Cloning and characterization of the glucosidase II alpha subunit gene of Trichoderma reesei: a frameshift mutation results in the aberrant glycosylation profile of the hypercellulolytic strain Rut-C30

We describe isolation and characterization of the gene encoding the glucosidase II alpha subunit (GIIα) of the industrially important fungus Trichoderma reesei. This subunit is the catalytic part of the glucosidase II heterodimeric enzyme involved in the structural modification within the endoplasmic reticulum (ER) of N-linked oligosaccharides present on glycoproteins. The gene encoding GIIα (gls2α) in the hypercellulolytic strain Rut-C30 contains a frameshift mutation resulting in a truncated gene product. Based on the peculiar monoglucosylated N-glycan pattern on proteins produced by the strain, we concluded that the truncated protein can still hydrolyze the first α-1,3-linked glucose residue but not the innermost α-1,3-linked glucose residue from the Glc(2)Man(9)GlcNAc(2) N-glycan ER structure. Transformation of the Rut-C30 strain with a repaired T. reesei gls2α gene changed the glycosylation profile significantly, decreasing the amount of monoglucosylated structures and increasing the amount of high-mannose N-glycans. Full conversion to high-mannose carbohydrates was not obtained, and this was probably due to competition between the endogenous mutant subunit and the introduced wild-type GIIα protein. Since glucosidase II is also involved in the ER quality control of nascent polypeptide chains, its transcriptional regulation was studied in a strain producing recombinant tissue plasminogen activator (tPA) and in cultures treated with the stress agents dithiothreitol (DTT) and brefeldin A (BFA), which are known to block protein transport and to induce the unfolded protein response. While the mRNA levels were clearly upregulated upon tPA production or BFA treatment, no such enhancement was observed after DTT addition

Engineering the yeast <it>Yarrowia lipolytica</it> for the production of therapeutic proteins homogeneously glycosylated with Man₈GlcNAc₂ and Man₅GlcNAc₂

<p>Abstract</p> <p>Background</p> <p>Protein-based therapeutics represent the fastest growing class of compounds in the pharmaceutical industry. This has created an increasing demand for powerful expression systems. Yeast systems are widely used, convenient and cost-effective. <it>Yarrowia lipolytica</it> is a suitable host that is generally regarded as safe (GRAS). Yeasts, however, modify their glycoproteins with heterogeneous glycans containing mainly mannoses, which complicates downstream processing and often interferes with protein function in man. Our aim was to glyco-engineer <it>Y. lipolytica</it> to abolish the heterogeneous, yeast-specific glycosylation and to obtain homogeneous human high-mannose type glycosylation.</p> <p>Results</p> <p>We engineered <it>Y. lipolytica</it> to produce homogeneous human-type terminal-mannose glycosylated proteins, <it>i.e.</it> glycosylated with Man₈GlcNAc₂ or Man₅GlcNAc₂. First, we inactivated the yeast-specific Golgi α-1,6-mannosyltransferases <it>Yl</it>Och1p and <it>Yl</it>Mnn9p; the former inactivation yielded a strain producing homogeneous Man₈GlcNAc₂ glycoproteins. We tested this strain by expressing glucocerebrosidase and found that the hypermannosylation-related heterogeneity was eliminated. Furthermore, detailed analysis of N-glycans showed that <it>Yl</it>Och1p and <it>Yl</it>Mnn9p, despite some initial uncertainty about their function, are most likely the α-1,6-mannosyltransferases responsible for the addition of the first and second mannose residue, respectively, to the glycan backbone. Second, introduction of an ER-retained α-1,2-mannosidase yielded a strain producing proteins homogeneously glycosylated with Man₅GlcNAc₂. The use of the endogenous LIP2pre signal sequence and codon optimization greatly improved the efficiency of this enzyme.</p> <p>Conclusions</p> <p>We generated a <it>Y. lipolytica</it> expression platform for the production of heterologous glycoproteins that are homogenously glycosylated with either Man₈GlcNAc₂ or Man₅GlcNAc₂ N-glycans. This platform expands the utility of <it>Y. lipolytica</it> as a heterologous expression host and makes it possible to produce glycoproteins with homogeneously glycosylated N-glycans of the human high-mannose-type, which greatly broadens the application scope of these glycoproteins.</p

MOESM1 of Pichia pastoris MutS strains are prone to misincorporation of O-methyl-l-homoserine at methionine residues when methanol is used as the sole carbon source

Additional file 1: Figure S1. MS/MS fragmentation of four peptides indicates that the Δ−16 Da modification was located at methionine residues. Typical peptide fragmentation generates b or y ions of different mass to charge ratios. Correspondence of the experimentally determined masses to the molecular masses of the amino acid residues can be used to derive the sequence of the parent ion. Fragmentation by collisional-induced dissociation of the peptides 2, 10 and 16 showed via the b and/or y ions that the modification of Δ−16 Da is indeed located at the methionine residue in all peptides. The fragmentation of peptide 8 was unsuccessful. However, an aspecific tryptic cleavage product of peptide 8 could be used for the fragmentation and the b and y ions showed again that the modification of Δ−16 Da is located at the methionine residue. A, C, E, G Show the theoretical spectral fragments of each peptide; B, D, F, H, show the observed spectral fragmentation of each peptide