Development of a Molasses-Based Medium for Agrobacterium tumefaciens Fermentation for Application in Plant-Based Recombinant Protein Production

The Agrobacterium-mediated transient gene expression system is a rapid and efficient method for heterologous recombinant protein expression in plants. The fermentation of genetically modified Agrobacterium tumefaciens is an important step in increasing the efficiency of recombinant protein production in plants. However, the limitation of this system that makes it economically non-competitive for industrial-scale applications is the Agrobacterium suspension production cost. In this study, the utilization of sugarcane molasses as an alternative low-cost source of carbon at a concentration of 8.7 g/L and nitrogen at a concentration of 2.4 g/L for Agrobacterium cultivation was investigated. Molasses pretreatment using sulfuric acid (SA) was applied before fermentation, and it resulted in a maximum specific growth rate of 0.232 ± 0.0063 h⁻¹ in the A. tumefaciens EHA105 culture. The supplementation of antibiotics in the molasses-based medium was shown to be unnecessary for plasmid maintenance during fermentation in both Agrobacterium strains, which helped to reduce the production cost. We evaluated recombinant protein production using an Agrobacterium culture without antibiotic supplementation in the growth media by demonstrating green fluorescent protein expression in wild-type Nicotiana benthamiana leaves. In the evaluation of the culture medium cost, the molasses-based medium cost was 6.1 times lower than that of LB. Finally, this study demonstrated that the newly developed molasses-based medium for Agrobacterium fermentation is a feasible and effective medium for transient recombinant protein production in plant tissues.