At-CycD2 Enhances Accumulation of Above-Ground Biomass and Recombinant Proteins in Transgenic Nicotiana benthamiana Plants

Abstract

Transient expression in Nicotiana benthamiana holds great potential for recombinant protein manufacturing due to its advantages in terms of speed and yield compared to stably transformed plants. To continue improving the quantity of recombinant proteins the plant host will need to be modified at both plant and cellular levels. In attempt to increase leaf mass fraction, we transformed N. benthamiana with the At-CycD2 gene, a positive regulator of the cell cycle. Phenotypic characterization of the T1 progeny plants revealed their accelerated above-ground biomass accumulation and enhanced rate of leaf initiation. In comparison to non-transgenic control the best performing line At-CycD2-15 provided 143 and 140% higher leaf and stem biomass fractions, respectively. The leaf area enlargement of the At-CycD2-15 genotype was associated with the increase of epidermal cell number compensated by slightly reduced cell size. The production capacity of the At-CycD2-15 transgenic line was superior to that of the non-transgenic N. benthamiana. The accumulation of transiently expressed GFP and scFv-TM43-E10 proteins per unit biomass was increased by 138.5 and 156.7%, respectively, compared to the wild type. With these results we demonstrate the potential of cell cycle regulator gene At-CycD2 to modulate both plant phenotype and intracellular environment of N. benthamiana for enhanced recombinant protein yield.

Effect of the At-CDC27a gene on Nicotiana benthamiana phenotype and accumulation of recombinant proteins

“…Similar increases in GFP and scFv-TM43-E10 yield were observed in N. benthamiana plants co-infiltrated with the plant-derived cell cycle regulators At-CycD2 and At-CDC27a and full TMV-based vector carrying GFP and scFv-TM43-E10 (Kopertekh and Schiemann, 2019b). Another study confirmed the positive effect of stably integrated At-CycD2 on production of GFP and scFv-TM43-E10 provided by the deconstructed TMV-based TRBO vector (Kopertekh and Reichardt, 2021).…”

“…Production of recombinant proteins in the At-CDC27a transgenic and nontransgenic N. benthamiana plants Agroinfiltration was performed following the procedure described by Kopertekh and Reichardt (2021). The samples for ELISA were collected from the middle leaves at 2, 4, 6 and 10 days after infiltration (dpi).…”

“…Transgenic N. benthamiana plants expressing the At-CycD2 positive cell cycle regulator were shown to produce 143% more leaf biomass than non-transgenic plants. In comparison to wild-type the yield of recombinant proteins in the At-CycD2 plants was increased by 139% for the green fluorescent protein (GFP) and 157% for single chain variable fragment (scFv-TM43-E10) ( Kopertekh and Reichardt, 2021 ).…”

Overexpression of PagSTOMAGEN, a Positive Regulator of Stomatal Density, Promotes Vegetative Growth in Poplar

“…SPSS software (IBM Corp., Armonk, NY, USA) was used to perform the statistical analyses, and the data are represented as the mean ± SD. The Student’s t -test and one-way ANOVA were used to determine variance among the mean values [ 77 ].…”

Ectopic Expression of Poplar PsnCYCD1;1 Reduces Cell Size and Regulates Flower Organ Development in Nicotiana tabacum

“…The Populus trichocarpa PtCYCB1 promoter is used to drive the expression of the CYCD gene in tobacco, which improves the cell division of vascular cambium and increases the differentiation of secondary xylem in tobacco ( Fujii et al, 2012 ). The ectopic expression of AtCYCD2 accelerates the growth and aboveground biomass accumulation rate of transgenic tobacco from the seedling stage to maturity ( Boucheron et al, 2005 ; Kopertekh and Reichardt, 2021 ). Moreover, overexpressing the CYCD3 gene into tobacco also accelerates the growth rate of leaves and changes the structure of the shoot apical meristem ( Boucheron et al, 2005 ).…”