In vitro cultured plant cells, in particular the tobacco BY-2 cell, have demonstrated their potential to provide a promising bioproduction platform for therapeutic proteins by integrating the merits of whole-plant cultivation systems with those of microbial and mammalian cell cultures. Over the past three decades, substantial progress has been made in improving the plant cell culture system, resulting in a few commercial success cases, such as taliglucerase alfa (Elelyso ® ), the first FDA-approved recombinant pharmaceutical protein derived from plant cells. However, compared to the major expression hosts (bacteria, yeast, and mammalian cells), plant cells are still largely underutilized, mainly due to low productivity and non-human glycosylation. Modern molecular biology tools, in particular RNAi and the latest genome editing technology CRISPR/Cas9, have been used to modulate the genome of plant cells to create new cell lines that exhibit desired “traits” for producing therapeutic proteins. This review highlights the recent advances in cellular engineering of plant cells towards improved recombinant protein production, including creating cell lines with deficient protease levels or humanized glycosylation, and considers potential development in the future.

Inflammatory bowel disease (IBD), including the two most common subtypes: Crohn’s disease (CD) and ulcerative colitis (UC), represents a group of intestinal disorders that cause prolonged inflammation of the digestive tract. The current treatment strategies, including the conventional anti‐inflammatory medications and the new biological drugs targeting the pro‐inflammatory cytokine tumor necrosis factor alpha (TNFα), have limited therapeutic efficacy and adverse drug reactions resulted from systemic administration. Colon‐targeted oral delivery of anti‐TNFα agents is highly desirable for the treatment of IBD, as it improves drugs’ efficacy while reducing systemic toxicity. Plant cell culture has emerged as a safe and cost‐effective bioproduction platform for therapeutic proteins. A unique feature of the plant cells is that they could serve not only as the “bio‐factory,” but also the oral delivery vehicle for recombinant biologics. Recent advances have demonstrated that plant cell walls, made primarily of cellulose microfibrils, can act as an excellent natural capsule for oral delivery of biologic drugs. This project aims to leverage two unique posttranslational modifications – “glycosyl‐phosphatidylinositol (GPI) anchor” and “plant‐specific hydroxyproline (Hyp)‐O‐glycosylation” – to strategically design and engineer novel anti‐TNFα biomolecules in plant cells to develop a new class of oral biologic drugs for the treatment of UC. The designer anti‐TNFα biomolecules consist of three functional domains: a N‐terminal single‐chain fragment variable (scFv) of an anti‐TNFα antibody, a proprietary Hyp‐O‐glycosylation module comprised of tandem repeats of the “Ser‐Pro” motif, or (SP)n (n= 5 to 30), and a C‐terminal GPI anchor. While the GPI anchor “displays” the expressed anti‐TNFα biomolecules at the plant cell surface to presumably create a high local concentration of the biologics, the (SP)n glycomodule stabilizes the protein from degradation during both the bioproduction and oral delivery processes. Designer anti‐TNFα biomolecules consisting of different sizes of the (SP)n glycomodule are investigated for their accumulations in tobacco BY‐2 cells, biological activity, and stability in a simulated gastric fluid, which determines the (SP)20 module as an optimal design for the biomolecules. The therapeutic effectiveness of the orally administrated designer anti‐TNFα biologic (optimal design) in mitigating the UC symptom is assessed in a dextran sulfate sodium (DSS)‐induced colitis mouse model. The immune‐modulatory effects of the anti‐TNFα biologics are determined by histopathological analysis and assay of the inflammatory markers. The research may develops a new platform to produce effective oral biologic drugs for the treatment of UC and other inflammatory diseases of colon.

Reconstructing the chemical and structural characteristics of the plant cell wall represents a promising solution to overcoming lignocellulosic biomass recalcitrance to biochemical deconstruction. This study aims to leverage hydroxyproline (Hyp)‐O‐glycosylation, a process unique to plant cell wall glycoproteins, as an innovative technology for de novo design and engineering in planta of Hyp‐O‐glycosylated biopolymers (HypGP) that facilitate plant cell wall reconstruction. HypGP consisting of 18 tandem repeats of “Ser–Hyp–Hyp–Hyp–Hyp” motif or (SP4)18 was designed and engineered into tobacco plants as a fusion peptide with either a reporter protein enhanced green fluorescence protein or the catalytic domain of a thermophilic E1 endoglucanase (E1cd) from Acidothermus cellulolyticus. The engineered (SP4)18 module was extensively Hyp‐O‐glycosylated with arabino‐oligosaccharides, which facilitated the deposition of the fused protein/enzyme in the cell wall matrix and improved the accumulation of the protein/enzyme in planta by 1.5–11‐fold. The enzyme activity of the recombinant E1cd was not affected by the fused (SP4)18 module, showing an optimal temperature of 80°C and optimal pH between 5 and 8. The plant biomass engineered with the (SP4)18‐tagged protein/enzyme increased the biomass saccharification efficiency by up to 3.5‐fold without having adverse impact on the plant growth.