Disclosed herein are novel Pichia pastoris strains for expression of exogenous proteins with substantially homogeneous N-glycans. The strains are genetically engineered to include a mutant OCH1 allele which is transcribed into an mRNA coding for a mutant OCH1 gene product (i.e., -1,6-mannosyltransferase, or OCH1 protein). The mutant OCH1protein contains a catalytic domain substantially identical to that of the wild type OCH1 protein, but lacks an N-terminal sequence necessary to target the OCH1 protein to the Golgi apparatus. The strains disclosed herein are robust, stable, and transformable, and the mutant OCH1 allele and the ability to produce substantially homogeneous N-glycans are maintained for generations after rounds of freezing and thawing and after subsequent transformations.

The invention relates to the field of glycoprotein production means and methods. More specifically, the present invention provides for a novel yeast strain with a mutant Och1 gene resulting in modified N-glycosylation properties of said strain. Even more specifically, said mutant Och1 gene comprises a sequence coding for a mutant OCH1 protein in which one amino acid near the catalytic site, corresponding to position 151 of the OCH1 protein of the methylotrophic wild type Pichia pastoris strain, is deleted. More specifically, the use of said mutant yeast strain for recombinant expression of glycoproteins results in more homogenous mammalian-like N-glycan structures on said heterologously produced glycoproteins.