PMT1 gene plays a major role in O-mannosylation of insulin precursor in Pichia pastoris

► Protein O-mannosyltransferase 1 is the key enzyme involved in O-mannosylation. ► Multiple PMT genes from Pichia were inactivated and checked for reduction in O-mannosylation of insulin precursor. ► PMT1 gene inactivation resulted in ∼60% reduction in O-mannosylation of insulin precursor. ► The mannosylation site was identified by peptide mapping using the protease Glu-C and mass spectroscopy.