Environment Control to Improve Recombinant Protein Yields in Plants Based on Agrobacterium-Mediated Transient Gene Expression

Abstract

Agrobacterium-mediated transient expression systems enable plants to produce a wide range of recombinant proteins on a rapid timescale. To achieve economically feasible upstream production and downstream processing, two yield parameters should be considered: (1) recombinant protein content per unit biomass and (2) recombinant protein productivity per unit area–time at the end of the upstream production. Because environmental factors in the upstream production have impacts on these parameters, environment control is important to maximize the recombinant protein yield. In this review, we summarize the effects of pre- and postinoculation environmental factors in the upstream production on the yield parameters and discuss the basic concept of environment control for plant-based transient expression systems. Preinoculation environmental factors associated with planting density, light quality, and nutrient supply affect plant characteristics, such as biomass and morphology, which in turn affect recombinant protein content and productivity. Accordingly, environment control for such plant characteristics has significant implications to achieve a high yield. On the other hand, postinoculation environmental factors, such as temperature, light intensity, and humidity, have been shown to affect recombinant protein content. Considering that recombinant protein production in Agrobacterium-mediated transient expression systems is a result of a series of complex biological events starting from T-DNA transfer from Agrobacterium tumefaciens to protein biosynthesis and accumulation in leaf tissue, we propose that dynamic environment control during the postinoculation process, i.e., changing environmental conditions at an appropriate timing for each event, may be a promising approach to obtain a high yield. Detailed descriptions of plant growth conditions and careful examination of environmental effects will significantly contribute to our knowledge to stably obtain high recombinant protein content and productivity, thus enhancing the utility of plant-based transient expression systems as recombinant protein factories.

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

“…This distribution pattern differed from that of the TSP content per unit biomass (Fig. Because Agrobacterium-mediated transient gene expression can be sensitive to temperature, PPFD and humidity after agroinfiltration (Fujiuchi et al, 2016a), such microclimate within the canopy at the high plant density might have had negative effects on HA accumulation in the young leaves. At the high plant density, the young leaves appeared to be overlapped by the old leaves (Fig.…”

“…Within a dense canopy, high relative humidity is generally observed as well as low light irradiation. Because Agrobacterium-mediated transient gene expression can be sensitive to temperature, PPFD and humidity after agroinfiltration (Fujiuchi et al, 2016a), such microclimate within the canopy at the high plant density might have had negative effects on HA accumulation in the young leaves.…”

“…In particular, decrease in recombinant protein content in young leaves at the high plant density, which might have been caused by microclimate in a dense canopy such as low PPFD in this study, might not be significant when using vectors and proteins other than those in this study. The effect of light intensity on recombinant protein accumulation in plant leaves in Agrobacterium-mediated transient expression systems significantly varies among researches using different vectors and subcellular localization (Fujiuchi et al, 2016a).…”

“…For the economically feasible manufacturing of recombinant proteins using Agrobacterium-mediated transient expression systems, there are two important yield-related quantities of upstream production needed to achieve a high recombinant protein yield (Fujiuchi et al, 2016a). The first is the recombinant protein content per unit fresh mass (FM) of harvested biomass (mg gFM À1 ), and the second is the recombinant protein productivity per unit area-time (unit: mg m À2 /month).…”

Development of Systems for the Production of Plant-Derived Biopharmaceuticals

“…Plant culture for transient expression is usually divided into two stages, the pre-inoculation and post-inoculation processes, which require different optimal environments. Cultivation conditions, such as the nutrient composition of the culture medium before agro-infiltration [55] and dehydration of plant material after agro-infiltration [70], are key factors. 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].…”