The Construction Of Low-mannose Type N-linked Glycosylation In Pichia Pastoris

P. pastorisis is a methylotrophic yeast that had already been used for production of a widevariety of heterologous proteins. Besides the advantages of protein processing, folding andpost-translational modifications in eukaryotic cells, it offers the benefits of cost-effective andeasy scale-up in E. coli. Though series of profits come from P. pastoris expression, but it isnot suitable to express glycogan proteins. Normaly, glycoproteins expressed from the yeastexpression systems contain high mannose-type N-glycans. The glycoproteins withyeast-specific mannosylation are significantly different from mammalian glycoproteins, andthey will cause strong immunogenicity and display unfavorable pharmacokinetic properties inhumans. The highly-mannosylated structures on N-linked oligosaccharides form the majorhindrance to develop the yeast for the human compatible glycoprotein production. To improvethe human source glycoprotein expression, we try to humanize the glycosylation process inPichia pastoris. In early studies, we had obtained an OCH1gene-deficient strain(X-33(och1)), and that strain can express low mannose-GM-CSF. The growth rate of X-33(och1) was very low and made it not suitable for further glycosylation engineering. In thisstudy, first, try to obtain a favorite culture condition suitable for the growth of X-33(och1).After that, try to knockout the α-1,3-mannose transferase gene (ALG3) and obtain an OCH1and ALG3gene double deletion strain (X-33(och1, alg3) or X33OA). Further, try to usethis engineered strain to express lower mannosylated GM-SCF.To improve the growth rate of X-33(och1), various external conditions that can affectthe state of growth of yeast, such as initial pH of the medium, temperature, ventilation, carbonsource content, inoculum amount, were investigated. Through the study of these conditions,we found that increasing the inoculation amount and reducing initial pH value can improvethe growth state of the X-33(och1) and this strain can be better applied to furtherglycosylation engineering.To knockout ALG3gene, we applied the temporary recombination system (Cre-LoxP)and made an ALG3knockout construct containing zeocin resistance. After zeocin resistancescreening, the positive transformants with ALG3gene knockout were obtained successfully.Further, zeocin resistance marker was removed by Cre recombinase. Finally, OCH1andALG3double gene deletion strain X33OA was obtained. The physiological studies of X33OAhave shown the poor growth state and the cell wall defects. When this strain was used toexpress the human glycoprotein GM-CSF, a lower glycosylated GM-CSF was expressed.These results further illustrate ALG3gene in X33OA has been successfully knockout.Thiswork laid the foundation for the subsequent humanize glycosylation engineering of Pichiapastoris X33strain.