A dual glycoengineering system: combining synthetic biology with an artificial Golgi column

Glycoengineering aims to generate proteins with defined glycosylation. Production of glycoengineered proteins can be performed in vivo, with modification of gene expression of the host glycosylation enzymes pre-protein purification, or in vitro, using glycan modifying enzymes post-protein purification. Glycoengineering in vivo is scalable but requires the combinatorial generation of genetic variants. Glycoengineering in vitro is more controllable but increases in cost as number of enzymes involved increases. Here a dual glycoengineering system, comprising of an in vivo glycoengineered Saccharomyces cerevisiae and an artificial Golgi column (AGC) was designed. When glycoengineered high-yield producing yeast was coupled to an in vitro glycosylating column, a scalable, glycan-defined glycoprotein production system was created. A target glycoprotein (Trastuzumab) is produced in S. cerevisiae engineered to synthesize the GlcNAcMan5GlcNAc2 hybrid glycan structure by disrupting genes OCH1 and MNN1, while inserting α-1,2-mannosidase and GnTI into the genome. Subsequently, the AGC performs in vitro glycosylation with immobilised B4GalT1 and ST6Gal1 on-column, forming galactosylated and sialylated glycans. Physical parameters such as flowrate and substrate concentrations were partially optimised for on-column glycosylation and the reusability of the column was demonstrated. This multi-faceted approach to glycoengineering shows promise for future industrial applications with potential for large-scale glycan modification of valuable therapeutics and glycoproteins -- abstract.