Gene Editing of Nicotiana benthamiana Architecture for Space-Efficient Production of Recombinant Proteins in Closed Environments.

Indoor vertical farming (VF) offers practical advantages for the cultivation of plant protein bio-factories including plant uniformity, product consistency, water/nutrient recycling and production cycles on a year-round basis. Much progress has been achieved toward the development of innovative systems for artificial lighting, automated irrigation, plant handling, environmental control and space use optimization in VF systems. Here, we used a CRISPR/Cas9 gene editing approach to generate mutant lines of protein expression host Nicotiana benthamiana presenting a compact, space-efficient phenotype suited to VF systems. Our strategy consisted of altering apical dominance by suppressing the synthesis of strigolactone, a negative regulator of axillary bud outgrowth. Strigolactone-depleted lines were generated by hindering the expression of either Carotenoid cleavage dioxygenase 7 (CCD7) or Carotenoid cleavage dioxygenase 8 (CCD8), two key enzymes of the strigolactone synthetic pathway. Knocking out of either enzyme had no impact on the plant's growth rate but drastically influenced its auxin/cytokinin ratio, leaf proteome and overall architecture. The ΔCCD mutants exhibited an axillary growth-oriented development pattern, altered glycolytic and malate-processing metabolic fluxes to drive pyruvate and cytokinin production, and a spatial footprint reduced by 45%-50% compared to the LAB strain commonly used for protein expression. Most importantly, recombinant protein yields per plant were maintained in the mutant lines, as illustrated with GFP and rituximab, a chimeric antibody of therapeutic value. Our data confirm the usefulness of ΔCCD7 and ΔCCD8 knockouts leading to strigolactone depletion for the generation of compact, space-efficient N. benthamiana lines better suited to VF systems.