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Applied and Environmental Microbiology, February 2008, p. 1076-1086, Vol. 74, No. 4
0099-2240/08/$08.00+0     doi:10.1128/AEM.01058-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

N-Glycan Modification in Aspergillus Species{triangledown}

Elke Kainz,1,§ Andreas Gallmetzer,1,§ Christian Hatzl,1,{dagger} Juergen H. Nett,2 Huijuan Li,2 Thorsten Schinko,1 Robert Pachlinger,1,{ddagger} Harald Berger,1 Yazmid Reyes-Dominguez,1 Andreas Bernreiter,1 Tillmann Gerngross,2,3 Stefan Wildt,2 and Joseph Strauss1*

Fungal Genomics Unit, Austrian Research Centers and BOKU Vienna, Muthgasse 18, A-1190 Vienna, Austria,1 GlycoFi, Inc., 21 Lafayette Street, Lebanon, New Hampshire 03766,2 Thayer School of Engineering, Dartmouth College, 8000 Cummings Hall, Hanover, New Hampshire 037553

Received 11 May 2007/ Accepted 2 December 2007

The production by filamentous fungi of therapeutic glycoproteins intended for use in mammals is held back by the inherent difference in protein N-glycosylation and by the inability of the fungal cell to modify proteins with mammalian glycosylation structures. Here, we report protein N-glycan engineering in two Aspergillus species. We functionally expressed in the fungal hosts heterologous chimeric fusion proteins containing different localization peptides and catalytic domains. This strategy allowed the isolation of a strain with a functional {alpha}-1,2-mannosidase producing increased amounts of N-glycans of the Man5GlcNAc2 type. This strain was further engineered by the introduction of a functional GlcNAc transferase I construct yielding GlcNAcMan5GlcNac2 N-glycans. Additionally, we deleted algC genes coding for an enzyme involved in an early step of the fungal glycosylation pathway yielding Man3GlcNAc2 N-glycans. This modification of fungal glycosylation is a step toward the ability to produce humanized complex N-glycans on therapeutic proteins in filamentous fungi.

* Corresponding author. Mailing address: Fungal Genomics Unit, Austrian Research Centers and BOKU Vienna, Muthgasse 18, A-1190 Vienna, Austria. Phone: 43-1-36006-6720. Fax: 43-1-36006-6392. E-mail: joseph.strauss{at}boku.ac.at

{triangledown} Published ahead of print on 14 December 2007.

§ E.K. and A.G. contributed equally to this work.

{dagger} Present address: Octapharma Produktionsges. m.b.H., Oberlaaerstrasse 235, A-1100 Vienna, Austria.

{ddagger} Present address: Baxter AG, Industriestrasse 67, A-1221 Vienna, Austria.

Applied and Environmental Microbiology, February 2008, p. 1076-1086, Vol. 74, No. 4
0099-2240/08/$08.00+0     doi:10.1128/AEM.01058-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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