title: Streamlined Construction of the Cyanobacterial CO2-Fixing Organelle via Protein Domain Fusions for Use in Plant Synthetic Biology.
abstract: Bacterial microcompartments (BMCs) are self-assembling organelles that sequester segments of biochemical pathways within a protein shell. Given their functional diversity, BMCs constitute a rich source of metabolic modules for applications in synthetic biology. The carboxysome, the cyanobacterial BMC for CO(2) fixation, has attracted significant attention as a target for installation into chloroplasts and serves as the foundation for introducing other types of BMCs into plants. Carboxysome assembly involves a series of protein-protein interactions among at least six gene products to form a metabolic core, around which the shell assembles. This complexity creates significant challenges for the transfer, regulation, and assembly of carboxysomes, or any of the myriad of functionally distinct BMCs, into heterologous systems. To overcome this bottleneck, we constructed a chimeric protein in the cyanobacterium Synechococcus elongatus that structurally and functionally replaces four gene products required for carboxysome formation. The protein was designed based on protein domain interactions in the carboxysome core. The resulting streamlined carboxysomes support photosynthesis. This strategy obviates the need to regulate multiple genes and decreases the genetic load required for carboxysome assembly in heterologous systems. More broadly, the reengineered carboxysomes represent a proof of concept for a domain fusion approach to building multifunctional enzymatic cores that should be generally applicable to the engineering of BMCs for new functions and cellular contexts.
authors: MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, Michigan 48824.
authors: C Raul
authors: http://orcid.org/0000-0003-3171-8106
authors: CR
authors: Gonzalez-Esquer
authors: MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, Michigan 48824.
authors: Tyler B
authors: TB
authors: Shubitowski
authors: MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, Michigan 48824 Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720 Department of Plant and Microbial Biology, UC Berkeley, Berkeley, California 94720 Berkeley Synthetic Biology Institute, UC Berkeley, Berkeley, California 94720 ckerfeld@lbl.gov.
authors: Cheryl A
authors: http://orcid.org/0000-0002-9977-8482
authors: CA
authors: Kerfeld
mesh_terms: D002245:Carbon Dioxide / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D015388:Organelles / Q000235:genetics* / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods; D017434:Protein Structure, Tertiary / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism*; D012273:Ribulose-Bisphosphate Carboxylase / Q000235:genetics; D046940:Synechococcus / Q000235:genetics / Q000378:metabolism*; D058615:Synthetic Biology / Q000379:methods
chemical_list: D011993:Recombinant Fusion Proteins; D002245:Carbon Dioxide; D049452:Green Fluorescent Proteins; C101625:RbcL protein, plastid; D012273:Ribulose-Bisphosphate Carboxylase