biostudies-literaturevan der Kaaij AThe International Coordination of Research on Infectious Animal DiseasesEurostarsNetherlands Organisation for Scientific Research, Veni grantDutch Research Council (NWO)The center for unusual collaboration (CUCo)224-238https://www.ebi.ac.uk/biostudies/studies/S-EPMC12854890biostudies-literatureUnknown24(1)Glycosylation is an important aspect for many biopharmaceuticals, including vaccines against parasitic helminths. Plants, especially Nicotiana benthamiana, have proven to be excellent production hosts for biopharmaceuticals with tailor-made glycosylation. If desired, galactosylation can be introduced on biopharmaceuticals through co-expression of the appropriate glycosyltransferase. However, achieving homogenous glycoforms with terminal galactose residues remains difficult as native N. benthamiana β-galactosidases (NbBGALs) truncate these glycans. Recently, the first NbBGAL has been identified, but a knockout line was insufficient to achieve near complete galactosylation, suggesting that other enzymes could have similar activity. In this study, we selected 10 NbBGALs for further investigatPlant biotechnology journalIdentification of β-galactosidases along the secretory pathway of Nicotiana benthamiana that collectively hamper engineering of galactose-extended glycans on recombinant glycoproteins.PMC1285489016740E! 115017Bunte MJMMokhtari SSchots AWilbers RHPNijhof LNibbering Pvan der Kaaij AOvermars HfalseIdentification of β-galactosidases along the secretory pathway of Nicotiana benthamiana that collectively hamper engineering of galactose-extended glycans on recombinant glycoproteins.Glycosylation is an important aspect for many biopharmaceuticals, including vaccines against parasitic helminths. Plants, especially Nicotiana benthamiana, have proven to be excellent production hosts for biopharmaceuticals with tailor-made glycosylation. If desired, galactosylation can be introduced on biopharmaceuticals through co-expression of the appropriate glycosyltransferase. However, achieving homogenous glycoforms with terminal galactose residues remains difficult as native N. benthamiana β-galactosidases (NbBGALs) truncate these glycans. Recently, the first NbBGAL has been identified, but a knockout line was insufficient to achieve near complete galactosylation, suggesting that other enzymes could have similar activity. In this study, we selected 10 NbBGALs for further investigat2026-01-01T00:00:00Z2026 Jan2026-06-09T06:47:30.796Z2026-06-09T03:11:52.113ZS-EPMC128548904033370610.1111/pbi.70126