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Modularity of a Carbon-Fixing Protein Organelle

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Modularity of a Carbon-Fixing Protein Organelle Modularity of a carbon-fixing protein organelle Walter Bonaccia, Poh K. Tengb, Bruno Afonsoa, Henrike Niederholtmeyera, Patricia Grobc, Pamela A. Silvera,d, and David F. Savageb,e,1 aDepartment of Systems Biology, Harvard Medical School, Boston, MA 02115; bDepartment of Molecular and Cell Biology and cHoward Hughes Medical Institute, University of California, Berkeley, CA 94720; dWyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115; and eDepartment of Chemistry, University of California, Berkeley, CA 94720 Edited* by Robert M. Stroud, University of California, San Francisco, CA, and approved November 17, 2011 (received for review May 31, 2011) Bacterial microcompartments are proteinaceous complexes that appear in roughly 20% of bacteria, suggesting the existence of catalyze metabolic pathways in a manner reminiscent of organelles. a large functional diversity of uncharacterized BMCs (10). Although microcompartment structure is well understood, much The CB was the first BMC to be discovered and remains a less is known about their assembly and function in vivo. We show model system for elucidating how BMCs assemble and function here that carboxysomes, CO2-fixing microcompartments encoded in the cell (11). Its shell is assembled from roughly 800 protein by 10 genes, can be heterologously produced in Escherichia coli. hexamers, which form the facets of the icosahedron, and 12 Expression of carboxysomes in E. coli resulted in the production pentamers, which form the vertices (8, 12). Inside the lumen of icosahedral complexes similar to those from the native host. In are the enzymes ribulose 1,5-bisphosphate carboxylase/oxygenase vivo, the complexes were capable of both assembling with carbox- (RuBisCO) and carbonic anhydrase (CA), which are directed to fi fi ysomal proteins and xing CO2. Characterization of puri ed syn- this location by protein–protein interactions with the shell and/or thetic carboxysomes indicated that they were well formed in shell interaction mediators (13). Depending on the organism, structure, contained the expected molecular components, and were genes for these shell proteins and luminal enzymes can occur fi capable of xing CO2 in vitro. In addition, we verify association of either together in a single operon or, alternatively, in several the postulated pore-forming protein CsoS1D with the carboxysome operons across the genome (9). and show how it may modulate function. We have developed a ge- Biochemically, the CB plays a central role in the carbon- netic system capable of producing modular carbon-fixing micro- concentrating mechanism (CCM) (14, 15). In the CCM, in- compartments in a heterologous host. In doing so, we lay the organic carbon (i.e., CO2 and bicarbonate) is actively transported groundwork for understanding these elaborate protein complexes into the cell. Bicarbonate, the major inorganic carbon species at and for the synthetic biological engineering of self-assembling pH ∼7, passively diffuses across the CB shell and is rapidly molecular structures. converted by CA into CO2. Alone, RuBisCO exhibits both low CO2 affinity and low selectivity against the competing substrate ribulose 1,5-bisphosphate carboxylase/oxygenase | synthetic biology | O2. In the context of the CCM, however, it is postulated that the metabolic engineering | self-assembly CB lumen contains elevated levels of CO2 and that RuBisCO is able to fixCO2 near its Vmax with high specificity. After fixation, he spatial organization of incompatible processes is a funda- the product 3-phosphoglycerate diffuses out of the carboxysome Tmental design principle of biological systems (1). For exam- and enters the central carbon metabolism (Fig. 1B). High local ple, metabolic pathways are confined to specific organelles as concentrations of CO2 and the possible selective nature of the a means of ensuring pathway fidelity, catalyzing reactions against shell proteins may be instrumental in excluding the competing the chemical equilibrium of other compartments, and limiting RuBisCO substrate O2 from the lumen, but this hypothesis exposure to toxic intermediates (2). Despite the generality of this remains unproven. In other BMCs, the shell is known to act as strategy in nature, the ability to rationally organize processes a barrier to prevent both the loss and toxicity of pathway inter- in vivo remains a major challenge to bioengineering (3). Recent mediates (16, 17). Finally, it was recently shown that a unique work has demonstrated that engineering protein–protein inter- class of trimeric shell proteins with pseudo-sixfold symmetry, actions using molecular scaffolds can be used to rewire signaling notably CsoS1D, forms larger pores and shows evidence of gat- networks and improve metabolic pathways (4, 5). A desirable ing, suggesting that these proteins may be crucial to substrate extension of this modularity would be the ability to rationally and product permeability (18, 19). organize pathways or networks in a topologically distinct com- Recent work has focused on the modularity of BMC assembly. partment, or synthetic organelle. If available, a synthetic organ- In the case of the CB from Halothiobacillus neapolitanus, in elle would have numerous applications in bioengineering, nano- which all genes occur in a single operon, deletion of RuBisCO- encoding genes was found to lead to empty, yet morphologically technology, and particularly metabolic engineering, where it could fi be used to improve the rate and yield of cellular chemical reactions well-formed shells (20). This nding suggests that BMCs form while lowering toxicity to the host. robust structures in which alternative proteins could be loaded One biological assembly that could form the basis of such inside the lumen. We recently reported that proteins can be a synthetic organelle is the bacterial microcompartment (BMC). targeted to the CB via fusion with CB proteins such as RuBisCO (21). Work on the PDU has confirmed this modularity. A genetic BMCs are enzyme-containing proteinaceous complexes that cat- screen in Citrobacter freundii led to the isolation and refinement alyze metabolic pathways incongruous with the cytoplasm (Fig. 1). of a single PDU regulon sequence for expressing functional Structurally, BMCs are formed from thousands of shell proteins that self-assemble into an icosahedral structure 100–200 nm in diameter (Fig. 1A)(6–8). These shell proteins surround a lumen fi Author contributions: W.B., P.K.T., B.A., P.A.S., and D.F.S. designed research; W.B., P.K.T., containing tens to hundreds of copies of two to four speci c H.N., P.G., and D.F.S. performed research; P.G. contributed new reagents/analytic tools; enzymes that constitute a short metabolic pathway. This metabolic W.B., P.K.T., P.A.S., and D.F.S. analyzed data; and W.B., P.A.S., and D.F.S. wrote the paper. pathway is the distinguishing feature between BMC compart- The authors declare no conflict of interest. ments of different organisms. There are currently three known *This Direct Submission article had a prearranged editor. fi 1 classes of BMCs: the CO2- xing carboxysome (CB), the 1,2-pro- To whom correspondence should be addressed. E-mail: [email protected]. panediol utilization microcompartment (PDU), and the ethanol- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. amine utilization microcompartment (9). BMC protein domains 1073/pnas.1108557109/-/DCSupplemental. 478–483 | PNAS | January 10, 2012 | vol. 109 | no. 2 www.pnas.org/cgi/doi/10.1073/pnas.1108557109 Downloaded by guest on September 30, 2021 Fig. 1. Physical and genetic organization of the carboxysome. (A) Molecular surface representation of a partial model of the carboxysome showing RuBisCO (green), carbon anhydrase (orange), CsoS1ABC (blue), and CsoS4AB (red). (B) Schematic of the carbon concentrating mechanism adapted from Kaplan and Reinhold (14). (C) Genomic organization of the carboxysome operon in H. neapolitanus. Colors match the structural model in A. compartments in Escherichia coli (22, 23). In addition, a putative galactopyranoside (IPTG)-inducible plasmid pNS3 (pHnCB) N-terminal signal sequence capable of loading new proteins into and investigated expression of the HnCB in E. coli. the PDU has been isolated; however, a targeting sequence has We observed the formation of CBs in E. coli expressing the H. not yet been elucidated for CB proteins (20, 24). Thus, although neapolitanus operon. Cells containing pHnCB were induced with the present work indicates that BMCs are capable of acting as IPTG and analyzed using ultrathin sectioning and electron mi- modular protein assemblies, more characterization is needed croscopy (EM). As expected, the presence and morphology of CBs was highly dependent on IPTG concentration. In the ab- before BMCs are ready for use in bioengineered systems. CELL BIOLOGY Ultimately, a synthetic organelle will require both simplicity, sence of IPTG, cells generally had between zero and two icosa- to facilitate the engineering process, and extensibility, to enable hedral structures per cell (Fig. 2A). This was presumably from a variety of functions. In the context of the BMC, these char- leaky expression of the pNS3 promoter, given that WT E. coli acteristics set molecular constraints, which must be clarified through genetic and protein engineering. These constraints in- clude a description of the genes necessary and sufficient for A B functional expression in an arbitrary host, an understanding of shell selectivity to allow tuning of permeability to the
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