LAUSR

The transient expression of recombinant proteins in plant tissues infiltrated with Agrobacterium tumefaciens is an established basic research method that is also widely used for the development and manufacture of plant-made pharmaceuticals (PMPs). However, large-scale automated production requires the synchronization of plant cultivation and bacterial fermentation. This is particularly important if the plant material has to be co-infiltrated with more than one bacterial strain. Therefore, we developed a ready to use approach that decouples bacterial fermentation and infiltration. We found that bacterial cultures can easily be reconstituted in infiltration medium at a user-defined time, optical density and quantity. This allows the process flow to be staggered, avoiding bottlenecks in process capacity and labor. Using the red fluorescent protein, DsRed, as a model product, the ready to use preparations achieved the same yields in infiltrated plant biomass as A. tumefaciens derived from regular fermentations. It was possible to store the ready to use stocks at -20°C and -80°C for more than two months without loss of activity. Using a consolidated cost model for our fermentation process, we found that the ready to use strategy can reduce operational costs by 20-95% and investment costs by up to 75%, which would otherwise offset the economic advantages of plants over mammalian expression systems during upstream production. Furthermore, the staggered cultivation of plants and bacteria reduces the likelihood of batch failure and thus increases the robustness and flexibility of transient expression for the production of recombinant proteins in plants. This article is protected by copyright. All rights reserved.