The Impacts of Environmental Factors and Culture Conditions on the Transient Expression of Recombinant Protein in <i>Nicotiana benthamiana</i> Under Controlled Environments

I summarise and discuss the effects of environmental factors and culture conditions on the productivity of recombinant proteins, including biopharmaceuticals, in Nicotiana benthamiana via transient gene expression platforms. A primary focus is on controlled-environment plant cultivation systems such as plant factories with artificial lighting. Before gene transfer, sufficient leaf biomass accumulation is suggested to contribute positively to subsequent target protein expression per unit of leaf biomass. Increasing the plant density and extending the photoperiod may be simple methods for increasing biomass productivity per unit of cultivation area; however, careful evaluation is needed to determine the effectiveness of these methods. Strictly regulated plant growth conditions should be crucial not only after but also before gene transfer to reduce batch-to-batch variation in target protein productivity. After gene transfer, target protein accumulation in leaves is highly sensitive to temperature. In this context, not only the air temperature but also the leaf temperature should be considered, given that the two temperatures can differ as a result of the leaf energy balance. Agroinfiltrated leaves often exhibit necrosis, which has been suggested to be triggered by endoplasmic reticulum stress, followed by oxidative stress. Lowering the air temperature or applying ascorbic acid can reduce the severity of necrosis. Light may only be necessary for specific timings after agroinfiltration, although more work is needed to draw general conclusions. A detached leaf-based system may be a promising choice because the handling of plant materials is easier than a whole plant-based system. Finally, current conclusions and future perspectives are provided.