Improving the activity and thermostability of PETase from Ideonella sakaiensis through modulating its post-translational glycan modification

The large-scale preparation of Polyehylene terephthalate (PET) hydrolysing enzymes in low-cost is critical for the biodegradation of PET in industry. In the present study, we demonstrate that the post-translational glycosylation of Pichia pastoris makes it a remarkable host for the heterologous expression of PETase from Ideonella sakaiensis 201-F6 ( Is PETase). Taking advantage of the abundant N- and O-linked glycosylation sites in Is PETase and the efficient post-translational modification in endoplasmic reticulum, Is PETase is heavily glycosylated during secretory expression with P. pastoris , which improves the specific activity and thermostability of the enzyme dramatically. Moreover, the specific activity of Is PETase increased further after the bulky N-linked polysaccharide chains were eliminated by Endo-β-N-acetylglucosaminidase H (Endo H). Importantly, the partially deglycosylated Is PETase still maintained high thermostability because of the remaining mono- and oligo-saccharide residues on the protein molecules. Consequently, the partially deglycosylated Is PETase was able to be applied at 50 °C and depolymerized raw, untreated PET flakes completely in 2 to 3 days. This platform was also applied for the preparation of a famous variant of Is PETase, Fast-PETase, and the same result was achieved. Partially deglycosylated Fast-PETase demonstrates elevated efficiency in degrading postconsumer-PET trays under 55 °C than 50 °C, the reported optimal temperature of Fast-PETase. The present study provides a strategy to modulate thermostable Is PETase through glycosylation engineering and paves the way for promoting PET biodegradation from laboratories to factories.