Glycoengineering of yeasts from the perspective of glycosylation efficiency

Piirainen, Mari A.; Ruijter, Jorg C. de; Koskela, Essi V.; Frey, Alexander D.

doi:10.1016/j.nbt.2014.03.001

Cited by 18 publications

(8 citation statements)

References 23 publications

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“…No doubt Nglycosylation is one of the most common forms of protein post-translational modification in P. pastoris, where approximately 70-90 % Asn residues included in potential Nglycosylation sites (Asn-Xaa-Ser/Thr) are N-glycosylated. This process is species-specific (Piirainen et al 2014) and often plays a crucial role in the secretion, stability, and/or activity of proteins expressed in P. pastoris (Han et al 2014;Skropeta 2009). Thus, glycosylation degree was essential for secretion of the fungi Volvariella volvacea acetyl xylan esterase (Tian et al 2012), thermal stability of the Candida rugosa lipase (Tang et al 2001), catalytic activity and stability of the horseradish peroxidase (Capone et al 2014), or the biochemical properties of β-glucuronidase GUS from Penicillium purpurogenum (Zou et al 2013).…”

Section: Discussionmentioning

confidence: 99%

Heterologous overproduction of β-fructofuranosidase from yeast Xanthophyllomyces dendrorhous, an enzyme producing prebiotic sugars

Gimeno-Pérez

Linde

Fernández‐Arrojo

et al. 2014

Appl Microbiol Biotechnol

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The β-fructofuranosidase Xd-INV from the yeast Xanthophyllomyces dendrorhous is the largest microbial enzyme producing neo-fructooligosaccharides (neo-FOS) known to date. It mainly synthesizes neokestose and neonystose, oligosaccharides with potentially improved prebiotic properties. The Xd-INV gene comprises an open reading frame of 1995 bp, which encodes a 665-amino acid protein. Initial N-terminal sequencing of Xd-INV pointed to a majority extracellular protein of 595 amino acids lacking the first 70 residues (potential signal peptide). Functionality of the last 1785 bp of Xd-INV gene was previously proved in Saccharomyces cerevisiae but only weak β-fructofuranosidase activity was quantified. In this study, different strategies to improve this enzyme level in a heterologous system have been used. Curiously, best results were obtained by increasing the protein N-terminus sequence in 39 amino acids, protein of 634 residues. The higher β-fructofuranosidase activity detected in this study, about 15 U/mL, was obtained using Pichia pastoris and represents an improvement of about 1500 times the level previously obtained in a heterologous organism and doubles the best level of activity obtained by the natural producer. Heterologously expressed protein was purified and characterized biochemically and kinetically. Except by its glycosylation degree (10 % lower) and thermal stability (4-5 °C lower in the 60-85 °C range), the properties of the heterologous enzyme, including ability to produce neo-FOS, remained unchanged. Interestingly, besides the neo-FOS referred before blastose was also detected (8-22 g/L) in the reaction mixtures, making Xd-INV the first yeast enzyme producing this non-conventional disaccharide reported to date.

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Section: Discussionmentioning

confidence: 99%

Heterologous overproduction of β-fructofuranosidase from yeast Xanthophyllomyces dendrorhous, an enzyme producing prebiotic sugars

Gimeno-Pérez

Linde

Fernández‐Arrojo

et al. 2014

Appl Microbiol Biotechnol

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“…Sc, S. cerevisiae; Pp, P. pastoris; Sp, S. pombe; Kl, K. lactis; Hp, H. polymorpha; Yl, Y. lipolytica. strains, which ensures the production of glycoproteins fully glycosylated with homogeneous N-glycans (Piirainen et al, 2014).…”

Section: Prb1mentioning

confidence: 99%

Yeast synthetic biology for the production of recombinant therapeutic proteins

2014

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The production of recombinant therapeutic proteins is one of the fast-growing areas of molecular medicine and currently plays an important role in treatment of several diseases. Yeasts are unicellular eukaryotic microbial host cells that offer unique advantages in producing biopharmaceutical proteins. Yeasts are capable of robust growth on simple media, readily accommodate genetic modifications, and incorporate typical eukaryotic post-translational modifications. Saccharomyces cerevisiae is a traditional baker's yeast that has been used as a major host for the production of biopharmaceuticals; however, several nonconventional yeast species including Hansenula polymorpha, Pichia pastoris, and Yarrowia lipolytica have gained increasing attention as alternative hosts for the industrial production of recombinant proteins. In this review, we address the established and emerging genetic tools and host strains suitable for recombinant protein production in various yeast expression systems, particularly focusing on current efforts toward synthetic biology approaches in developing yeast cell factories for the production of therapeutic recombinant proteins.

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“…Various studies have measured glycosylation occupancy in clinical samples such as plasma , saliva , and tissue for the purpose of biomarker discovery for diverse diseases. Heterologous expression of glycoproteins often results in inefficient site‐specific glycosylation, with deleterious impacts on glycoprotein production quality and efficiency . Analytical measurement of the site‐specific extent of glycosylation is therefore required for monitoring and quality control of such processes.…”

Section: Introductionmentioning

confidence: 99%

Automated measurement of site‐specific N‐glycosylation occupancy with SWATH‐MS

2015

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Asparagine-linked glycosylation is a common post-translational modification of proteins catalyzed by oligosaccharyltransferase that is important in regulating many aspects of protein function. Analysis of protein glycosylation, including glycoproteomic measurement of the site-specific extent of glycosylation, remains challenging. Here, we developed methods combining enzymatic deglycosylation and protease digestion with SWATH-MS to enable automated measurement of site-specific occupancy at many glycosylation sites. Deglycosylation with peptide-endoglycosidase H, leaving a remnant N-acetylglucosamine on asparagines previously carrying high-mannose glycans, followed by trypsin digestion allowed robust automated measurement of occupancy at many sites. Combining deglycosylation with the more general peptide-N-glycosidase F enzyme with AspN protease digest allowed robust automated differentiation of nonglycosylated and deglycosylated forms of a given glycosylation site. Ratiometric analysis of deglycosylated peptides and the total intensities of all peptides from the corresponding proteins allowed relative quantification of site-specific glycosylation occupancy between yeast strains with various isoforms of oligosaccharyltransferase. This approach also allowed robust measurement of glycosylation sites in human salivary glycoproteins. This method for automated relative quantification of site-specific glycosylation occupancy will be a useful tool for research with model systems and clinical samples.

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Glycoengineering of yeasts from the perspective of glycosylation efficiency

Cited by 18 publications

References 23 publications

Heterologous overproduction of β-fructofuranosidase from yeast Xanthophyllomyces dendrorhous, an enzyme producing prebiotic sugars

Heterologous overproduction of β-fructofuranosidase from yeast Xanthophyllomyces dendrorhous, an enzyme producing prebiotic sugars

Yeast synthetic biology for the production of recombinant therapeutic proteins

Automated measurement of site‐specific N‐glycosylation occupancy with SWATH‐MS

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