","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100019235","name":"MIT | Abdul Latif Jameel Water and Food Systems Lab, Massachusetts Institute of Technology","doi-asserted-by":"publisher","award":["Grand Challenge Award"],"award-info":[{"award-number":["Grand Challenge Award"]}],"id":[{"id":"10.13039\/100019235","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Martin Family Society Fellowship for Sustainability","award":["Graduate Fellowship"],"award-info":[{"award-number":["Graduate Fellowship"]}]},{"DOI":"10.13039\/100000057","name":"HHS | NIH | National Institute of General Medical Sciences","doi-asserted-by":"publisher","award":["1R35GM136354"],"award-info":[{"award-number":["1R35GM136354"]}],"id":[{"id":"10.13039\/100000057","id-type":"DOI","asserted-by":"publisher"}]},{"name":"MIT Robert J. Sibley Fellowship","award":["Graduate Fellowship"],"award-info":[{"award-number":["Graduate Fellowship"]}]},{"name":"MIT Future of Science Fellowship","award":["Graduate Fellowship"],"award-info":[{"award-number":["Graduate Fellowship"]}]},{"name":"BroadIgnite Fellowship","award":["Graduate Fellowship"],"award-info":[{"award-number":["Graduate Fellowship"]}]}],"content-domain":{"domain":["www.pnas.org"],"crossmark-restriction":true},"short-container-title":["Proc. Natl. Acad. Sci. U.S.A."],"published-print":{"date-parts":[[2025,7,8]]},"abstract":"\n Carbon dioxide (CO\n 2<\/jats:sub>\n ) assimilation by the enzyme Ribulose-1,5-bisphosphate Carboxylase\/Oxygenase (Rubisco) underpins biomass accumulation in photosynthetic bacteria and eukaryotes. Despite its pivotal role, Rubisco has a slow carboxylation rate (\n \n \n \n k<\/mml:mi>\n \n cat<\/mml:mi>\n <\/mml:mrow>\n \n \n CO<\/mml:mi>\n <\/mml:mrow>\n 2<\/mml:mn>\n <\/mml:msub>\n <\/mml:msubsup>\n <\/mml:math>\n <\/jats:inline-formula>\n ) and is competitively inhibited by oxygen (O\n 2<\/jats:sub>\n ). These traits impose limitations on photosynthetic efficiency, making Rubisco a compelling target for improvement. Interest in Form II Rubisco from\n Gallionellaceae<\/jats:italic>\n bacteria, which comprise a dimer or hexamer of large subunits, arises from their nearly fivefold higher\n \n \n \n k<\/mml:mi>\n \n cat<\/mml:mi>\n <\/mml:mrow>\n \n \n CO<\/mml:mi>\n <\/mml:mrow>\n 2<\/mml:mn>\n <\/mml:msub>\n <\/mml:msubsup>\n <\/mml:math>\n <\/jats:inline-formula>\n than the average Rubisco enzyme. As well as having a fast\n \n \n \n k<\/mml:mi>\n \n cat<\/mml:mi>\n <\/mml:mrow>\n \n \n CO<\/mml:mi>\n <\/mml:mrow>\n 2<\/mml:mn>\n <\/mml:msub>\n <\/mml:msubsup>\n <\/mml:math>\n <\/jats:inline-formula>\n (25.8 s\n \n \u2212<\/jats:sup>\n <\/jats:bold>\n 1<\/jats:sup>\n at 25 \u00b0C), we show that\n Gallionellaceae<\/jats:italic>\n Rubisco (GWS1B) is extremely sensitive to O\n 2<\/jats:sub>\n inhibition, consistent with its evolution under semianaerobic environments. We therefore used an in vivo mutagenesis-mediated screening pipeline to evolve GWS1B over six rounds under oxygenic selection, identifying three catalytic point mutants with improved ambient carboxylation efficiency: Thr-29-Ala (T29A), Glu-40-Lys (E40K), and Arg-337-Cys (R337C). Full kinetic characterization showed that each substitution enhanced the CO\n 2<\/jats:sub>\n affinity of GWS1B under oxygenic conditions by subduing oxygen affinity, leading to 25% (E40K), 11% (T29A), and 8% (R337C) enhancements in carboxylation efficiency under ambient O\n 2<\/jats:sub>\n at 25 \u00b0C. By contrast, under the near anaerobic natural environment of\n Gallionellaceae<\/jats:italic>\n , the carboxylation efficiency of each mutant was impaired ~16%. These findings demonstrate the efficacy of artificial directed evolution to access distinctive regions of catalytic space in Rubisco.\n <\/jats:p>","DOI":"10.1073\/pnas.2505083122","type":"journal-article","created":{"date-parts":[[2025,6,30]],"date-time":"2025-06-30T15:45:31Z","timestamp":1751298331000},"update-policy":"https:\/\/doi.org\/10.1073\/pnas.cm10313","source":"Crossref","is-referenced-by-count":21,"title":["In vivo directed evolution of an ultrafast Rubisco from a semianaerobic environment imparts oxygen resistance"],"prefix":"10.1073","volume":"122","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-3715-9619","authenticated-orcid":false,"given":"Julie L.","family":"McDonald","sequence":"first","affiliation":[{"name":"Department of Chemistry, Massachusetts Institute of Technology","place":["Cambridge"]},{"name":"Department of Biology, Massachusetts Institute of Technology","place":["Cambridge"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nathan P.","family":"Shapiro","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Massachusetts Institute of Technology","place":["Cambridge"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Amanuella A.","family":"Mengiste","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Massachusetts Institute of Technology","place":["Cambridge"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1875-0024","authenticated-orcid":false,"given":"Sarah","family":"Kaines","sequence":"additional","affiliation":[{"name":"Plant Science Division, Research School of Biology, The Australian National University","place":["Canberra, Australia"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2954-2359","authenticated-orcid":false,"given":"Spencer M.","family":"Whitney","sequence":"additional","affiliation":[{"name":"Plant Science Division, Research School of Biology, The Australian National University","place":["Canberra, Australia"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Robert H.","family":"Wilson","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Massachusetts Institute of Technology","place":["Cambridge"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6511-3431","authenticated-orcid":false,"given":"Matthew D.","family":"Shoulders","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Massachusetts Institute of Technology","place":["Cambridge"]},{"name":"Broad Institute of Massachusetts Institute of Technology and Harvard","place":["Cambridge"]},{"name":"Koch Institute for Integrative Cancer Research at Massachusetts Institute of Technology","place":["Cambridge"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"341","published-online":{"date-parts":[[2025,6,30]]},"reference":[{"key":"e_1_3_4_1_2","doi-asserted-by":"publisher","DOI":"10.1146\/annurev-biochem-040320-101244"},{"key":"e_1_3_4_2_2","doi-asserted-by":"publisher","DOI":"10.15252\/embj.2019104081"},{"key":"e_1_3_4_3_2","doi-asserted-by":"publisher","DOI":"10.1104\/pp.110.164814"},{"key":"e_1_3_4_4_2","doi-asserted-by":"publisher","DOI":"10.1093\/jxb\/erm361"},{"key":"e_1_3_4_5_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.abq1416"},{"key":"e_1_3_4_6_2","doi-asserted-by":"publisher","DOI":"10.1021\/acs.biochem.9b00237"},{"key":"e_1_3_4_7_2","doi-asserted-by":"publisher","DOI":"10.1146\/annurev-arplant-043015-111709"},{"key":"e_1_3_4_8_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.2012288117"},{"key":"e_1_3_4_9_2","doi-asserted-by":"publisher","DOI":"10.1111\/j.1472-4669.2007.00127.x"},{"key":"e_1_3_4_10_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.copbio.2017.07.017"},{"key":"e_1_3_4_11_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.2321050121"},{"key":"e_1_3_4_12_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0911663107"},{"key":"e_1_3_4_13_2","doi-asserted-by":"publisher","DOI":"10.1038\/ncomms10382"},{"key":"e_1_3_4_14_2","doi-asserted-by":"publisher","DOI":"10.3389\/fmicb.2019.00603"},{"key":"e_1_3_4_15_2","doi-asserted-by":"publisher","DOI":"10.1186\/s40643-021-00439-6"},{"key":"e_1_3_4_16_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.pbi.2016.04.002"},{"key":"e_1_3_4_17_2","doi-asserted-by":"publisher","DOI":"10.1111\/nph.14351"},{"key":"e_1_3_4_18_2","doi-asserted-by":"publisher","DOI":"10.1093\/jxb\/eraa075"},{"key":"e_1_3_4_19_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.semcdb.2023.04.003"},{"key":"e_1_3_4_20_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.pecs.2024.101184"},{"key":"e_1_3_4_21_2","doi-asserted-by":"publisher","DOI":"10.1038\/nchembio.1715"},{"key":"e_1_3_4_22_2","doi-asserted-by":"publisher","DOI":"10.1038\/srep22284"},{"key":"e_1_3_4_23_2","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M117.810861"},{"key":"e_1_3_4_24_2","doi-asserted-by":"publisher","DOI":"10.3390\/ijms20205019"},{"key":"e_1_3_4_25_2","doi-asserted-by":"publisher","DOI":"10.1042\/BJ20070071"},{"key":"e_1_3_4_26_2","doi-asserted-by":"publisher","DOI":"10.1007\/s13238-014-0072-x"},{"key":"e_1_3_4_27_2","doi-asserted-by":"publisher","DOI":"10.1021\/bi9006385"},{"key":"e_1_3_4_28_2","doi-asserted-by":"publisher","DOI":"10.1111\/febs.13774"},{"key":"e_1_3_4_29_2","doi-asserted-by":"publisher","DOI":"10.1128\/mBio.01537-19"},{"key":"e_1_3_4_30_2","doi-asserted-by":"crossref","unstructured":"N. Prywes A map of the Rubisco biochemical landscape. Nature 638 823\u2013828 (2025).","DOI":"10.1038\/s41586-024-08455-0"},{"key":"e_1_3_4_31_2","doi-asserted-by":"publisher","DOI":"10.1042\/BJ20080668"},{"key":"e_1_3_4_32_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0022-2836(03)00786-1"},{"key":"e_1_3_4_33_2","doi-asserted-by":"publisher","DOI":"10.1093\/protein\/gzj010"},{"key":"e_1_3_4_34_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41598-018-23869-3"},{"key":"e_1_3_4_35_2","doi-asserted-by":"publisher","DOI":"10.1111\/pce.12335"},{"key":"e_1_3_4_36_2","doi-asserted-by":"publisher","DOI":"10.1038\/s44318-024-00119-z"},{"key":"e_1_3_4_37_2","doi-asserted-by":"publisher","DOI":"10.1111\/1462-2920.13138"},{"key":"e_1_3_4_38_2","doi-asserted-by":"publisher","DOI":"10.1126\/sciadv.adc9440"},{"key":"e_1_3_4_39_2","doi-asserted-by":"publisher","DOI":"10.1038\/s44318-025-00419-y"},{"key":"e_1_3_4_40_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-020-20230-z"},{"key":"e_1_3_4_41_2","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkaa1231"},{"key":"e_1_3_4_42_2","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkad003"},{"key":"e_1_3_4_43_2","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkad266"},{"key":"e_1_3_4_44_2","doi-asserted-by":"publisher","DOI":"10.1021\/acssynbio.4c00316"},{"key":"e_1_3_4_45_2","doi-asserted-by":"publisher","DOI":"10.1021\/jacs.8b04001"},{"key":"e_1_3_4_46_2","doi-asserted-by":"publisher","DOI":"10.1038\/s43586-022-00119-5"},{"key":"e_1_3_4_47_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41592-021-01090-x"},{"key":"e_1_3_4_48_2","doi-asserted-by":"publisher","DOI":"10.1021\/bi700820a"},{"key":"e_1_3_4_49_2","doi-asserted-by":"publisher","DOI":"10.1007\/978-3-319-50413-1_4"},{"key":"e_1_3_4_50_2","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-60761-925-3_27"},{"key":"e_1_3_4_51_2","doi-asserted-by":"publisher","DOI":"10.1104\/pp.114.237941"},{"key":"e_1_3_4_52_2","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/btp324"},{"key":"e_1_3_4_53_2","doi-asserted-by":"publisher","DOI":"10.1093\/gigascience\/giab008"},{"key":"e_1_3_4_54_2","doi-asserted-by":"publisher","DOI":"10.1071\/PP9940449"},{"key":"e_1_3_4_55_2","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-62703-995-6_15"},{"key":"e_1_3_4_56_2","doi-asserted-by":"publisher","DOI":"10.1046\/j.1365-313x.2001.01056.x"},{"key":"e_1_3_4_57_2","unstructured":"M. D. Shoulders In vivo directed evolution of an ultrafast Rubisco from a semianaerobic environment imparts oxygen resistance. NCBI. https:\/\/www.ncbi.nlm.nih.gov\/bioproject\/PRJNA1275035\/. Deposited 11 June 2025."},{"key":"e_1_3_4_58_2","doi-asserted-by":"crossref","unstructured":"J. L. McDonald et al. Plasmids from \u201cIn vivo directed evolution of an ultrafast Rubisco from a semi-anaerobic environment imparts oxygen resistance.\u201d Addgene. https:\/\/www.addgene.org\/browse\/article\/28253576\/. Accessed 5 April 2025.","DOI":"10.1101\/2025.02.17.638297"}],"container-title":["Proceedings of the National Academy of Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pnas.org\/doi\/pdf\/10.1073\/pnas.2505083122","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,6,30]],"date-time":"2025-06-30T15:45:41Z","timestamp":1751298341000},"score":1,"resource":{"primary":{"URL":"https:\/\/pnas.org\/doi\/10.1073\/pnas.2505083122"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,6,30]]},"references-count":58,"journal-issue":{"issue":"27","published-print":{"date-parts":[[2025,7,8]]}},"alternative-id":["10.1073\/pnas.2505083122"],"URL":"https:\/\/doi.org\/10.1073\/pnas.2505083122","relation":{},"ISSN":["0027-8424","1091-6490"],"issn-type":[{"value":"0027-8424","type":"print"},{"value":"1091-6490","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,6,30]]},"assertion":[{"value":"2025-03-11","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication History"}},{"value":"2025-05-19","order":2,"name":"accepted","label":"Accepted","group":{"name":"publication_history","label":"Publication History"}},{"value":"2025-06-30","order":3,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}],"article-number":"e2505083122"}}