N-glycan modification in Aspergillus species.
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.
Kainz
Elke
E
Fungal Genomics Unit, Austrian Research Centers and BOKU Vienna, Muthgasse 18, A-1190 Vienna, Austria.
Gallmetzer
Andreas
A
Hatzl
Christian
C
Nett
Juergen H
JH
Li
Huijuan
H
Schinko
Thorsten
T
Pachlinger
Robert
R
Berger
Harald
H
Reyes-Dominguez
Yazmid
Y
Bernreiter
Andreas
A
Gerngross
Tillmann
T
Wildt
Stefan
S
Strauss
Joseph
J
D001230:Aspergillus / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D018014:Gene Transfer Techniques; D008364:Mannosyltransferases / Q000235:genetics; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000096:biosynthesis* / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014169:Transformation, Bacterial / Q000235:genetics*; D043323:alpha-Mannosidase / Q000378:metabolism
D017931:DNA Primers; D011134:Polysaccharides; D011993:Recombinant Fusion Proteins; D008364:Mannosyltransferases; D043323:alpha-Mannosidase