Modification of Pichia kudriavzevii cells for humanized glycosylation

Abstract:

[Background] Proteins produced by yeast usually undergo hyper glycosylation and form high mannose-type N-glycans, which can induce immune reactions in humans. As a result, the use of yeast is limited in the production of glycoprotein in drugs. Modifying the glycosylation pathway in yeast is a feasible approach for the production of glycoproteins used in drugs. Pichia kudriavzevii, a non-typical yeast species with strong stress resistance and rapid growth, has attracted much attention in recent years. By humanized glycosylation, P. kudriavzevii will serve as a cell factory to produce glycoproteins. [Objective] To obtain Man₅GlcNAc₂, the core structure of human N-glycan, is the key step to modify the glycosylation pathway in yeast. To achieve this goal, we deleted och1 of P. kudriavzevii and introduced msdS of Aspergillus into P. kudriavzevii. [Methods] The CRISPR-Cas9 system was employed to modify the N-glycosylation pathway of P. kudriavzevii, and the modified strain Δura3Δoch1::msdS was obtained. Furthermore, the N-glycocome of the glycoproteins secreted by Δura3Δoch1::msdS was analyzed. [Results] The N-linked Man₅GlcNAc₂ was detected on the glycoproteins secreted by Δura3Δoch1::msdS. [Conclusion] After the deletion of och1 and the introduction of msdS from Aspergillus,P. kudriavzevii cells can secrete the proteins with N-linked Man₅GlcNAc₂, which underpins the further humanized glycosylation in P. kudriavzevii.