Preliminary Study Of The Humanization Of N-glycosylation Pathways In Trichoderma Reesei

Filamentous fungi have long been used for the production of metabolites and enzymes,expecially the proteins that cannot be actively expressed in Escherichia coli or require glycosylation for proper folding and biological activity.The benefits of the filamentous fungus Trichoderma reesei include its simplicity,rapidity,and ability to glycosylate recombinant proteins.However,for the production of therapeutic glycoproteins intended for use in humans,fungi glycosylation is of the high-mannose type,which confers a short in vivo half-life to the protein and may render it less efficacious or even immunogenic.An enzymatically modulation of host glycosylation pathways in vivo is needed to change their ability to modify proteins with human glycosylation structures.Thinking about the cost of manufacturing and purification,the conversion of the fungi-type high-mannose glycans to mammalian N-type complex glycans is showing a significant promise and challenging the current dominance of therapeutic protein production based on mammalian cell culture.In this perspective,we have constructed an expression plasmid vector pS△M containing Palg3-ST6-Talg3 expressive box-a ratα-2,6-sialyltransferase(ST6GalI) cDNA was subcloned between the promoter and terminator of T.reeseiα-1,3-mannosyltransferase gene(alg3).The resulting construct was used to knockout T.reesei alg3 and express rat ST6GalI gene at the same time at the case of homointergration.Co-transformation of the auxotroph strains T.reesei M23 with plasmid vectors pS△M and pAB4-1 containing the A.niger pyrG selectable gene has been performed to isolate a stably-transformed T.reesei variant.Five homointergrants were identified from about 3000 heterointergrants,while four of them can not survive no matter what effort we have attempted.It supposed that a lethal knockout happened. The only survival designated T.reesei b-8-1 was identitied partial homointergrant at the downstream side of alg3 gene.The expression activity of alg3 gene was suppessed at a certain extent,while ST6GalI gene was found an expression activity in T.reesei b-8-1. Then we constructed the second expression plasmid vector pGG containing Pebh1-GnTI-Tcbh1-PpgdA-GalT-TtrpC expressive box-a N-acetylglucosaminyl transferaseⅠ(GnTI)gene and a bovineβ-1,4-galactosyltransferase(GalTI)eDNA were subcloned into the fungous promoter and terminator.The resulting construct was used to express GnTI gene and GalTI gene at the same time.Co-transformation of T.reesei b-8-1 with plasmid vectors pGG and pAN7-1 carrying the Escherichia coliβ-glucuronidase gusA gene has been performed to isolate a stably-transformed T. reesei variant.Two transformants were identified and designated T.reesei GF4 and T. reesei GF52.The expression activity of GnTI and GalTI gene was found through RT-PCR experiment and a declined expression activity of alg3 gene was found through Real Time RT-PCR in T.reesei GF4 and GF52.Analysis to N-glycan of secret protein CBHI in T.reesei GF4 is ongoing.