(582bn) Fermentation of Agrobacterium Tumefaciens for 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 is induced by introducing a gene construct to the nucleus plant cell, where it is expressed for a period of time without being stably integrated into the plant genome. A common method for inducing transient expression is to use the gene transfer capability of Agrobacterium tumefaciens. Agrobacterium-mediated transient expression can be induced in whole plants or in pre-harvested plant tissue by “agroinfiltration,” a process that introduces an Agrobacterium suspension to the interior of a plant leaf, allowing the bacteria to transfer the target gene to the plant cells using its natural virulence machinery. 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 Agrobacterium for agroinfiltration at a large-scale. We are investigating the effects of growth conditions including temperature, pH, and media on Agrobacterium growth kinetics and gene transfer capability, with the goal of identifying optimal processing conditions for growing Agrobacterium at large scale in a 100L working volume bioreactor. Specific growth rates and biomass yield parameters for a range of laboratory Agrobacterium strains have been identified, and  the effect of growth temperature and media composition on recombinant protein expression level in plants have been examined. Maximum specific growth rates were found to vary from 0.13 to 0.38 h^-1, and biomass yield was found to be in the range of 0.66-0.76 g biomass/g sucrose, depending on growth conditions and strain. It was determined that growth temperature and bacterial harvest time did not significantly affect transgenic protein expression level after agroinfiltration, allowing greater flexibility in large scale operation design. A defined media for Agrobacterium fermentation has been developed and direct agroinfiltration with the bacterial growth media has been demonstrated to be effective, which may allow the elimination of a costly processing step when preparing Agrobacterium for large scale vacuum infiltration.