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1. The 2008 update of the Aspergillus nidulans genome annotation: A community effort

Author

Wortman, Jennifer Russo, Gilsenan, Jane Mabey, Joardar, Vinita, Deegan, Jennifer, Clutterbuck, John, Andersen, Mikael R., Archer, David, Bencina, Mojca, Braus, Gerhard, Coutinho, Pedro, Döhren, Hans von, Doonan, John, Driessen, Arnold J.M., Durek, Pawel, Espeso, Eduardo, Fekete, Erzsébet, Flipphi, Michel, Estrada, Carlos Garcia, Geysens, Steven, Goldman, Gustavo, de Groot, Piet W.J., Hansen, Kim, Harris, Steven D., Heinekamp, Thorsten, Helmstaedt, Kerstin, Henrissat, Bernard, Hofmann, Gerald, Homan, Tim, Horio, Tetsuya, Horiuchi, Hiroyuki, James, Steve, Jones, Meriel, Karaffa, Levente, Karányi, Zsolt, Kato, Masashi, Keller, Nancy, Kelly, Diane E., Kiel, Jan A.K.W., Kim, Jung-Mi, van der Klei, Ida J., Klis, Frans M., Kovalchuk, Andriy, Kraševec, Nada, Kubicek, Christian P., Liu, Bo, MacCabe, Andrew, Meyer, Vera, Mirabito, Pete, Miskei, Márton, Mos, Magdalena, Mullins, Jonathan, Nelson, David R., Nielsen, Jens, Oakley, Berl R., Osmani, Stephen A., Pakula, Tiina, Paszewski, Andrzej, Paulsen, Ian, Pilsyk, Sebastian, Pócsi, István, Punt, Peter J., Ram, Arthur F.J., Ren, Qinghu, Robellet, Xavier, Robson, Geoff, Seiboth, Bernhard, van Solingen, Piet, Specht, Thomas, Sun, Jibin, Taheri-Talesh, Naimeh, Takeshita, Norio, Ussery, Dave, vanKuyk, Patricia A., Visser, Hans, van de Vondervoort, Peter J.I., de Vries, Ronald P., Walton, Jonathan, Xiang, Xin, Xiong, Yi, Zeng, An Ping, Brandt, Bernd W., Cornell, Michael J., van den Hondel, Cees A.M.J.J., Visser, Jacob, Oliver, Stephen G., and Turner, Geoffrey

Published
2009
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4. 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

Author

Geysens, Steven, Pakula, Tiina, Uusitalo, Jaana, Dewerte, Isabelle, Penttila, Merja, and Contreras, Roland

Subjects
Celluloid -- Research, Gene mutations -- Research, Genetic code -- Research, Biological sciences
Abstract

The isolation and characterization of the gene encoding the glycosidase II alpha subunit of the industrially important fungus Trichoderma reesei is described. The gene encoding the glucosidase II alpha subunit in the hypercellulolytic strain Rut-C30 contains a frameshift mutation resulting in a truncated gene product.

Published
2005

10. Parallel glycoprotein N-glycan analysis

Author

Callewaert, Nico and Geysens, Steven

Subjects
Pharmacokinetics -- Research, Glycoproteins -- Research, Protein research -- Analysis, Biotechnology industry, Business
Abstract

The effects of N-glycan structural profiling tools on pharmacokinetics of drugs during clinical trails are presented.

Published
2007

11. In vivo synthesis of mammalian-like, hybrid-type N-glycans in pichia pastoris

Author

Vervecken, Wouter, Kiagorodov, Vladimir, Callewaert, Nico, Geysens, Steven, Vusser, Kristof De, and Contreras, Roland

Subjects
Glycosylation -- Research, Transferases -- Research, Endoplasmic reticulum -- Research, Microbiology -- Research, Biological sciences
Abstract

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.

Published
2004

13. Genome sequencing and analysis of the versatile cell factory Aspergillus niger CBS 513.88

Author

Pel, Herman J., de Winde, Johannes H., Archer, David B., Dyer, Paul S., Hofmann, Gerald, Schaap, Peter J., Turner, Geoffrey, de Vries, Ronald P., Albang, Richard, Albermann, Kaj, Andersen, Mikael R., Bendtsen, Jannick D., Benen, Jacques A. E., van den Berg, Marco, Breestraat, Stefaan, Caddick, Mark X., Contreras, Roland, Cornell, Michael, Coutinho, Pedro M., Danchin, Etienne G. J., Debets, Alfons J. M., Dekker, Peter, van Dijck, Piet W. M., van Dijk, Alard, Dijkhuizen, Lubbert, Driessen, Arnold J. M., d'Enfert, Christophe, Geysens, Steven, Goosen, Coenie, Groot, Gert S. P., de Groot, Piet W. J., Guillemette, Thomas, Henrissat, Bernard, Herweijer, Marga, van den Hombergh, Johannes P. T. W., van den Hondel, Cees A. M. J. J., van der Heijden, Rene T. J. M., van der Kaaij, Rachel M., Klis, Frans M., Kools, Harrie J., Kubicek, Christian P., van Kuyk, Patricia A., Lauber, Juergen, Lu, Xin, van der Maarel, Marc J. E. C., Meulenberg, Rogier, Menke, Hildegard, Mortimer, Martin A., Nielsen, Jens, Oliver, Stephen G., Olsthoorn, Maurien, Pal, Karoly, van Peij, Noel N. M. E., Ram, Arthur F. J., Rinas, Ursula, Roubos, Johannes A., Sagt, Cees M. J., Schmoll, Monika, Sun, Jibin, Ussery, David, Varga, Janos, Vervecken, Wouter, de Vondervoort, Peter J. J. van, Wedler, Holger, Wosten, Han A. B., Zeng, An-Ping, van Ooyen, Albert J. J., Visser, Jaap, Stam, Hein, Pel, Herman J., de Winde, Johannes H., Archer, David B., Dyer, Paul S., Hofmann, Gerald, Schaap, Peter J., Turner, Geoffrey, de Vries, Ronald P., Albang, Richard, Albermann, Kaj, Andersen, Mikael R., Bendtsen, Jannick D., Benen, Jacques A. E., van den Berg, Marco, Breestraat, Stefaan, Caddick, Mark X., Contreras, Roland, Cornell, Michael, Coutinho, Pedro M., Danchin, Etienne G. J., Debets, Alfons J. M., Dekker, Peter, van Dijck, Piet W. M., van Dijk, Alard, Dijkhuizen, Lubbert, Driessen, Arnold J. M., d'Enfert, Christophe, Geysens, Steven, Goosen, Coenie, Groot, Gert S. P., de Groot, Piet W. J., Guillemette, Thomas, Henrissat, Bernard, Herweijer, Marga, van den Hombergh, Johannes P. T. W., van den Hondel, Cees A. M. J. J., van der Heijden, Rene T. J. M., van der Kaaij, Rachel M., Klis, Frans M., Kools, Harrie J., Kubicek, Christian P., van Kuyk, Patricia A., Lauber, Juergen, Lu, Xin, van der Maarel, Marc J. E. C., Meulenberg, Rogier, Menke, Hildegard, Mortimer, Martin A., Nielsen, Jens, Oliver, Stephen G., Olsthoorn, Maurien, Pal, Karoly, van Peij, Noel N. M. E., Ram, Arthur F. J., Rinas, Ursula, Roubos, Johannes A., Sagt, Cees M. J., Schmoll, Monika, Sun, Jibin, Ussery, David, Varga, Janos, Vervecken, Wouter, de Vondervoort, Peter J. J. van, Wedler, Holger, Wosten, Han A. B., Zeng, An-Ping, van Ooyen, Albert J. J., Visser, Jaap, and Stam, Hein

Abstract

The filamentous fungus Aspergillus niger is widely exploited by the fermentation industry for the production of enzymes and organic acids, particularly citric acid. We sequenced the 33.9-megabase genome of A. niger CBS 513.88, the ancestor of currently used enzyme production strains. A high level of synteny was observed with other aspergilli sequenced. Strong function predictions were made for 6,506 of the 14,165 open reading frames identified. A detailed description of the components of the protein secretion pathway was made and striking differences in the hydrolytic enzyme spectra of aspergilli were observed. A reconstructed metabolic network comprising 1,069 unique reactions illustrates the versatile metabolism of A. niger. Noteworthy is the large number of major facilitator superfamily transporters and fungal zinc binuclear cluster transcription factors, and the presence of putative gene clusters for fumonisin and ochratoxin A synthesis.

Published
2007

14. Genome sequencing and analysis of the versatile cell factory Aspergillus niger CBS 513.88

Author

Sub Molecular Microbiology, Molecular Microbiology, Pel, Herman J., de Winde, Johannes H., Archer, David B., Dyer, Paul S., Hofmann, Gerald, Schaap, Peter J., Turner, Geoffrey, de Vries, Ronald P., Albang, Richard, Albermann, Kaj, Andersen, Mikael R., Bendtsen, Jannick D., Benen, Jacques A. E., van den Berg, Marco, Breestraat, Stefaan, Caddick, Mark X., Contreras, Roland, Cornell, Michael, Coutinho, Pedro M., Danchin, Etienne G. J., Debets, Alfons J. M., Dekker, Peter, van Dijck, Piet W. M., van Dijk, Alard, Dijkhuizen, Lubbert, Driessen, Arnold J. M., d'Enfert, Christophe, Geysens, Steven, Goosen, Coenie, Groot, Gert S. P., de Groot, Piet W. J., Guillemette, Thomas, Henrissat, Bernard, Herweijer, Marga, van den Hombergh, Johannes P. T. W., van den Hondel, Cees A. M. J. J., van der Heijden, Rene T. J. M., van der Kaaij, Rachel M., Klis, Frans M., Kools, Harrie J., Kubicek, Christian P., van Kuyk, Patricia A., Lauber, Juergen, Lu, Xin, van der Maarel, Marc J. E. C., Meulenberg, Rogier, Menke, Hildegard, Mortimer, Martin A., Nielsen, Jens, Oliver, Stephen G., Olsthoorn, Maurien, Pal, Karoly, van Peij, Noel N. M. E., Ram, Arthur F. J., Rinas, Ursula, Roubos, Johannes A., Sagt, Cees M. J., Schmoll, Monika, Sun, Jibin, Ussery, David, Varga, Janos, Vervecken, Wouter, de Vondervoort, Peter J. J. van, Wedler, Holger, Wosten, Han A. B., Zeng, An-Ping, van Ooyen, Albert J. J., Visser, Jaap, Stam, Hein, Sub Molecular Microbiology, Molecular Microbiology, Pel, Herman J., de Winde, Johannes H., Archer, David B., Dyer, Paul S., Hofmann, Gerald, Schaap, Peter J., Turner, Geoffrey, de Vries, Ronald P., Albang, Richard, Albermann, Kaj, Andersen, Mikael R., Bendtsen, Jannick D., Benen, Jacques A. E., van den Berg, Marco, Breestraat, Stefaan, Caddick, Mark X., Contreras, Roland, Cornell, Michael, Coutinho, Pedro M., Danchin, Etienne G. J., Debets, Alfons J. M., Dekker, Peter, van Dijck, Piet W. M., van Dijk, Alard, Dijkhuizen, Lubbert, Driessen, Arnold J. M., d'Enfert, Christophe, Geysens, Steven, Goosen, Coenie, Groot, Gert S. P., de Groot, Piet W. J., Guillemette, Thomas, Henrissat, Bernard, Herweijer, Marga, van den Hombergh, Johannes P. T. W., van den Hondel, Cees A. M. J. J., van der Heijden, Rene T. J. M., van der Kaaij, Rachel M., Klis, Frans M., Kools, Harrie J., Kubicek, Christian P., van Kuyk, Patricia A., Lauber, Juergen, Lu, Xin, van der Maarel, Marc J. E. C., Meulenberg, Rogier, Menke, Hildegard, Mortimer, Martin A., Nielsen, Jens, Oliver, Stephen G., Olsthoorn, Maurien, Pal, Karoly, van Peij, Noel N. M. E., Ram, Arthur F. J., Rinas, Ursula, Roubos, Johannes A., Sagt, Cees M. J., Schmoll, Monika, Sun, Jibin, Ussery, David, Varga, Janos, Vervecken, Wouter, de Vondervoort, Peter J. J. van, Wedler, Holger, Wosten, Han A. B., Zeng, An-Ping, van Ooyen, Albert J. J., Visser, Jaap, and Stam, Hein

Published
2007

15. A bacterial glycosidase enables mannose-6-phosphate modification and improved cellular uptake of yeast-produced recombinant human lysosomal enzymes

Author

Tiels, Petra, primary, Baranova, Ekaterina, additional, Piens, Kathleen, additional, De Visscher, Charlotte, additional, Pynaert, Gwenda, additional, Nerinckx, Wim, additional, Stout, Jan, additional, Fudalej, Franck, additional, Hulpiau, Paco, additional, Tännler, Simon, additional, Geysens, Steven, additional, Van Hecke, Annelies, additional, Valevska, Albena, additional, Vervecken, Wouter, additional, Remaut, Han, additional, and Callewaert, Nico, additional

Published
2012
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18. Genome sequencing and analysis of the versatile cell factory Aspergillus niger CBS 513.88

Author

Pel, Herman J, primary, de Winde, Johannes H, additional, Archer, David B, additional, Dyer, Paul S, additional, Hofmann, Gerald, additional, Schaap, Peter J, additional, Turner, Geoffrey, additional, de Vries, Ronald P, additional, Albang, Richard, additional, Albermann, Kaj, additional, Andersen, Mikael R, additional, Bendtsen, Jannick D, additional, Benen, Jacques A E, additional, van den Berg, Marco, additional, Breestraat, Stefaan, additional, Caddick, Mark X, additional, Contreras, Roland, additional, Cornell, Michael, additional, Coutinho, Pedro M, additional, Danchin, Etienne G J, additional, Debets, Alfons J M, additional, Dekker, Peter, additional, van Dijck, Piet W M, additional, van Dijk, Alard, additional, Dijkhuizen, Lubbert, additional, Driessen, Arnold J M, additional, d'Enfert, Christophe, additional, Geysens, Steven, additional, Goosen, Coenie, additional, Groot, Gert S P, additional, de Groot, Piet W J, additional, Guillemette, Thomas, additional, Henrissat, Bernard, additional, Herweijer, Marga, additional, van den Hombergh, Johannes P T W, additional, van den Hondel, Cees A M J J, additional, van der Heijden, Rene T J M, additional, van der Kaaij, Rachel M, additional, Klis, Frans M, additional, Kools, Harrie J, additional, Kubicek, Christian P, additional, van Kuyk, Patricia A, additional, Lauber, Jürgen, additional, Lu, Xin, additional, van der Maarel, Marc J E C, additional, Meulenberg, Rogier, additional, Menke, Hildegard, additional, Mortimer, Martin A, additional, Nielsen, Jens, additional, Oliver, Stephen G, additional, Olsthoorn, Maurien, additional, Pal, Karoly, additional, van Peij, Noël N M E, additional, Ram, Arthur F J, additional, Rinas, Ursula, additional, Roubos, Johannes A, additional, Sagt, Cees M J, additional, Schmoll, Monika, additional, Sun, Jibin, additional, Ussery, David, additional, Varga, Janos, additional, Vervecken, Wouter, additional, van de Vondervoort, Peter J J, additional, Wedler, Holger, additional, Wösten, Han A B, additional, Zeng, An-Ping, additional, van Ooyen, Albert J J, additional, Visser, Jaap, additional, and Stam, Hein, additional

Published
2007
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19. Modification of the N-Glycosylation Pathway to Produce Homogeneous, Human-Like Glycans Using GlycoSwitch Plasmids.

Author

Walker, John M., Cregg, James M., Vervecken, Wouter, Callewaert, Nico, Kaigorodov, Vladimir, Geysens, Steven, and Contreras, Roland

Abstract

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. [ABSTRACT FROM AUTHOR]

Published
2007
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21. Engineering Yarrowia lipolytica to Produce Glycoproteins Homogeneously Modified with the Universal Man3GlcNAc2 N-Glycan Core.

Author

De Pourcq, Karen, Tiels, Petra, Van Hecke, Annelies, Geysens, Steven, Vervecken, Wouter, and Callewaert, Nico

Subjects
YEAST, GLYCOPROTEINS, MANNOSE, BIOSYNTHESIS, GENE expression, GLYCOSYLATION
Abstract

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]

Published
2012
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22. Ultrasensitive profiling andsequencing of N-linked oligosaccharides using standard DNA-sequencingequipment.

Author

Callewaert, Nico, Geysens, Steven, Molemans, Francis, and Contreras, Roland

Abstract

The analysis of protein-linked glycans is of increasing importance,both in basic glycobiological research and during the productionprocess of glycoprotein pharmaceuticals. In many cases, the amountof glycoprotein available for typing the glycans is very low. This,combined with the high branching complexity typical for this classof compounds, makes glycan typing a challenging task. We presenthere methodology allowing the medium-throughput analysis of N-glycansderived from low picomole amounts of glycoproteins using the standardDNA-sequencing equipment available in any life sciences laboratory.The high sensitivity of the overall analytical process (from glycoproteinto results) is obtained using state-of-the-art deglycosylation procedurescombined with a highly efficient and reproducible novel postderivatizationcleanup step involving Sephadex G10 packed 96-well filterplates. Allsample preparation steps (enzymatic deglycosylation with PNGaseF, desalting, derivatization with 8-amino-1,3,6-pyrenetrisulfonicacid, and postderivatization cleanup) are performed using 96-well-basedplates. This integrated sample preparation scheme is also compatible withcapillary electrophoresis and MALDI-TOF-MS platforms already inuse in some glycobiology labs and anticipates the higher throughputthat will be offered by the capillary-array-based DNA sequencerscurrently penetrating the market. The described technology should bringhigh-performance glycosylation analysis within reach of each lifesciences lab and thus help expedite the pace of discovery in thefield of glycobiology. [ABSTRACT FROM PUBLISHER]

Published
2001
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23. Engineering complex-type N-glycosylation in Pichia pastoris using GlycoSwitch technology

Author

Jacobs, Pieter P, Geysens, Steven, Vervecken, Wouter, Contreras, Roland, and Callewaert, Nico

Abstract

Here we provide a protocol for engineering the N-glycosylation pathway of the yeast Pichia pastoris. The general strategy consists of the disruption of an endogenous glycosyltransferase gene (OCH1) and the stepwise introduction of heterologous glycosylation enzymes. Each engineering step results in the introduction of one glycosidase or glycosyltransferase activity into the Pichia endoplasmic reticulum or Golgi complex and consists of a number of stages: transformation with the appropriate GlycoSwitch vector, small-scale cultivation of a number of transformants, sugar analysis and heterologous protein expression analysis. If desired, the resulting clone can be further engineered by repeating the procedure with the next GlycoSwitch vector. Each engineering step takes ∼3 weeks. The conversion of any wild-type Pichia strain into a strain that modifies its glycoproteins with Gal2GlcNAc2Man3GlcNAc2N-glycans requires the introduction of five GlycoSwitch vectors. Three examples of the full engineering procedure are provided to illustrate the results that can be expected.

Published
2009
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24. Use of HDEL‐tagged Trichoderma reeseimannosyl oligosaccharide 1,2‐α‐D‐mannosidase for N‐glycan engineering in Pichia pastoris

Author

Callewaert, Nico, Laroy, Wouter, Cadirgi, Hasan, Geysens, Steven, Saelens, Xavier, Min Jou, Willy, and Contreras, Roland

Abstract

Therapeutic glycoprotein production in the widely used expression host Pichia pastorisis hampered by the differences in the protein‐linked carbohydrate biosynthesis between this yeast and the target organisms such as man. A significant step towards the generation of human‐compatible N‐glycans in this organism is the conversion of the yeast‐type high‐mannose glycans to mammalian‐type high‐mannose and/or complex glycans. In this perspective, we have co‐expressed an endoplasmic reticulum‐targeted Trichoderma reesei1,2‐α‐D‐mannosidase with two glycoproteins: influenza virus haemagglutinin and Trypanosoma cruzi trans‐sialidase. Analysis of the N‐glycans of the two purified proteins showed a >85% decrease in the number of α‐1,2‐linked mannose residues. Moreover, the human‐type high‐mannose oligosaccharide Man5GlcNAc2was the major N‐glycan of the glyco‐engineered trans‐sialidase, indicating that N‐glycan engineering can be effectively accomplished in P. pastoris.

Published
2001
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25. Glykosylering Af Molekyler

Author

Nico Callewaert, Vervecken Wouter, De Pourq Karen Jacqueline Marcel, Geysens Steven Christian Jozef, and Guerfal Mouna

33. Glycosylation Of Molecules

Author

Nico Callewaert, Vervecken Wouter, Marcel De Pourcq Karen Jacqueline, Geysens Steven Christian Jozef, and Guerfal Mouna

35. Modification of the N-glycosylation pathway to produce homogeneous, human-like glycans using GlycoSwitch plasmids.

Author

Vervecken W, Callewaert N, Kaigorodov V, Geysens S, and Contreras R

Subjects
Endoplasmic Reticulum enzymology, Glycosylation, Golgi Apparatus enzymology, Humans, Mannosidases metabolism, Mannosyltransferases genetics, N-Acetylglucosaminyltransferases metabolism, Pichia enzymology, Plasmids genetics, Polysaccharides biosynthesis
Abstract

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.

Published
2007
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