Glyco-Engineering of Plant-Based Expression Systems

Abstract

Most secreted proteins in eukaryotes are glycosylated, and after a number of common biosynthesis steps the glycan structures mature in a species-dependent manner. Therefore, human therapeutic proteins produced in plants often carry plant-like rather than human-like glycans, which can affect protein stability, biological function, and immunogenicity. The glyco-engineering of plant-based expression systems began as a strategy to eliminate plant-like glycans and produce human proteins with authentic or at least compatible glycan structures. The precise replication of human glycans is challenging, owing to the absence of a pathway in plants for the synthesis of sialylated proteins and the necessary precursors, but this can now be achieved by the coordinated expression of multiple human enzymes. Although the research community has focused on the removal of plant glycans and their replacement with human counterparts, the presence of plant glycans on proteins can also provide benefits, such as boosting the immunogenicity of some vaccines, facilitating the interaction between therapeutic proteins and their receptors, and increasing the efficacy of antibody effector functions. Graphical Abstract Typical structures of native mammalian and plant glycans with symbols indicating sugar residues identified by their short form and single-letter codes. Both glycans contain fucose, albeit with different linkages.

Recent Developments in Bioprocessing of Recombinant Proteins: Expression Hosts and Process Development

“…Human therapeutic proteins produced in plants often exhibit a plantlike rather than a humanlike glycosylation pattern. Glycoengineering is being used to solve this issue (Fischer et al, 2018;Owczarek et al, 2019). Rozov and Deineko (2019) discussed in detail the classical strategies for optimizing the synthesis of recombinant proteins and also new approaches, including gene-editing tools associated with the insertion of target genes in euchromatin genome regions.…”

Potential Applications of Plant Biotechnology against SARS-CoV-2

“…These established platforms utilize the bacterium Escherichia coli and a few other microbes, and various mammalian cell lines, mainly due to the robust regulatory framework that exists for these systems and the historic industry investment in corresponding production technologies [6]. However, plants have carved a niche in a small number of cases because they can produce biologics with favorable glycan configurations (such as taliglucerase alfa) [4,7], they allow production on a massive scale (as required for HIV microbicides) [8,9], and, most relevant to the current situation, when transient expression systems are used, they can be scaled up rapidly to meet sudden and unforeseen demand [10]. This is ideal for the production of diagnostic reagents, vaccine candidates, and antiviral drugs in the face of a rapidly spreading pandemic disease (Figure 1).…”

Targeted genome editing of plants and plant cells for biomanufacturing

“…Because RNAi only downregulates genes and is sensitive to variable experimental conditions, leading to inconsistent and heterogeneous glycosylation patterns (Kallolimath et al 2020), it is important to achieve stable gene disruption at the DNA level by genome editing to generate a robust chassis. The full integration of the different steps needed to generate a plant with no plant-specific b(1,2)xylose and a(1,3)fucose and no degradation of terminal GlcNAc has yet to be achieved (Fischer et al 2018). Once such a chassis is available, the enzymatic machinery for the synthesis, transport and addition of galactosylated and sialylated N-linked glycans (Kallolimath et al 2016) can be introduced by conventional transformation to ultimately produce recombinant proteins with a homogeneous, human-like glycosylation profiles.…”

“…Product yields in plants can also vary substantially within the biomass (Sack et al 2015;Buyel and Fischer 2012;Knödler et al 2019), especially if host reactions, such as the response to infiltrating bacteria during transient expression, lead to the activation of endogenous proteases (Grosse-Holz et al 2017). Proteins expressed in plants and plant cells gain non-human glycosylation profiles (Fischer et al 2018;Strasser 2016), and further unwanted product modifications or degradation may occur during downstream processing (DSP) due to oxidation or proteolysis in the crude extract. DSP in general can be difficult to develop and operate for plant-based systems due to the large quantities of host cell proteins (HCPs) and potentially toxic metabolites in the extracts, including nicotine if whole tobacco plants are used as the production host (Buyel et al 2015b).…”