Development of the Photoheterotroph Rhodobacter for Functional Membrane Protein Expression
Brandon S. Curtis1, Philip D. Laible2, Deborah K. Hanson2, and Wayne Curtis1. (1) Chemical Engineering Department, Pennsylvania State University, Fenske Building, University Park, PA 16802, (2) Biosciences Division, Argonne National Laboratory, Argonne, IL 60439
This paper presents progress on the development of Rhodobacter sphaeroides as a platform for high-level expression of functional membrane proteins. The high capacity of Rhodobacter for membrane protein expression stems from the dense intracellular membranes synthesized by this organism under anaerobic photoheterotrophic conditions. Using plasmids with promoters from the light-harvesting pigments, inducible heterologous expression is accomplished simultaneously with membrane formation when the organism experiences reduced oxygen tension and low-light conditions. Unique growth characteristics of this organism are presented: although anaerobic growth ceases in the dark, anaerobic photoheterotrophic growth is as fast as aerobic growth and results in nearly 3-times greater biomass yield as a result of photosynthetic ATP production. Growth yield (on carbon basis) is also several-fold higher on defined media as compared to complex media – which is of particular interest for the application of isotopic labeling for membrane protein structural studies. Although transformed lines display dramatically reduced growth under low light conditions, this can be overcome with high light, even with ‘knockout' strains where the dominant light harvesting protein (LH2) has been removed to facilitate greater capacity for functional heterologous expression. Preliminary growth testing in a thin film photobioreactor combined with mass-balance rationalized fed-batch operation are shown to facilitate photoheterotrophic growth to nearly 10 grams DW/L (an order of magnitude higher than current practice in flask culture). Preliminary results demonstrate high-level membrane protein expression.