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

Abstract Protein mannosyltransferases (PMTs) catalyze the O -mannosylation of serine and threonine residues of proteins in the endoplasmic reticulum. The five PMT genes coding for protein mannosyltransferases, designated as PMT1, 2, 4, 5 and 6, were identified from Pichia pastoris genome based on the homology to PMT genes in Saccharomyces cerevisiae genome, which has seven PMT genes. The homologues of S. cerevisiae PMT 3 & 7 genes are absent in P. pastoris genome. Approximately 5% of the recombinant insulin precursor expressed in P. pastoris is O -mannosylated. In this study, we attempted to prevent O -mannosylation of insulin precursor in vivo , through inactivation of the Pichia PMT genes. Since multiple PMTs are found to be expressed, it was important to understand which of these are involved in O -mannosylation of the insulin precursor. The genes encoding PMT1 , 4 , 5 and 6 were knocked out by insertional inactivation method. Inactivation of PMT genes 4, 5 and 6 showed ∼16–28% reductions in the O -mannosylation of insulin precursor. The PMT1 gene disrupted Pichia clone showed ∼60% decrease in O -mannosylated insulin precursor, establishing its role as an important enzyme for insulin precursor O -mannosylation.