title: Production and Characterization of Synthetic Carboxysome Shells with Incorporated Luminal Proteins.
abstract: Spatial segregation of metabolism, such as cellular-localized CO2 fixation in C4 plants or in the cyanobacterial carboxysome, enhances the activity of inefficient enzymes by selectively concentrating them with their substrates. The carboxysome and other bacterial microcompartments (BMCs) have drawn particular attention for bioengineering of nanoreactors because they are self-assembling proteinaceous organelles. All BMCs share an architecturally similar, selectively permeable shell that encapsulates enzymes. Fundamental to engineering carboxysomes and other BMCs for applications in plant synthetic biology and metabolic engineering is understanding the structural determinants of cargo packaging and shell permeability. Here we describe the expression of a synthetic operon in Escherichia coli that produces carboxysome shells. Protein domains native to the carboxysome core were used to encapsulate foreign cargo into the synthetic shells. These synthetic shells can be purified to homogeneity with or without luminal proteins. Our results not only further the understanding of protein-protein interactions governing carboxysome assembly, but also establish a platform to study shell permeability and the structural basis of the function of intact BMC shells both in vivo and in vitro. This system will be especially useful for developing synthetic carboxysomes for plant engineering.
authors.affiliation: Department of Plant and Microbial Biology, University of California, Berkeley, California 94720 (F.C., S.L.B., S.C.W., C.A.K.); Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720 (F.C., S.L.B., S.C.W., C.A.K.); and MSU-DOE Plant Research Laboratory and Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824 (C.A.K.).
authors.forename: Fei
authors.identifier: http://orcid.org/0000-0002-3742-6245
authors.initials: F
authors.lastname: Cai
authors.affiliation: Department of Plant and Microbial Biology, University of California, Berkeley, California 94720 (F.C., S.L.B., S.C.W., C.A.K.); Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720 (F.C., S.L.B., S.C.W., C.A.K.); and MSU-DOE Plant Research Laboratory and Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824 (C.A.K.).
authors.forename: Susan L
authors.initials: SL
authors.lastname: Bernstein
authors.affiliation: Department of Plant and Microbial Biology, University of California, Berkeley, California 94720 (F.C., S.L.B., S.C.W., C.A.K.); Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720 (F.C., S.L.B., S.C.W., C.A.K.); and MSU-DOE Plant Research Laboratory and Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824 (C.A.K.).
authors.forename: Steven C
authors.identifier: http://orcid.org/0000-0002-7382-3388
authors.initials: SC
authors.lastname: Wilson
authors.affiliation: Department of Plant and Microbial Biology, University of California, Berkeley, California 94720 (F.C., S.L.B., S.C.W., C.A.K.); Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720 (F.C., S.L.B., S.C.W., C.A.K.); and MSU-DOE Plant Research Laboratory and Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824 (C.A.K.) ckerfeld@lbl.gov.
authors.forename: Cheryl A
authors.identifier: http://orcid.org/0000-0002-9977-8482
authors.initials: CA
authors.lastname: Kerfeld
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000458:Cyanobacteria / Q000235:genetics* / Q000378:metabolism; D003594:Cytoplasmic Granules / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D015151:Immunoblotting; D046529:Microscopy, Electron, Transmission; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D009876:Operon; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D058615:Synthetic Biology / Q000379:methods
chemical_list: D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins