Search

Abstract Background 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. Yarrowia lipolytica 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 Y. lipolytica to abolish the heterogeneous, yeast-specific glycosylation and to obtain homogeneous human high-mannose type glycosylation. Results We engineered Y. lipolytica to produce homogeneous human-type terminal-mannose glycosylated proteins, i.e. glycosylated with Man8GlcNAc2 or Man5GlcNAc2. First, we inactivated the yeast-specific Golgi α-1,6-mannosyltransferases YlOch1p and YlMnn9p; 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 YlOch1p and YlMnn9p, 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. Conclusions We generated a Y. lipolytica 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 Y. lipolytica 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.

An engineering of the P. pastoris N-glycosylation pathway to produce nonhyperglycosylated hybrid glycans are described. This is done by inactivation of OCH1 and over expression of an alpha-1,2-mannosidase retained in the endoplasmic reticulum and N-acetyglucosaminyltransferase I and beta-1,4-hybrid-type N-linked oligosaccharide structure on its glycoproteins.

Yarrowia lipolytica is a dimorphic yeast that efficiently secretes various heterologous proteins and is classified as "generally recognized as safe." Therefore, it is an attractive protein production host. However, yeasts modify glycoproteins with nonhuman high mannose-type N-glycans. These structures reduce the protein half-life in vivo and can be immunogenic in man. Here, we describe how we genetically engineered N-glycan biosynthesis in Yarrowia lipolytica so that it produces Man3GlcNAc2 structures on its glycoproteins. We obtained unprecedented levels of homogeneity of this glycanstructure. This is the ideal starting point for building human-like sugars. Disruption of the ALG3 gene resulted in modification of proteins mainly with Man5GlcNAc2 and GlcMan5GlcNAc2 glycans, and to a lesser extent with Glc2Man5GlcNAc2 glycans. To avoid underoccupancy of glycosylation sites, we concomitantly overexpressed ALG6. We also explored several approaches to remove the terminal glucose residues, which hamper further humanization of N-glycosylation; overexpression of the heterodimeric Apergillus niger glucosidase II proved to be the most effective approach. Finally, we overexpressed an α-1,2- mannosidase to obtain Man3GlcNAc2 structures, which are substrates for the synthesis of complex-type glycans. The final Yarrowia lipolytica strain produces proteins glycosylated with the trimannosyl core N-glycan (Man3GlcNAc2), which is the common core of all complex-type N-glycans. All these glycans can be constructed on the obtained trimannosyl N-glycan using either in vivo or in vitro modification with the appropriate glycosyltransferases. The results demonstrate the high potential of Yarrowia lipolytica to be developed as an efficient expression system for the production of glycoproteins with humanized glycans. [ABSTRACT FROM AUTHOR]

Background: 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. Yarrowia lipolytica 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 Y. lipolytica to abolish the heterogeneous, yeast-specific glycosylation and to obtain homogeneous human highmannose type glycosylation. Results: We engineered Y. lipolytica to produce homogeneous human-type terminal-mannose glycosylated proteins, i.e. glycosylated with Man8GlcNAc2 or Man5GlcNAc2. First, we inactivated the yeast-specific Golgi α-1,6- mannosyltransferases YlOch1p and YlMnn9p; 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 YlOch1p and YlMnn9p, 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. Conclusions: We generated a Y. lipolytica 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 Y. lipolytica 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. [ABSTRACT FROM AUTHOR]

Background: 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. Yarrowia lipolytica 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 Y. lipolytica to abolish the heterogeneous, yeast-specific glycosylation and to obtain homogeneous human highmannose type glycosylation. Results: We engineered Y. lipolytica to produce homogeneous human-type terminal-mannose glycosylated proteins, i.e. glycosylated with Man8GlcNAc2 or Man5GlcNAc2. First, we inactivated the yeast-specific Golgi α-1,6- mannosyltransferases YlOch1p and YlMnn9p; 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 YlOch1p and YlMnn9p, 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. Conclusions: We generated a Y. lipolytica 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 Y. lipolytica 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. [ABSTRACT FROM AUTHOR]

Background: Non-invasive staging of human liver fibrosis is a desirable objective that remains under extensive evaluation. Animal model systems are often used for studying human liver disease and screening antifibrotic compounds. The aim of the present study was to investigate the potential use of serum N-glycan profiles to evaluate liver fibrosis in a rat model. Methods: Liver fibrosis and cirrhosis were induced in rats by oral administration of CCl4. Liver injury was assessed biochemically (alanine aminotransferase [ALT] activity, aspartate aminotransferase [AST] activity and total bilirubin) and histologically. The N-glycan profile (GlycoTest) was performed using DNA sequencer-assisted–fluorophore-assisted carbohydrate electrophoresis technology. In parallel, the effect of cotreatment with antifibrotic interferon-γ (IFN-γ) was studied. Results: The biopsy scoring system showed that CCl4 induced early fibrosis ( F < 1–2) in rats after 3 weeks of treatment, and cirrhosis (F4) after 12 weeks. Significant increases in ALT activity, AST activity and total bilirubin levels were detected only after 12 weeks of CCl4 treatment. GlycoTest showed three glycans were significantly altered in the CCl4-goup. Peak 3 started at week 6, at an early stage in fibrosis development ( F < 1–2), whereas peaks 4 and 5 occurred at week 9, at which time mild liver fibrosis ( F = 1–2) had developed. The changes in the CCl4-IFN-γ group were intermediate between the CCl4- and the control groups. Conclusion: The GlycoTest is much more sensitive than biochemical tests for evaluating liver fibrosis/cirrhosis in the rat model. The test can also be used as a non-invasive marker for screening and monitoring the antifibrotic activity of potential therapeutic compounds. [ABSTRACT FROM AUTHOR]

A correction to the article "Human Gut Bacteroidetes Can Utilize Yeast Mannan Through A Selfish Mechanism" by Fiona Cuskin, Max J. Temple and Yanping Zhu published in "Nature" Vol 517 2015 issue is presented.

Glycosylation is an important issue in heterologous protein production for therapeutic applications. Glycoproteins produced in Pichia pastoris contain high mannose glycan structures that can hamper downstream processing, might be immunogenic, and cause rapid clearance from the circulation. This chapter describes a method that helps solving these glycosylation-related problems by inactivation of OCH1, overexpression of an HDEL-tagged mannosidase, and overexpression of a Kre2/GlcNAc-transferase I chimeric enzyme. Different plasmids are described as well as glycan analysis methods.