(2h) Development of Rhodobacter Sphaeroides for the Production of Functional Membrane Proteins

In this paper, we present progress on the development of Rhodobacter sphaeroides as a platform for high-level expression of functional membrane proteins. The enhanced capacity of Rhodobacter for membrane protein expression when compared to traditional hosts results from the dense intracellular membrane infoldings synthesized by this organism under anaerobic photoheterotrophic growth conditions. Using plasmids with promoters from the light-harvesting pigments, membrane protein expression is induced simultaneously with membrane formation when the organism experiences anaerobic conditions in the presence of light. We present unique growth characteristics of this organism: although anaerobic growth ceases in the dark, anaerobic photoheterotrophic growth in the presence of light is faster than aerobic growth and results in up to 5-times greater biomass yield as a result of light-based energy production. Although genetically-engineered strains display dramatically reduced growth under low light conditions, this can be overcome with high-intensity light, even in ?knockout' strains where the dominant light harvesting protein has been deleted to increase functional membrane protein production capacity. Scaling up to a proprietary thin-film photobioreactor, we demonstrate photoheterotrophic growth to nearly 10 grams of cells per liter, an order of magnitude higher than current practice in flask culture. Membrane protein expression levels in the unoptimized system approach three times those of comparable systems from the literature.