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This activity is interesting for the synthesis of the disialylated oligosaccharide disialyllacto-\n                      <jats:italic>N<\/jats:italic>\n                      -tetraose (DSLNT). Human sialyltransferases ST6GalNAc5 and ST6GalNAc6 produced in HEK293 cells are commercially available at a smaller scale. In this study, we demonstrated that ST6GalNAc5 and ST6GalNAc6 can be functionally expressed in\n                      <jats:italic>Pichia pastoris<\/jats:italic>\n                      X-33. The level of ST6GalNAc5 and ST6GalNAc6 expression and activity largely depended on the type of construct, as well as on expression conditions, namely temperature, methanol feeding regime, and supplements. Insertion of a (GGGS)\u2082 linker peptide between the gene and the \u03b1 secretion factor improved the secretion of active enzyme in\n                      <jats:italic>P. pastoris<\/jats:italic>\n                      X-33. The use of media supplemented with MgCl\n                      <jats:sub>2<\/jats:sub>\n                      and Casamino acids led to increased cell growth and, importantly, enhanced ST6GalNAc5 and ST6GalNAc6 production. Under optimized conditions, the\n                      <jats:italic>P. pastoris<\/jats:italic>\n                      X-33 strain could secrete up to 10\u00a0mg of active sialyltransferase protein per liter of culture. Compared to their wild-type counterparts, mutants of ST6GalNAc5 and ST6GalNAc6 devoid of\n                      <jats:italic>N<\/jats:italic>\n                      -glycosylation sites exhibited reduced enzymatic activity and stability. Apart from contributing to successful\n                      <jats:italic>P. pastoris<\/jats:italic>\n                      expression, our findings also contribute to a deeper understanding of the role of\n                      <jats:italic>N<\/jats:italic>\n                      -glycosylation in the activity and stability of sialyltransferases.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Key points<\/jats:title>\n                    <jats:p>\n                      \u2022\u00a0\n                      <jats:italic>Expression of functional human ST6GalNAc5 and ST6GalNAc6 in Pichia pastoris<\/jats:italic>\n                    <\/jats:p>\n                    <jats:p>\n                      \u2022\u00a0\n                      <jats:italic>Mutants devoid of N-glycosylations lack activity<\/jats:italic>\n                    <\/jats:p>\n                    <jats:p>\n                      \u2022\u00a0\n                      <jats:italic>Media supplementation with MgCl2 and Casamino acids improves expression<\/jats:italic>\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1007\/s00253-025-13607-x","type":"journal-article","created":{"date-parts":[[2025,10,15]],"date-time":"2025-10-15T01:57:02Z","timestamp":1760493422000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Expression of functional human sialyltransferases ST6GalNAc5 and ST6GalNAc6 in Pichia pastoris"],"prefix":"10.1007","volume":"109","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-1538-3115","authenticated-orcid":false,"given":"Ganna","family":"Krasnoselska","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8473-2361","authenticated-orcid":false,"given":"Marton","family":"Lengyel","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Martin","family":"Matwiejuk","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1776-7844","authenticated-orcid":false,"given":"Marlene","family":"Vuillemin","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Dora","family":"Molnar-Gabor","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8910-9931","authenticated-orcid":false,"given":"Anne S.","family":"Meyer","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8593-6742","authenticated-orcid":false,"given":"Birgitte","family":"Zeuner","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"297","published-online":{"date-parts":[[2025,10,15]]},"reference":[{"key":"13607_CR1","doi-asserted-by":"publisher","first-page":"9","DOI":"10.1016\/j.ejbt.2016.01.007","volume":"21","author":"RP Ang","year":"2016","unstructured":"Ang RP, Teoh LS, Chan MK et al (2016) Comparing the expression of human DNA topoisomerase I in KM71H and X33 strains of Pichia pastoris. 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