Effects of plant density on recombinant hemagglutinin yields in an Agrobacterium‐mediated transient gene expression system using Nicotiana benthamiana plants

Abstract

Agrobacterium-mediated transient expression systems enable plants to rapidly produce a wide range of recombinant proteins. To achieve economically feasible upstream production and downstream processing, it is beneficial to obtain high levels of two yield-related quantities of upstream production: recombinant protein content per fresh mass of harvested biomass (g gFM ) and recombinant protein productivity per unit area-time (g m /month). Here, we report that the density of Nicotiana benthamiana plants during upstream production had significant impacts on the yield-related quantities of recombinant hemagglutinin (HA). The two quantities were smaller at a high plant density of 400 plants m than at a low plant density of 100 plants m . The smaller quantities at the high plant density were attributed to: (i) a lower HA content in young leaves, which usually have high HA accumulation potentials; (ii) a lower biomass allocation to the young leaves; and (iii) a high area-time requirement for plants. Thus, plant density is a key factor for improving upstream production in Agrobacterium-mediated transient expression systems. Biotechnol. Bioeng. 2017;114: 1762-1770. © 2017 Wiley Periodicals, Inc.

Effects of environmental conditions before gene transfer on the amount of influenza hemagglutinin transiently expressed in <I>Nicotiana benthamiana</I> leaves

“…This suggests that the average PPFD, rather than the PPFD during a particular period e.g., for a few days before gene transfer , affected the accumulation level of HA in leaves. These results for leaf HA content per unit FM are consistent with results from our recent study in which leaf HA content per unit FM was significantly lowered when plant density was high 400 plants m 2 , Fujiuchi et al, 2017 , as dense planting decreases the amount of light received by a plant. In contrast, Shang et al 2018 reported that supplemental lighting with LEDs and dense planting in a greenhouse before gene transfer had little or no effect on leaf HA content per unit of biomass.…”

Development of Systems for the Production of Plant-Derived Biopharmaceuticals

“…Changing environmental conditions at appropriate times during each stage may be required to obtain a high yield [56]; a high-nitrate nutrient solution during the pre-inoculation state results in high recombinant protein content [55]. Very recently, interest in the surrounding environment has extended beyond research into the plant itself [57,58,71]. Nitrate-enriched fertilizers that enable plant growth [55], immediate desiccation after agro-infiltration [56], and slightly lower plant density [57], are critical factors for improving plant biomass.…”

“…Very recently, interest in the surrounding environment has extended beyond research into the plant itself [57,58,71]. Nitrate-enriched fertilizers that enable plant growth [55], immediate desiccation after agro-infiltration [56], and slightly lower plant density [57], are critical factors for improving plant biomass. Plant density, the number of plants per unit growth area, and the effect of leaf position affect harvested biomass, and are therefore, important factors for achieving economically feasible production [57].…”

“…Nitrate-enriched fertilizers that enable plant growth [55], immediate desiccation after agro-infiltration [56], and slightly lower plant density [57], are critical factors for improving plant biomass. Plant density, the number of plants per unit growth area, and the effect of leaf position affect harvested biomass, and are therefore, important factors for achieving economically feasible production [57]. Removal of residual water from a bacterial suspension on detached leaves significantly affects the yield of recombinant hemagglutinin (HA) protein, and recovery from detached leaves in a transient over-expression system is comparable with that from intact leaves [56].…”

Production of Biopharmaceuticals in Nicotiana benthamiana—Axillary Stem Growth as a Key Determinant of Total Protein Yield

“…Other studies have described the expression of a viral proton channel to stabilize labile proteins in the cell secretory pathway (Jutras et al., 2015, 2018), the expression of a human convertase to promote the post-translational proteolytic processing of clinically useful proteins in vivo (Wilbers et al., 2016; Mamedov et al., 2019), or the exogenous induction of the jasmonic acid defense pathway to reduce endogenous protein content in leaf tissue prior to recombinant protein purification (Robert et al., 2015). In parallel, studies have documented the effects of cultural practices on N. benthamiana growth and leaf biomass production before agroinfiltration (Fujiuchi et al., 2014; Shang et al., 2018), the influence of environmental parameters in growth chambers following agroinfiltration (Matsuda et al., 2017, 2018), or the impact of plant density on overall protein yield in specific culture settings (Fujiuchi et al., 2017; Shang et al., 2018).…”