Agrobacterium tumefaciens Production to Enable the Large-Scale Transient Expression of Recombinant Proteins in Plants

Production of proteins through in planta transient expression offers an alternative to conventional microbial and mammalian cell culture systems. This platform is particularly appealing because of its rapid and relatively low-cost implementation and its ease of scale-up. Transient expression occurs when a functional gene construct is inserted into a plant cell, where it is expressed over a short period of time without being stably integrated into the plant genome. Large-scale transient expression of recombinant proteins in plants is a relatively new area, and studies are underway to optimize the stages of the process in order to make it economically competitive. One area that has not been examined is the fermentation of A. tumefaciens for agroinfiltration at a large-scale. This research investigated the effects of growth conditions including temperature, pH, and media composition on Agrobacterium growth kinetics and gene transfer capability, with the goal of identifying optimal process conditions for growing Agrobacterium at large scale for use in transient agroinfiltration. Growth temperature was found to affect bacterial growth rate but not gene transfer capability, and a growth temperature of 28°C was selected as optimal. Growth in Lysogeny Broth (LB) and Yeast Extract Peptone (YEP) media was examined and subsequent transient gene expression was measured. A defined media was developed and optimized for growing Agrobacterium and growth and gene transfer capability with this media was found to be comparable to LB and YEP media. Growth of Agrobacterium strain C58C1 pTFS40 in LB, YEP, and defined media resulted in maximum specific growth rates of 0.36 ± 0.01, 0.37 ± 0.03, and 0.33 ±0.01 h−1 and maximum biomass concentrations of 1.9, 3.6, and 3.9 grams dry cell weight per liter after 12, 16, and 20 hours, respectively. It was demonstrated that direct infiltration with Agrobacterium in diluted growth media was an effective method of inducing transient expression. Batch fermentation of Agrobacterium was scaled up to benchtop (5 L) scale with the three types of media. Finally, production was scaled up to a 100 L working volume reactor