Plants are being recognized as promising hosts for molecular farming and several molecular tools have been developed over the last two decades to optimize recombinant protein yields. However, the effects of basic growth factors on protein yield have been much less studied. Here, we investigated the effects of supplemental light emitting diode (LED) inter-lighting, CO2 enrichment, and plant density on growth and recombinant protein yield of Nicotiana benthamiana used as a host to express the vaccine antigen influenza virus hemagglutinin H1. LED inter-lighting improved plant growth and recombinant protein yield on a per-plant basis. CO2 enrichment also enhanced plant growth, but its effect on recombinant protein yield was not significant. By comparison, high plant density decreased recombinant protein production per plant, mainly because of its negative impact on protein accumulation on a per-plant basis. On a whole-crop area basis, supplemental lighting, CO2 enrichment, and high plant density improved plant growth, while only LED inter-lighting and high plant density positively impacted recombinant protein yield. We suggest that LED inter-lighting and an elevated plant density should be used to maximize H1 antigen yield in large-scale protein production systems using N. benthamiana.
