Use of combinatorial genetic libraries to humanize N-linked glycosylation in the yeastPichiapastoris

Abstract

The secretory pathway of Pichia pastoris was genetically re-engineered to perform sequential glycosylation reactions that mimic early processing of N-glycans in humans and other higher mammals. After eliminating nonhuman glycosylation by deleting the initiating α-1,6-mannosyltransferase gene from P. pastoris, several combinatorial genetic libraries were constructed to localize active α-1,2-mannosidase and human β-1,2-N-acetylglucosaminyltransferase I (GnTI) in the secretory pathway. First, >32 N-terminal leader sequences of fungal type II membrane proteins were cloned to generate a leader library. Two additional libraries encoding catalytic domains of α-1,2-mannosidases and GnTI from mammals, insects, amphibians, worms, and fungi were cloned to generate catalytic domain libraries. In-frame fusions of the respective leader and catalytic domain libraries resulted in several hundred chimeric fusions of fungal targeting domains and catalytic domains. Although the majority of strains transformed with the mannosidase/leader library displayed only modest in vivo [i.e., low levels of mannose (Man)5-(GlcNAc)2] activity, we were able to isolate several yeast strains that produce almost homogenous N-glycans of the (Man)5-(GlcNAc)2 type. Transformation of these strains with a UDP-GlcNAc transporter and screening of a GnTI leader fusion library allowed for the isolation of strains that produce GlcNAc-(Man)5-(GlcNAc)2 in high yield. Recombinant expression of a human reporter protein in these engineered strains led to the formation of a glycoprotein with GlcNAc-(Man)5-(GlcNAc)2 as the primary N-glycan. Here we report a yeast able to synthesize hybrid glycans in high yield and open the door for engineering yeast to perform complex human-like glycosylation

In Vivo Synthesis of Mammalian-Like, Hybrid-Type N-Glycans inPichia pastoris

“…Surprisingly, there were also quantitative and qualitative differences in the oligosaccharide compositions of the nonmodified strains. In our untransformed strain (GS115) mainly Man 8-9 GlcNAc 2 and small amounts of Man 10-15 Glc NAc 2 were present (in the size range of 5 to 20 glucose units), whereas Choi et al (11) reported that Man 10 GlcNAc 2 is the smallest glycan. The differences in oligosaccharide composition may have been due to the strains used (GS115 and JC308) or the nature of the expressed glycoprotein.…”

“…It was speculated that this was due to trimming of the high-mannose structures, which would have resulted in the availability of more substrate for elongation of mannosyltransferases. However, this explanation is speculative, as Choi et al (11) used the same Mns1p signal sequence to direct a Caenorhabditis elegans mannosidase successfully to the ER, resulting in Man 5 GlcNAc 2 on the glycoproteins. The two situations, however, are not completely comparable, because Choi et al worked with a strain with a defective OCH1 gene.…”

Heterologous protein production using the Pichia pastoris expression system

“…The immunogenicity of Pichia-derived proteins is an issue that has attracted interest in the literature with regard to humanizing the glycosylation patterns [2,24]. One group of researchers disrupted the PNO1 gene of P. pastoris, resulting in a reduction of N -liked glycosylation of a recombinant human antithrombin.…”

Recombinant human lactoferrin expressed in glycoengineered Pichia pastoris: effect of terminal N-acetylneuraminic acid on in vitro secondary humoral immune response

“…N-glycans were released and separated from hLF as described earlier [8,12]. Molecular weight was determined by using a Voyager linear matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometer (Applied Biosystems) with delayed extraction.…”

“…The application of a combinatorial library approach has been essential to generate P. pastoris strains harboring combinations of mannosidases, glycosyltransferases, GlcNAc/Gal transporters, and Gal epimerase [24]. Over the past years we have created a library of glycoengineered P. pastoris strains each capable of displaying defined N-linked glycans at high uniformity [5,8,12,17,24]. This growing library of strains has the potential to allow elucidation of the relationship between specific N-linked glycans and the function of glycoproteins.…”