{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T09:02:19Z","timestamp":1785402139561,"version":"3.56.0"},"posted":{"date-parts":[[2025,2,17]]},"group-title":"Biochemistry","reference-count":56,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,2,17]],"date-time":"2025-02-17T00:00:00Z","timestamp":1739750400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"funder":[{"award":["2244770"],"award-info":[{"award-number":["2244770"]}],"id":[{"id":"https:\/\/ror.org\/021nxhr62","id-type":"ROR","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000057","name":"National Institute of General Medical Sciences","doi-asserted-by":"crossref","award":["1R35GM136354"],"award-info":[{"award-number":["1R35GM136354"]}],"id":[{"id":"10.13039\/100000057","id-type":"DOI","asserted-by":"crossref"}]},{"name":"Abdul Latif Jameel Water and Food Systems Lab"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,5,5]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  Carbon dioxide (CO\n                  <jats:sub>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                  <jats:inline-formula>\n                    <jats:inline-graphic xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" xlink:href=\"638297v4_inline1.gif\"\/>\n                  <\/jats:inline-formula>\n                  and is competitively inhibited by oxygen (O\n                  <jats:sub>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                  <jats:italic>Gallionellaceae<\/jats:italic>\n                  bacteria, which comprise a dimer or hexamer of large subunits, arises from their nearly 5-fold higher\n                  <jats:inline-formula>\n                    <jats:inline-graphic xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" xlink:href=\"638297v4_inline2.gif\"\/>\n                  <\/jats:inline-formula>\n                  than the average Rubisco enzyme. As well as having a fast\n                  <jats:inline-formula>\n                    <jats:inline-graphic xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" xlink:href=\"638297v4_inline3.gif\"\/>\n                  <\/jats:inline-formula>\n                  (25.8 s\n                  <jats:bold>\n                    <jats:sup>\u2212<\/jats:sup>\n                  <\/jats:bold>\n                  <jats:sup>1<\/jats:sup>\n                  at 25 \u00b0C), we show that\n                  <jats:italic>Gallionellaceae<\/jats:italic>\n                  Rubisco (GWS1B) is extremely sensitive to O\n                  <jats:sub>2<\/jats:sub>\n                  inhibition, consistent with its evolution under semi-anaerobic environments. We therefore used a novel\n                  <jats:italic>in vivo<\/jats:italic>\n                  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                  <jats:sub>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                  <jats:sub>2<\/jats:sub>\n                  at 25 \u00b0C. By contrast, under the near anaerobic natural environment of\n                  <jats:italic>Gallionellaceae<\/jats:italic>\n                  , the carboxylation efficiency of each mutant was impaired \u223c16%. These findings demonstrate the efficacy of artificial directed evolution to access novel regions of catalytic space in Rubisco.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>Significance<\/jats:title>\n                  <jats:p>\n                    Given Rubisco\u2019s crucial role in carbon dioxide assimilation, addressing its slow carboxylation rate and oxygen inhibition is a significant challenge. Utilizing one of the fastest known, yet also highly oxygen-sensitive, Rubisco \u2013 from the bacteria\n                    <jats:italic>Gallionellaceae<\/jats:italic>\n                    \u2013 we applied a novel\n                    <jats:italic>in vivo<\/jats:italic>\n                    directed evolution pipeline in\n                    <jats:italic>Escherichia coli<\/jats:italic>\n                    to discover mutations that specifically enhance carboxylation efficiency under ambient oxygen, a condition distinct from\n                    <jats:italic>Gallionellaceae\u2019s<\/jats:italic>\n                    natural semi-anaerobic environment. Our findings underscore the potential of directed evolution to unlock new catalytic capabilities for Rubisco, with implications for both fundamental research and practical agricultural applications.\n                  <\/jats:p>\n                <\/jats:sec>","DOI":"10.1101\/2025.02.17.638297","type":"posted-content","created":{"date-parts":[[2025,2,17]],"date-time":"2025-02-17T22:20:34Z","timestamp":1739830834000},"source":"Crossref","is-referenced-by-count":5,"title":["<i>In Vivo<\/i>\n                  Directed Evolution of an Ultra-Fast Rubisco from a Semi-Anaerobic Environment Imparts Oxygen Resistance"],"prefix":"10.64898","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, Cambridge, MA, 02139"},{"name":"Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, 02139"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nathan P.","family":"Shapiro","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA, 02139"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Amanuella A.","family":"Mengiste","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA, 02139"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sarah","family":"Kaines","sequence":"additional","affiliation":[{"name":"Plant Science Division, Research School of Biology, The Australian National University, Canberra, ACT 0200, 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, Canberra, ACT 0200, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Robert H.","family":"Wilson","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA, 02139"}],"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, Cambridge, MA, 02139"},{"name":"Broad Institute of MIT and Harvard, Cambridge, MA, 02139"},{"name":"Koch Institute for Integrative Cancer Research at MIT, Cambridge, MA, 02139"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026041120370145000_2025.02.17.638297v4.1","doi-asserted-by":"publisher","DOI":"10.1146\/annurev-biochem-040320-101244"},{"key":"2026041120370145000_2025.02.17.638297v4.2","doi-asserted-by":"publisher","DOI":"10.15252\/embj.2019104081"},{"key":"2026041120370145000_2025.02.17.638297v4.3","doi-asserted-by":"publisher","DOI":"10.1104\/pp.110.164814"},{"key":"2026041120370145000_2025.02.17.638297v4.4","doi-asserted-by":"publisher","DOI":"10.1093\/jxb\/erm361"},{"key":"2026041120370145000_2025.02.17.638297v4.5","doi-asserted-by":"publisher","DOI":"10.1126\/science.abq1416"},{"key":"2026041120370145000_2025.02.17.638297v4.6","doi-asserted-by":"publisher","DOI":"10.1021\/ACS.BIOCHEM.9B00237\/SUPPL_FILE\/BI9B00237_SI_005.XLSX"},{"key":"2026041120370145000_2025.02.17.638297v4.7","doi-asserted-by":"publisher","DOI":"10.1146\/annurev-arplant-043015-111709"},{"key":"2026041120370145000_2025.02.17.638297v4.8","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.2012288117"},{"key":"2026041120370145000_2025.02.17.638297v4.9","doi-asserted-by":"publisher","DOI":"10.1111\/j.1472-4669.2007.00127.x"},{"key":"2026041120370145000_2025.02.17.638297v4.10","doi-asserted-by":"crossref","unstructured":"T. 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