Production and Characterization of Synthetic Carboxysome Shells

33 Spatial segregation of metabolism, such as cellular-localized CO2 fixation in C4 34 plants or in the cyanobacterial carboxysome, enhances the activity of inefficient 35 enzymes by selectively concentrating them with their substrates. The 36 carboxysome and other bacterial microcompartments (BMCs) have drawn 37 particular attention for bioengineering of nanoreactors because they are self38 assembling proteinaceous organelles. All BMCs share an architecturally similar, 39 selectively permeable shell that encapsulates enzymes. Fundamental to 40 engineering carboxysomes and other BMCs for applications in plant synthetic 41 biology and metabolic engineering is understanding the structural determinants 42 of cargo packaging and shell permeability. Here we describe the expression of a 43 synthetic operon in Escherichia coli that produces carboxysome shells. Protein 44 domains native to the carboxysome core were used to encapsulate foreign cargo 45 (GFP) into the synthetic shells. These synthetic shells can be purified to 46 homogeneity with or without luminal proteins. Our results not only further the 47 understanding of protein-protein interactions governing carboxysome assembly, 48 but also establish a platform to study shell permeability and the structural basis of 49 the function of intact BMC shells both in vivo and in vitro. This system will be 50 especially useful for developing synthetic carboxysomes for plant engineering. 51