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As photosynthetic unicellular organisms, they do not require costly and complex media for growing and are able to secrete proteins and perform protein glycosylation. Some biologics have been successfully produced in the green microalgae <jats:italic>Chlamydomonas reinhardtii.<\/jats:italic> However, post\u2010translational modifications like glycosylation of these Chlamydomonas\u2010made biologics have poorly been investigated so far. Therefore, in this study, we report on the first structural investigation of glycans linked to human erythropoietin (hEPO) expressed in a wild\u2010type <jats:italic>C. reinhardtii<\/jats:italic> strain and mutants impaired in key Golgi glycosyltransferases. The glycoproteomic analysis of recombinant hEPO (rhEPO) expressed in the wild\u2010type strain demonstrated that the three <jats:italic>N<\/jats:italic>\u2010glycosylation sites are 100% glycosylated with mature <jats:italic>N<\/jats:italic>\u2010glycans containing four to five mannose residues and carrying core xylose, core fucose and <jats:italic>O<\/jats:italic>\u2010methyl groups. Moreover, expression in <jats:italic>C. reinhardtii<\/jats:italic> insertional mutants defective in <jats:italic>xylosyltransferases A<\/jats:italic> and <jats:italic>B<\/jats:italic> and <jats:italic>fucosyltransferase<\/jats:italic> resulted in drastic decreases of core xylosylation and core fucosylation of glycans <jats:italic>N<\/jats:italic>\u2010linked to the rhEPOs, thus demonstrating that this strategy offers perspectives for humanizing the <jats:italic>N<\/jats:italic>\u2010glycosylation of the Chlamydomonas\u2010made biologics.<\/jats:p>","DOI":"10.1111\/pbi.14424","type":"journal-article","created":{"date-parts":[[2024,7,5]],"date-time":"2024-07-05T17:40:20Z","timestamp":1720201220000},"page":"3018-3027","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":10,"title":["Fine\u2010tuning the <i>N<\/i>\u2010glycosylation of recombinant human erythropoietin using <i>Chlamydomonas reinhardtii<\/i> mutants"],"prefix":"10.1111","volume":"22","author":[{"given":"S.","family":"Leprovost","sequence":"first","affiliation":[{"name":"Universit\u00e9 de Rouen Normandie, Normandie Univ, GlycoMEV UR 4358, SFR Normandie V\u00e9g\u00e9tal FED 4277, Innovation Chimie Carnot, IRIB, GDR CNRS Chemobiologie  Rouen France"},{"name":"Institute for Plant Biology and Biotechnology (IBBP), University of M\u00fcnster  M\u00fcnster Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"C.","family":"Plasson","sequence":"additional","affiliation":[{"name":"Universit\u00e9 de Rouen Normandie, Normandie Univ, GlycoMEV UR 4358, SFR Normandie V\u00e9g\u00e9tal FED 4277, Innovation Chimie Carnot, IRIB, GDR CNRS Chemobiologie  Rouen France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"J.","family":"Balieu","sequence":"additional","affiliation":[{"name":"Universit\u00e9 de Rouen Normandie, Normandie Univ, GlycoMEV UR 4358, SFR Normandie V\u00e9g\u00e9tal FED 4277, Innovation Chimie Carnot, IRIB, GDR CNRS Chemobiologie  Rouen France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"M\u2010L.","family":"Walet\u2010Balieu","sequence":"additional","affiliation":[{"name":"Infrastructure de Recherche HeRacLeS, Plate\u2010forme prot\u00e9omique PISSARO, Universit\u00e9 de Rouen Normandie  Rouen France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"P.","family":"Lerouge","sequence":"additional","affiliation":[{"name":"Universit\u00e9 de Rouen Normandie, Normandie Univ, GlycoMEV UR 4358, SFR Normandie V\u00e9g\u00e9tal FED 4277, Innovation Chimie Carnot, IRIB, GDR CNRS Chemobiologie  Rouen France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"M.","family":"Bardor","sequence":"additional","affiliation":[{"name":"Universit\u00e9 de Rouen Normandie, Normandie Univ, GlycoMEV UR 4358, SFR Normandie V\u00e9g\u00e9tal FED 4277, Innovation Chimie Carnot, IRIB, GDR CNRS Chemobiologie  Rouen France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3377-6169","authenticated-orcid":false,"given":"E.","family":"Mathieu\u2010Rivet","sequence":"additional","affiliation":[{"name":"Universit\u00e9 de Rouen Normandie, Normandie Univ, GlycoMEV UR 4358, SFR Normandie V\u00e9g\u00e9tal FED 4277, Innovation Chimie Carnot, IRIB, GDR CNRS Chemobiologie  Rouen France"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2024,7,5]]},"reference":[{"key":"e_1_2_8_2_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0300-9084(01)01297-4"},{"key":"e_1_2_8_3_1","doi-asserted-by":"publisher","DOI":"10.3390\/md21060342"},{"key":"e_1_2_8_4_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pgen.1008944"},{"key":"e_1_2_8_5_1","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gky532"},{"key":"e_1_2_8_6_1","doi-asserted-by":"publisher","DOI":"10.3390\/molecules27165119"},{"key":"e_1_2_8_7_1","doi-asserted-by":"publisher","DOI":"10.1093\/glycob\/cwg024"},{"key":"e_1_2_8_8_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0257089"},{"key":"e_1_2_8_9_1","doi-asserted-by":"publisher","DOI":"10.3389\/fpls.2020.610032"},{"key":"e_1_2_8_10_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41587-021-00884-w"},{"key":"e_1_2_8_11_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M112.402685"},{"key":"e_1_2_8_12_1","doi-asserted-by":"publisher","DOI":"10.1186\/s13007-017-0183-5"},{"key":"e_1_2_8_13_1","doi-asserted-by":"publisher","DOI":"10.34172\/bi.2020.33"},{"key":"e_1_2_8_14_1","doi-asserted-by":"publisher","DOI":"10.1007\/s00425-008-0879-x"},{"key":"e_1_2_8_15_1","doi-asserted-by":"publisher","DOI":"10.1021\/acssynbio.0c00632"},{"key":"e_1_2_8_16_1","first-page":"90","article-title":"Engineering a powerful green cell factory for robust photoautotrophic diterpenoid production","volume":"82","author":"Einhaus A.","year":"2022","journal-title":"Metab. 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