Posttranslational modifications of heterologous proteins expressed in Nicotiana benthamiana.

The success of Nicotiana benthamiana as a workhorse for heterologous protein production is closely linked to its accessibility and tolerance to genetic manipulation, allowing efficient engineering of posttranslational protein modifications (PTMs) that are critical for the function and stability of heterologous proteins. Therefore, control over PTMs has a significant impact on the quality of a product. Most recombinant protein therapeutics are glycosylated, and glycosylation is the most common and complex PTM. The machinery for initiating N-glycosylation is largely conserved in N. benthamiana, and there are generally fewer glycosyltransferases involved in modifying N-glycans compared to human cells. This results in less processed and more homogeneous complex N-glycans, which serve as acceptors for various extensions and the generation of tailored N-glycans. O-glycosylation is different and quite diverse in plants. Recent advances in genome editing have resulted in N. benthamiana with greatly reduced plant-specific modifications, making it a valuable tool for studying O-glycosylation and the production of heterologous proteins with human-type O-glycans. In contrast to glycosylation, there are far fewer studies focusing on other PTMs, and the engineering of these modifications in plants is still in its infancy. Noteworthy exceptions include the successful tyrosine sulfation of antibodies and the use of the human protease furin for the activation of recombinant proteins, achieved through a controlled proteolytic processing approach. In summary, recent advances in genome editing and pathway engineering by transient or stable co-expression of multiple foreign genes in N. benthamiana lay the foundation for novel protein-based products with optimized functions.