Morphology and bioproduction potential of DCB-induced cell wall-deficient tobacco BY-2 cells and transcriptomic...

Plant cell cultures, especially tobacco BY-2 cells, represent a promising platform for recombinant protein production. However, their full potential is hindered by several challenges. Some of these challenges, such as inefficient protein secretion, the presence of large vacuoles, and difficulties in cryopreservation can be attributed to the rigidity of the plant cell wall. We developed and characterized cell wall-deficient BY-2 cells by gradually adapting them to the cellulose biosynthesis inhibitor 2,6-dichlorobenzonitrile (DCB). The DCB-adapted cells exhibited marked morphological changes, including cell clumping due to pectin overaccumulation and an ∼80 % reduction in cellulose content. Despite these alterations, the DCB-adapted BY-2 cells maintained robust growth and demonstrated efficient Agrobacterium-mediated transformation. The production and secretion levels of the model protein EGFP and the therapeutic protein human EPO were 2.1- to 2.8-fold higher than those observed in native BY-2 cells. However, secretion of EGFP fused with a hydroxyproline-O-glycosylated module (SP)₃₂ was dramatically enhanced by up to 35-fold. Transcriptomic analysis revealed broad gene expression changes, including down-regulation of genes involved in cellulose biosynthesis and upregulation of genes associated with branched pectic polysaccharide synthesis. These results provide a proof of concept that cell wall-deficient plant cell lines can serve as effective platforms for recombinant protein production, laying foundation for further engineering of plant cells as next-generation biofactories.