455721 Agrobacterium Tumefaciens Production to Enable the Large-Scale Transient Expression of Recombinant Proteins in Plants

Monday, November 14, 2016
Grand Ballroom B (Hilton San Francisco Union Square)
Ingrid Leth, Chemical Engineering & Materials Science, University of California at Davis, Davis, CA and Karen A McDonald, Chemical Engineering and Materials Science, University of California, Davis, Davis, CA

Production of recombinant 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. Agrobacterium-mediated transient expression in plants has been shown to be a viable method of producing functional human therapeutic proteins, vaccines, and commercial enzymes. Large-scale transient expression of recombinant proteins in plants is a developing area, and studies are underway to optimize the stages of the process. One area that has not been examined is the fermentation of Agrobacterium for the process at large-scale. We investigated the effects of growth conditions including temperature, pH, and media on Agrobacterium growth kinetics and gene transfer capability, with the goal of identifying optimal conditions for growing Agrobacterium at large scale. Growth temperature was found to affect bacterial growth rate but not gene transfer capability, and a growth temperature of 28oC 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 successfully scaled up to a 100 L working volume bioreactor.

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