Growth kinetics and scale-up of Agrobacterium tumefaciens

Abstract

Production of recombinant proteins in plants through Agrobacterium-mediated transient expression is a promising method of producing human therapeutic proteins, vaccines, and commercial enzymes. This process has been shown to be viable at a large scale and involves growing large quantities of wild-type plants and infiltrating the leaf tissue with a suspension of Agrobacterium tumefaciens bearing the genes of interest. This study examined one of the steps in this process that had not yet been optimized: the scale-up of Agrobacterium production to sufficient volumes for large-scale plant infiltration. Production of Agrobacterium strain C58C1 pTFS40 was scaled up from shake flasks (50-100 mL) to benchtop (5 L) scale with three types of media: Lysogeny broth (LB), yeast extract peptone (YEP) media, and a sucrose-based defined media. The maximum specific growth rate (μ ) of the strain in the three types of media was 0.46 ± 0.04 h in LB media, 0.43 ± 0.03 h in YEP media, and 0.27 ± 0.01 h in defined media. The maximum biomass concentration reached at this scale was 2.0 ± 0.1, 2.8 ± 0.1, and 2.6 ± 0.1 g dry cell weight (DCW)/L for the three media types. Production was successfully scaled up to a 100-L working volume reactor with YEP media, using k a as the scale-up parameter.

Media development for large scale Agrobacterium tumefaciens culture

“…A few additional considerations regarding the use of defined media: this media is more economical in terms of material cost, but the labor cost of preparation may be higher since there are more steps required. The overall ionic character of the defined media is also a consideration, as higher ionic strength may correspond to higher cell maintenance costs, and previous work has indicated that Agrobacterium grown in chemically defined media may be more sensitive to shear stress . In this study sucrose and ammonium concentration were observed to have a negative effect on final biomass concentration, but higher biomass concentrations may be possible if the concentration of these nutrients can be increased without this negative effect.…”

“…With no supplemental sugars added to LB or YEP media the most abundant nutrient source available in these media is amino acids. To determine if certain amino acids were preferentially consumed, free amino acid concentration was measured at three points over an Agrobacterium batch growth in LB media grown at a 5 L scale . Measurements were taken on media directly before inoculation (0 h), during the exponential phase of growth (8 h), and when growth had reached stationary phase (12.5 h) and a final biomass concentration of 3.3 OD 600 (2.0 g DCW/L).…”

“…Free amino acid concentration was also measured at three points over an 18‐h Agrobacterium growth period in YEP media at a 100 L volume. The 100 L Agrobacterium fermentation was carried out as previously described . The overall concentration of free amino acids was lower in the YEP media than in LB media, but l ‐asparagine and l ‐serine were again the first amino acids to be depleted (Figure ).…”

“…Readings were adjusted to correspond to a 1 cm pathlength. Optical density was converted to dry cell weight per milliliter based on linear correlations determined for each type of media; correlations were 0.60 ± 0.01 (g DCW/L)/OD 600 for LB, 0.46 ±0.01 (g DCW/L)/OD 600 for YEP, and 0.66 ± 0.02 (g DCW/L)/OD 600 for the defined media.…”

Production of recombinant butyrylcholinesterase from transgenic rice cell suspension cultures in a pilot‐scale bioreactor

“…Wild-type plant cell suspension cultures in pilot-scale and industrial-scale STBs for the production of plant metabolites have been widely reported (Katō et al, 1972;Noguchi et al, 1977;Schiel & Berlin, 1987;Ulbrich et al, 1985), including Taxus chinensis suspended cells grown in a 75,000-L STB at Phyton Biotech® for the production of paclitaxel, a potent cancer treatment chemical (Imseng et al, 2014). However, the studies of pilot-scale and commercialscale bioreactors for recombinant therapeutic protein productions from transgenic plant cell cultures are scarce, especially in STBs.…”