Engineered bacterial microcompartments: apps for programming metabolism

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Appendix 1: Model Details Appendix 2 Thermodynamics of assembly around a fluid cargo References Decision letter Author response Article and author information Metrics Abstract We computationally study how an icosahedral shell assembles around hundreds of molecules. Such a process occurs during the formation of the carboxysome, a bacterial microcompartment that assembles around many copies of the enzymes ribulose 1,5-bisphosphate carboxylase/ oxygenase and carbonic anhydrase to facilitate carbon fixation in cyanobacteria. Our simulations identify two classes of assembly pathways leading to encapsulation of many-molecule cargoes. In one, shell assembly proceeds concomitantly with cargo condensation. In the other, the cargo first forms a dense globule; then, shell proteins assemble around and bud from the condensed cargo complex. Although the model is simplified, the simulations predict intermediates and closure mechanisms not accessible in experiments, and show how assembly can be tuned between these two pathways by modulating protein interactions. In addition to elucidating assembly pathways and critical control parameters for microcompartment assembly, our results may guide the reengineering of viruses as nanoreactors that self-assemble around their reactants. https://doi.org/10.7554/eLife.14078.001 eLife digest Bacterial microcompartments are protein shells that are found inside bacteria and enclose enzymes and other chemicals required for certain biological reactions. For example, the carboxysome is a type of microcompartment that enables the bacteria to convert the products of photosynthesis into sugars. During the formation of a microcompartment, the outer protein shell assembles around hundreds of enzymes and chemicals. This formation process is tightly controlled and involves multiple interactions between the shell proteins and the cargo – the enzymes and other reaction ingredients – they will enclose. Understanding how to control which enzymes are encapsulated within microcompartments could help researchers to re-engineer the microcompartments so that they contain drugs or other useful products. Recent studies have used microscopy to visualize how microcompartments are assembled. However, most of the intermediate structures that form during assembly are too small and short-lived to be seen. It has therefore not been possible to explore in detail how shell proteins collect the necessary cargo and then assemble into an ordered shell with the cargo on the inside. Experiments alone are probably not enough to understand the process, especially since microcompartment assembly can currently only be studied within live cells or cellular extract. Within these complex environments it is difficult to determine the effect of any individual factor on the overall assembly process. Perlmutter, Mohajerani and Hagan have now taken a different approach by developing computational and theoretical models to explore how microcompartments assemble. Computer simulations showed that microcompartments could assemble by two pathways. In one pathway, the protein shell and cargo coalesce at the same time. In the other pathway, the cargo molecules first assemble into a large disordered complex, with the shell proteins attached on the outside. The shell proteins then assemble, carving out a piece of the cargo complex. The simulations showed that many factors affect how the shell assembles, such as the strengths of the interactions between the shell proteins and the cargo. They also identified a factor that controls how much cargo ends up inside the assembled shell. Perlmutter, Mohajerani and Hagan found that, in addition to revealing how microcompartments may assemble within their natural setting, the simulations provided guidance on how to re-engineer microcompartments to assemble around other components. This would enable researchers to create customizable compartments that self-assemble within bacteria or other host organisms, for example to carry out carbon fixation or make biofuels. A future challenge will be to investigate other aspects of microcompartment assembly, such as the factors that control the size of these compartments. https://doi.org/10.7554/eLife.14078.002 Introduction Encapsulation is a hallmark of biology. A cell must co-localize high concentrations of enzymes and reactants to perform the reactions that sustain life, and it must safely store genetic material to ensure long-term viability. While lipid-based organelles primarily fulfill these functions in eukaryotes, self-assembling protein shells take the lead in simpler organisms. For example, viruses surround their genomes with a protein capsid, while bacteria use large icosahedral shells known as bacterial microcompartments (BMCs) to sequester the enzymes and reactions responsible for particular metabolic pathways (Kerfeld et al., 2010; Axen et al., 2014; Shively et al., 1998; Bobik et al., 1999; Erbilgin et al., 2014; Petit et al., 2013; Price and Badger, 1991; Shively et al., 1973; Shively et al., 1973; Kerfeld and Erbilgin, 2015). Within diverse bacteria, BMC functions have been linked to bacterial growth, carbon fixation, symbiosis, or pathogenesis (Kerfeld and Erbilgin, 2015). Other protein-based compartments are found in bacteria and archea (e.g. encapsulins (Sutter et al., 2008) and gas vesicles (Pfeifer, 2012; Sutter et al., 2008)) and even eukaryotes (e.g. vault particles (Kickhoefer et al., 1998)), while some viruses may assemble around lipidic globules (Lindenbach and Rice, 2013; Faustino et al., 2014). Thus, understanding the factors that control microcompartment assembly and encapsulation is a central question in modern cell biology. From the perspectives of synthetic biology and nanoscience, there is great interest in reengineering BMCs or viruses as nanoreactors that spontaneously encapsulate enzymes and reagents in vitro (e.g. Luque et al., 2014; Douglas and Young, 1998; Rurup et al., 2014; Patterson et al., 2014; Patterson et al., 2012; Zhu et al., 2014; Rhee et al., 2011; Rurup et al., 2014; Wörsdörfer et al., 2012; Comas-Garcia et al., 2014), or as customizable organelles that assemble around a programmable set of core enzymes in vivo, introducing capabilities such as carbon fixation or biofuel production into bacteria or other organisms (e.g. Kerfeld and Erbilgin, 2015; Bonacci et al., 2012; Parsons et al., 2010; Choudhary et al., 2012; Lassila et al., 2014). However, the principles controlling such co-assembly processes have yet to be established, and it is not clear how to design systems to maximize encapsulation. In this article we take a step toward this goal, by developing theoretical and computational models that describe the dynamical encapsulation of hundreds of cargo molecules by self-assembling icosahedral shells. Although our models are general, we are motivated by recent experiments on a type of BMC known as the carboxysome (Kerfeld et al., 2010; Schmid et al., 2006; Iancu et al., 2007; Tanaka et al., 2008). Carboxysomes are large (40–400 nm), roughly icosahedral shells that encapsulate a dense complex of the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and other proteins to facilitate the Calvin-Bensen-Bassham cycle in autotrophic bacteria (Price and Badger, 1991; Shively et al., 1973; Shively et al., 1973; Iancu et al., 2007; 2010; Kerfeld et al., 2010; Tanaka et al., 2008). Recently, striking microscopy experiments visualized β-carboxysome shells assembling on and budding from procarboxysomes (the condensed complex of RuBisCO and other proteins found in the interior of carboxysomes) (Cameron et al., 2013; Chen et al., 2013). Genomic analysis suggests that many BMCs with diverse functions assemble via similar pathways (Cameron et al., 2013; Kerfeld and Erbilgin, 2015). However, the mechanisms of budding and pinch-off to close the shell remain incompletely understood because of the small size and transient nature of assembly intermediates. Moreover, experiments suggest that α-carboxysomes (another form of carboxysome) assemble by a different mechanism, in which shell assembly encapsulates an initially diffuse pool of RuBisCO (Iancu et al., 2010; Cai et al., 2015). The factors determining which of these assembly pathways occurs are unknown. BMC assembly is driven by a complex interplay of interactions among the proteins forming the external shell and the interior cargo. It is difficult, with experiments alone, to parse these interactions for those mechanisms and factors that critically influence assembly pathways, especially due to the lack of an in vitro assembly system. Models which can correlate individual factors to their effect on assembly are therefore an important complement to experiments. Previous experimental and theoretical studies of encapsulation by icosahedral shells, e.g. the assembly of viral capsids around their nucleic acid genomes (e.g. Hu and Shklovskii, 2007; Kivenson and Hagan, 2010; Elrad and Hagan, 2010; Perlmutter et al., 2013; 2014; Mahalik and Muthukumar, 2012; Zhang et al., 2013; Zhang and Linse, 2013; Hagan, 2008; Devkota et al., 2009; Dixit et al., 2006; Borodavka et al., 2012; Dykeman et al., 2013; 2014; Zlotnick et al., 2013; Johnson et al., 2004; Patel et al., 2015; Cadena-Nava et al., 2012; Comas-Garcia et al., 2012; 2014; Garmann et al., 2014a; 2014b; Malyutin and Dragnea, 2013), have demonstrated that the structure of the cargo can strongly influence assembly pathways and products. However, BMCs assemble around a cargo which is topologically different from a nucleic acid — a fluid complex comprising many, noncovalently linked molecules. We demonstrate here that changing the cargo topology leads to new assembly pathways and different critical control parameters. We present phase diagrams and analysis of dynamical simulation trajectories showing how the thermodynamics, assembly pathways, and emergent structures depend on the interactions among shell proteins and cargo molecules. Within distinct parameter ranges, we observe two classes of assembly pathways, which resemble those suggested for respectively α- or β-carboxysomes. We find that tunability of cargo loading is a key functional difference between the two classes of pathways. Shells assembled around a diffuse cargo can be varied from empty (containing almost no cargo) to completely full, whereas assembly around a condensed, procarboxysome-like complex invariably produces full shells. While we find that the encapsulated cargo becomes ordered due to confinement, complete crystalline order in the globule before encapsulation inhibits budding. We discuss these results in the context of recent observations on carboxysome assembly, and their implications for engineering BMCs, viruses or drug delivery vehicles that assemble around a fluid cargo (e.g. Refs. [Kerfeld and Erbilgin, 2015; Parsons et al., 2010; Choudhary et al., 2012; Lassila et al., 2014; Luque et al., 2014; Douglas and Young, 1998; Rurup et al., 2014; Patterson et al., 2014; Patterson et al., 2012; Zhu et al., 2014; Rhee et al., 2011; Rurup et al., 2014; Wörsdörfer et al., 2012]). Results Our model system is motivated by icosahedral viral capsids and BMCs (Tanaka et al., 2008; Kerfeld et al., 2010). Since icosahedral symmetry can accommodate at most 60 identical subunits, formation of large icosahedral structures requires subunits to assemble into different local environments. The subunits can be grouped into pentamers and hexamers, with 12 pentamers at the icosahedron vertices and the remaining subunits in hexamers. Viruses typically assemble from small oligomers of the capsid protein, which we refer to as the basic assembly unit (Hagan, 2014). Recent AFM experiments demonstrated that hexamers are the basic assembly unit during the assembly of BMC shell facets (Sutter et al., 2016), and the carboxysome major shell proteins crystallize as pentamers and hexamers (Tanaka et al., 2008). Motivated by these observations, our model considers two basic assembly units, one a pentamer and the other a hexamer, with interactions designed so that the lowest energy structure corresponds to a truncated icosahedron with 12 pentamers and 20 hexamers (Figure 1). While BMCs generally have more hexamers, our model is intended to explore the general principles of assembly around a fluid cargo rather than model a specific system. Further details of the model and a thermodynamic analysis are given in section 3 and the appendices. Figure 1 Download asset Open asset Description of the model. (A) Each shell subunit contains 'Attractors' (green circles) on the perimeter, a 'Top' (tan circle, 'T') in the center above the plane, and a 'Bottom' (purple circle, 'B' below the plane). (B) Interactions between complementary Attractors drive subunit dimerization, with the Top-Top repulsions (tan arrow) tuned to favor the subunit-subunit angle in a complete shell. Complementary pairs of attractors are indicated by green arrows in (A) for the pentamer-hexamer interface and in (B) for the hexamer-hexamer interface. (C) Bottom psuedoatoms bind cargo molecules (terra cotta circles, 'C'), while excluder atoms (blue and brown pseudoatoms in (D)) placed in the plane of the pentagon experience excluded volume interactions with the cargo. (D) The positions of excluder atoms in the lowest energy shell geometry, a truncated icosahedron with 12 pentamers (blue) and 20 hexamers (brown). https://doi.org/10.7554/eLife.14078.003 To understand how assembly around multiple cargo molecules depends on the relative strengths of interactions between components, we performed dynamical simulations as a function of the parameters controlling shell subunit-subunit (εSS), shell subunit-cargo (εSC), and cargo-cargo (εCC) interaction strengths. All energy values are given in units of the thermal energy, kB⁢T. We focus on parameters for which shell subunit-subunit interactions are too weak to drive assembly in the absence of cargo (εSS≤4.5). Except where mentioned otherwise, the cargo diameter is set equal to the circumradius of a shell subunit. For the simulated density of cargo particles, the phase behavior (in the absence of shells) corresponds to a vapor at εCC=1.3, liquid-vapor phase coexistence for εCC∈[1.6,2.0] (the phase coexistence boundary is slightly below εCC=1.6), and a solid phase at εCC=3.0. We find that tuning εCC through phase coexistence dramatically alters the typical assembly process. Strong cargo interactions (εCC≥1.6) drive formation of a globule followed by assembly and budding of a shell, such as observed for β-carboxysomes (Figure 2A, Simulation Video 1), while under weak interactions (εCC<1.6) shell assembly usually proceeds in concert with cargo encapsulation (Figure 2B, Simulation Video 2), as suggested for assembly of α-carboxysomes. We now elaborate on these classes of assembly pathways, and how the resulting assembly products depend on parameter values. Figure 2 with 2 supplements see all Download asset Open asset Snapshots illustrating typical assembly trajectories. (A) Multi-step assembly involving an amorphous globule of cargo and shell subunits. (B) Single-step assembly, in which shell assembly drives local cargo condensation. and (C) when shell-cargo interactions are too weak to condense the cargo. The values of the cargo-cargo (εCC), shell subunit-cargo (εSC), and subunit-subunit (εSS) interaction strengths are listed above each panel (all energies are in units of the thermal energy kB⁢T), and the time (in units of 106 timesteps) is noted below each image. The color scheme here and throughout the manuscript is: Red=Cargo, Blue=Pentagon Excluder, Brown=Hexagon Excluder. Attractor and Bottom pseudoatoms are omitted to aid visibility. Videos of assembly trajectories are included below. https://doi.org/10.7554/eLife.14078.004 Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Animation of a typical simulation showing assembly around a cargo globule. Parameters are εCC=1.6, εSC=7, and εSS=2.5. https://doi.org/10.7554/eLife.14078.007 Video 2 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Animation of a typical simulation showing simultaneous assembly and cargo condensation. Parameters are εCC=1.3, εSC=9, and εSS=3.5. https://doi.org/10.7554/eLife.14078.008 Assembly and budding from a cargo globule We begin by discussing assembly behavior when the cargo-cargo interactions are strong enough to drive equilibrium phase coexistence (εCC≥1.6). Near the phase boundary (εCC=1.6) a system of pure cargo particles is metastable on the timescales we simulate. However, for εSC>4, adding shell subunits drives nucleation of a cargo globule with shell subunits adsorbed on the surface. The subsequent fate of the globule depends on parameter values; typical simulation end-states are shown as a function of parameter values in Figure 3. For moderate interaction strengths (2.5≤εSS≤3.5) the globule grows to a large size, typically containing at least twice the cargo molecules that can be packaged within a complete shell. Adsorbed shell subunits then reversibly associate to form ordered clusters. Once a cluster acquires enough inter-subunit interactions to be a stable nucleus, it grows by coagulation of additional subunits or other adsorbed clusters. For the parameter set corresponding to Figure 2A, nucleation is fast in comparison to cluster growth, and thus two nuclei grow simultaneously. The last three images show the system immediately preceding and following detachment of the lower shell. Missing only one of its 32 subunits, the shell is connected to the remainder of the droplet only by a narrow neck of cargo. Insertion of the final subunit breaks the neck and completes shell detachment. The complete shell contains 120–130 cargo particles, which is slighty above random close packing (≈120 particles) but below fcc density (≈150 particles, see appendix 1.2). Figure 3 with 3 supplements see all Download asset Open asset Results of assembly around a cargo globule. (A) The most frequently observed assembly outcome is overlaid on a color map of the theoretical free energy density difference Δ⁢fassem (Equation (3)) between assembled shells and the unassembled globule. Results are plotted against the shell-cargo adsorption strength εSC and the shell-shell interaction strength εSS for indicated values of the cargo-cargo interaction strength εCC. (B) Representative snapshots of the predominant assembly outcomes shown in (A). https://doi.org/10.7554/eLife.14078.009 Figure 3—source data 1 List of all simulation outcomes for Figures 3A,5A. https://doi.org/10.7554/eLife.14078.010 Download elife-14078-fig3-data1-v3.zip Figure 3—source data 2 Criteria used to categorize assembly outcomes. The sizes of each cargo globule and shell assemblage, and associations between shell assemblages and cargo globules, were determined by clustering. The outcome was then categorized according to the criteria listed in this table. https://doi.org/10.7554/eLife.14078.011 Download elife-14078-fig3-data2-v3.zip Increasing the shell-shell interaction strength drives faster shell assembly and closure, thus limiting the size of the globule before budding. For the largest interaction strength we simulated (εSS=4.5) the globule typically does not exceed the size of a single shell, and multiple globules nucleate within the simulation box (Figure 2—figure supplement 1). This observation could place an upper bound on shell-shell interaction strengths, since multiple nucleation events were rare in the carboxysome assembly experiments (Cameron et al., 2013) (however, we discuss potential complicating factors within the cellular environment below). To quantify the relationship between assembly mechanism and parameter values, we calculate an assembly order parameter, defined as the maximum number of unassembled subunits adsorbed onto a globule during an assembly trajectory. The order parameter is shown as a function of the interaction strengths in Figure 4. For εCC≥1.6 and εSS≤3 we observe large values of the order parameter (e.g. >32, the red and yellow regions in Figure 4), which formation of a large amorphous globule with the to carboxysome shell assembly (Cameron et al., 2013). Figure with 1 supplement see all Download asset Open asset of assembly on shell-cargo and shell-shell interaction The assembly order parameter, defined as the maximum number of unassembled shell subunits adsorbed on a globule at any during a is shown as a function of εSC and εSS for indicated values of the cargo-cargo interaction εCC. of adsorbed unassembled subunits the two step assembly mechanism (Figure whereas values to simultaneous assembly and cargo (Figure Other assembly products of the parameter ranges, we observe classes of outcomes. weak shell-shell interactions to drive For and the cargo vapor phase is and the system no cargo on simulated timescales discuss below). cargo-cargo or shell-cargo interactions in unassembled where a cargo globule forms but the shell subunits on its to εSS we observe assembly on the leading to complete shells or two classes of In the first one or more shells almost but to from the droplet within simulated for when the subunit-cargo interaction does not a strong enough for the last to the cargo and close the shell. strong interactions drive the other of in which an of assembled shells the system of free subunits before any shells are In this it is also to observe in which within shells. the cargo-cargo interaction multiple narrow the parameter that leads to complete assembly and detachment. cargo globules nucleate at multiple within the simulation the of the the of εSC required for cargo resulting in shells a parameter We also observe a we refer to as in which shell assembly to the globule thus does not even to the attached The is especially for when the cargo even in the absence of shell encapsulation. For and of the number of nucleation we typically observe into a large globule. shell assembly and cargo For the cargo forms an equilibrium vapor phase in the absence of shell subunits. However, above values of εSS and the diffuse cargo molecules drive nucleation of shell The subsequent assembly depends on the shell-cargo interaction For εSC (Figure assembly only a cargo leading to but empty shells. For εSC (Figure 2B, and Simulation Video 2), the shell-cargo interactions drive local of cargo molecules. assembly and cargo then in the mechanism for assembly of α-carboxysomes (Iancu et al., 2010). Thus, tuning the shell-cargo interaction dramatically cargo with a from empty to shells around This the equilibrium (Figure by a complete shell to cargo molecules. This effect is to the of vapor below its inside of In assembly around a globule only full shells. Figure with 2 supplements see all Download asset Open asset Results of assembly around a cargo with weak interactions (A) The most frequently observed assembly outcome as a function of εSS and The of outcomes for is shown in Figure supplement and a data containing the outcome for each at each parameter set is included (Figure 3—source data 1). (B) Representative snapshots for the outcomes shown in (A). The complete shell outcomes are shown with the and to enable the encapsulated cargo. (C) The number of cargo molecules encapsulated by shells assembled in simulations is to the results of equilibrium simulations The results are all complete shells any assembled at each of the simulations were performed for simulations with and assembled shells at and were up to Assembly of full shells pathway, Figure or Figure is typically two of faster than assembly of empty shells (Figure This the key that the cargo in shell during all of molecules initially shell nucleation by interactions among clusters. the of a condensed globule a large for adsorption of additional subunits, the of subunits to the capsid, thus its The condensed cargo of the last subunits, which are by as noted for simulations of empty capsids et al., Figure how the products of assembly around cargo with weak interactions depends on parameters. While moderate parameter values lead to complete assembly, weak εSC and εSS of Figure shell leading to the In the of large εSC but weak εSS the shell-cargo interaction small disordered globules cargo particles, lower of Figure while under strong subunit and weak cargo interactions shells nucleate but cannot condense the leading to the complete but assembly for assembly around a strong interactions lead to and shells. However, the predominant of is now shell the cargo is below its the condensed globule leads to a on the shell subunits, which can the shell and thus closure of a shell. model The free energy model the parameter values required for shell assembly with no parameters map in Figure Since it is an equilibrium model and only considers the free energy difference between complete and unassembled it cannot between parameter values that lead to complete assembly or at the but simulation However, the thermodynamic does suggest that the simulations resulting in shells would on a We not show Δ⁢fassem in Figure because the globule is than assembled shells for εCC=1.3, but the of shells in our simulations roughly the of the equilibrium (Figure supplement 1). of other parameters or To investigate the results above depend on within our we performed of additional we performed simulations in which the between cargo diameter in shell subunit size was shown in Figure supplement assembly is most for our cargo diameter which the model was but assembly occurs for cargo varied a factor of we performed assembly simulations with cargo molecules with a

We have discovered a new cluster of genes that is found exclusively in the Actinobacteria phylum. This locus includes genes for the 2-aminophenol meta-cleavage pathway and the shell proteins of a bacterial microcompartment (BMC) and has been named aromatics (ARO) for its putative role in the breakdown of aromatic compounds. In this study, we provide details about the distribution and composition of the ARO BMC locus and conduct phylogenetic, structural, and functional analyses of the first two enzymes in the catabolic pathway: a unique 2-aminophenol dioxygenase, which is exclusively found alongside BMC shell genes in Actinobacteria, and a semialdehyde dehydrogenase, which works downstream of the dioxygenase. Genomic analysis reveals variations in the complexity of the ARO loci across different orders. Some loci are simple, containing shell proteins and enzymes for the initial steps of the catabolic pathway, while others are extensive, encompassing all the necessary genes for the complete breakdown of 2-aminophenol into pyruvate and acetyl-CoA. Furthermore, our analysis uncovers two subtypes of ARO BMC that likely degrade either 2-aminophenol or catechol, depending on the presence of a pathway-specific gene within the ARO locus. The precise precursor of 2-aminophenol, which serves as the initial substrate and/or inducer for the ARO pathway, remains unknown, as our model organism Micromonospora rosaria cannot utilize 2-aminophenol as its sole energy source. However, using enzymatic assays, we demonstrate the dioxygenase's ability to cleave both 2-aminophenol and catechol in vitro, in collaboration with the aldehyde dehydrogenase, to facilitate the rapid conversion of these unstable and toxic intermediates. IMPORTANCE Bacterial microcompartments (BMCs) are proteinaceous organelles that are widespread among bacteria and provide a competitive advantage in specific environmental niches. Studies have shown that the genetic information necessary to form functional BMCs is encoded in loci that contain genes encoding shell proteins and the enzymatic core. This allows the bioinformatic discovery of BMCs with novel functions and expands our understanding of the metabolic diversity of BMCs. ARO loci, found only in Actinobacteria, contain genes encoding for phylogenetically remote shell proteins and homologs of the meta-cleavage degradation pathway enzymes that were shown to convert central aromatic intermediates into pyruvate and acetyl-CoA in gamma Proteobacteria. By analyzing the gene composition of ARO BMC loci and characterizing two core enzymes phylogenetically, structurally, and functionally, we provide an initial functional characterization of the ARO BMC, the most unusual BMC identified to date, distinctive among the repertoire of studied BMCs.

<p indent="0mm">Traditionally, bacteria were considered primitive organisms that lack intracellular organization. However, this knowledge was overturned by studies that showed bacteria have a high level of organization including research on bacterial microcompartments. Microcompartments are self-assembling organelles composed of a protein shell, and internal enzymes encapsulated by the shell. The overall morphology of bacterial microcompartments is similar to viruses. They are polyhedral in shape and about 100 to <sc>200 nm</sc> in size. The common feature of bacterial microcompartments is that they use a protein shell as a diffusion barrier to help channel toxic or volatile pathway intermediates to the next pathway enzyme. Gene clusters of bacterial microcompartments are widespread, and their specific physiological roles are diverse. 68 types were found in the genome of bacteria across 45 phyla. However, the functions of many types of bacterial microcompartments remain unclear, and only several types have been characterized experimentally. One of the best-studied microcompartments is used for 1,2-propanediol catabolism in <italic>Salmonella typhimurium</italic>. Genes encoding enzymes that are responsible for 1,2-propanediol catabolism and shell proteins form a <italic>pdu</italic> operon. Enzymes and shell proteins of <italic>pdu</italic> operon form a Propanediol utilization (Pdu) microcompartment. 1,2-Propanediol is transported into the lumen of the microcompartment, where it is converted to propionaldehyde by propanediol dehydratase. Propionaldehyde is a toxic chemical and high levels of propionaldehyde inhibit the activity of the cell. The shell of the Pdu microcompartment prevents the propionaldehyde transported into the cytoplasm and reduces its toxicity to the cell. Propionaldehyde is converted to propanol, a less toxic chemical, and finally transported into the cytoplasm. The disruption of the Pdu microcompartment increases propionaldehyde levels in the culture broth, and slows the growth of cells. The carboxysome microcompartment is another type of highly investigated microcompartment. It is used to enhance autotrophic CO₂ fixation and is found in nearly all cyanobacteria and some chemoautotrophs. Bicarbonate crosses the protein shell of the carboxysome and enters the lumen, where bicarbonate is converted to CO₂. The protein shell of the carboxysome helps to maintain a high local concentration of CO₂ by inhibiting its outward diffusion. If the shell of the carboxysome is disrupted, cells are unable to grow autotrophically at atmospheric CO₂. levels. Besides these two types of microcompartments, the ethanolamine utilization (Eut) microcompartment, glycyl-radical propanediol (Grp) microcompartment, choline utilization (Cut) microcompartment, <italic>Planctomycete</italic> and <italic>Verrucomicrobia</italic> (PV) microcompartment, taurine metabolism microcompartment, and ethanol utilization (Eut) microcompartment are microcompartments whose specific function is known. Bacterial microcompartments are interesting structures, and they have potential applications in biomedicine and biotechnology. Some heterologous pathways have been encapsulated into microcompartments and shown potential biotechnology advantages. Research on bacterial microcompartments is in its infancy, but these tiny organelles have big potential.

Bacterial microcompartments (BMCs) are protein-based organelles found in diverse bacteria that encapsulate a sequence of enzymes to accelerate specific metabolic pathways and limit toxicity by confining intermediate products. To facilitate catalysis in BMCs, reactants and products must transit across the protein shells, which are made up of multimeric protein tiles. Quantifying the permeability for transport of small molecules across the shell is a key engineering consideration to design novel catalytic microcompartments. We examine the permeability of reactants and products for two reaction pathways, the degradation of 2-aminophenol by AmnAB and the reduction of nitrite to ammonium by NrfA, through a computational lens to quantify permeability for these metabolites at the nanoscale. From Hamiltonian replica exchange umbrella sampling simulations, we determine that the energetic barriers to permeation for the relevant metabolites along both pathways are small, with permeability coefficients in the range of 0.1-4 cm s-1. The high permeabilities calculated for each metabolite through the inhomogeneous solubility diffusion model are corroborated by enzyme activity measurements in vitro that point to enzymatic activity within the shell system. We expand on these findings by quantifying the scale of transport and catalysis that can be supported by in vitro BMC systems. The results highlight the importance of substrate permeability across BMCs within a biological context, and represent steps toward generating synthetic shells or bioengineering novel nanoreactors.

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.

Biologists have long thought that the internal workings of prokaryotes—the smallest and simplest organisms, including bacteria—are well understood, and have accordingly considered eukaryotic organisms and their cells to be more fascinating objects for study. During the past decade, however, the focus of some research has begun to turn back to prokaryotes—particularly because genomic analyses have shown the enormous flexibility and adaptability of these seemingly simple organisms. Moreover, recent structural research has shown that the cell components of prokaryotes might also be more complex than previously thought. It has long been held that prokaryotic cells lack the internal structure and organization of eukaryotic cells, which have various membrane‐bound organelles, such as mitochondria, chloroplasts and the nucleus. Indeed, prokaryotes are assumed to be the predecessors of some eukaryotic organelles—permanently captured after a period of endosymbiosis. > …there is no rule that says an organelle must be enclosed by a membrane The view that bacterial cells are structurally simpler than those of ‘higher’ organisms is reflected in many text books and web sites with comments such as: “the ribosome is the only prokaryotic organelle”—if, indeed, this highly complex and sophisticated protein‐synthesizing machinery is an organelle in its own right. These types of statement are correct in the sense that prokaryotes contain no membrane‐bound organelles; but there is no rule that says an organelle must be enclosed by a membrane. Many eukaryotic ribosomes are freely suspended in the cytoplasm where they manufacture cytosolic proteins. Furthermore, the word ‘organelle’ itself is being replaced by the terms ‘functional unit’ or ‘compartment’, which are increasingly used to describe coherent structures within a cell that perform clearly defined sets of tasks—such as mitochondria, which create energy by aerobic respiration, or chloroplasts, which do so by photosynthesis. By using this definition, a series of recent discoveries show that prokaryotes also …

Bacterial microcompartments are large supramolecular structures comprising an outer proteinaceous shell that encapsulates various enzymes in order to optimize metabolic processes. The outer shells of bacterial microcompartments are made of several thousand protein subunits, generally forming hexameric building blocks based on the canonical bacterial microcompartment (BMC) domain. Among the diverse metabolic types of bacterial microcompartments, the structures of those that use glycyl radical enzymes to metabolize choline have not been adequately characterized. Here, six structures of hexameric shell proteins from type I and type II choline-utilization microcompartments are reported. Sequence and structure analysis reveals electrostatic surface properties that are shared between the four types of shell proteins described here.

Bacterial microcompartments (BMCs) are proteinaceous organelles that carry out specific metabolic reactions. Using domain representations of the BMC shell proteins, we identified BMCs in genomes of 358 bacterial species including human gut microbes, bioremediation agents, cellulosic ethanol producers, and pathogens. Multiple BMCs of different metabolic types are present in 40% of the BMC-containing genomes. BMC genes frequently clustered at a single locus that includes enzymes related to the compartment’s metabolic function. The distribution of BMC-containin species was mapped onto a phylogenetic tree constructed from 16S rRNA sequences. The presence of BMCs was sporadically distributed across the phylogenetic tree. All bacterial families that contained species with BMCs also had species without them. Even within a species, BMC number varied, indicative of frequent horizontal transfer and gene loss. Similarly, phylogenetic trees constructed from individual BMC genes indicated that horizontal gene transfer of the BMC loci is a common occurrence.

Bacterial microcompartments (MCPs) are subcellular organelles that are composed of a protein shell and encapsulated metabolic enzymes. It has been suggested that MCPs can be engineered to encapsulate protein cargo for use as in vivo nanobioreactors or carriers for drug delivery. Understanding the stability of the MCP shell is critical for such applications. Here, we investigate the integrity of the propanediol utilization (Pdu) MCP shell of Salmonella enterica over time, in buffers with various pH, and at elevated temperatures. The results show that MCPs are remarkably stable. When stored at 4°C or at room temperature, Pdu MCPs retain their structure for several days, both in vivo and in vitro. Furthermore, Pdu MCPs can tolerate temperatures up to 60°C without apparent structural degradation. MCPs are, however, sensitive to pH and require conditions between pH 6 and pH 10. In nonoptimal conditions, MCPs form aggregates. However, within the aggregated protein mass, MCPs often retain their polyhedral outlines. These results show that MCPs are highly robust, making them suitable for a wide range of applications.

This article describes dynamical simulations of the assembly of an icosahedral protein shell around a bicomponent fluid cargo. Our simulations are motivated by bacterial microcompartments, which are protein shells found in bacteria that assemble around a complex of enzymes and other components involved in certain metabolic processes. The simulations demonstrate that the relative interaction strengths among the different cargo species play a key role in determining the amount of each species that is encapsulated, their spatial organization, and the nature of the shell assembly pathways. However, the shell protein–shell protein and shell protein–cargo component interactions that help drive assembly and encapsulation also influence cargo composition within certain parameter regimes. These behaviors are governed by a combination of thermodynamic and kinetic effects. In addition to elucidating how natural microcompartments encapsulate multiple components involved within reaction cascades, these results have implications for efforts in synthetic biology to colocalize alternative sets of molecules within microcompartments to accelerate specific reactions. More broadly, the results suggest that coupling between self-assembly and multicomponent liquid–liquid phase separation may play a role in the organization of the cellular cytoplasm.

This article describes dynamical simulations of the assembly of an icosahedral protein shell around a bicomponent fluid cargo. Our simulations are motivated by bacterial microcompartments, which are protein shells found in bacteria that assemble around a complex of enzymes and other components involved in certain metabolic processes. The simulations demonstrate that the relative interaction strengths among the different cargo species play a key role in determining the amount of each species that is encapsulated, their spatial organization, and the nature of the shell assembly pathways. However, the shell protein–shell protein and shell protein–cargo component interactions that help drive assembly and encapsulation also influence cargo composition within certain parameter regimes. These behaviors are governed by a combination of thermodynamic and kinetic effects. In addition to elucidating how natural microcompartments encapsulate multiple components involved within reaction cascades, these results have implications for efforts in synthetic biology to colocalize alternative sets of molecules within microcompartments to accelerate specific reactions. More broadly, the results suggest that coupling between self-assembly and multicomponent liquid–liquid phase separation may play a role in the organization of the cellular cytoplasm.

The carboxysome is a bacterial microcompartment (BMC) which plays a central role in the cyanobacterial CO2-concentrating mechanism. These proteinaceous structures consist of an outer protein shell that partitions Rubisco and carbonic anhydrase from the rest of the cytosol, thereby providing a favorable microenvironment that enhances carbon fixation. The modular nature of carboxysomal architectures makes them attractive for a variety of biotechnological applications such as carbon capture and utilization. In silico approaches, such as molecular dynamics (MD) simulations, can support future carboxysome redesign efforts by providing new spatio-temporal insights on their structure and function beyond in vivo experimental limitations. However, specific computational studies on carboxysomes are limited. Fortunately, all BMC (including the carboxysome) are highly structurally conserved which allows for practical inferences to be made between classes. Here, we review simulations on BMC architectures which shed light on (1) permeation events through the shell and (2) assembly pathways. These models predict the biophysical properties surrounding the central pore in BMC-H shell subunits, which in turn dictate the efficiency of substrate diffusion. Meanwhile, simulations on BMC assembly demonstrate that assembly pathway is largely dictated kinetically by cargo interactions while final morphology is dependent on shell factors. Overall, these findings are contextualized within the wider experimental BMC literature and framed within the opportunities for carboxysome redesign for biomanufacturing and enhanced carbon fixation.

Biomimetic engineering opens unprecedented possibilities of combining biomolecules (i.e. proteins, DNA, polysaccharides) with synthetic materials (i.e. synthetic polymers). This combination results in unique hybrid systems with functionalities that mimic processes in living organisms. While the translational value of functional biomimetically engineered structures is of exceptional importance in fields such as technology, engineering, chemistry, biology and medicine, due to the properties the structures inherit from both the synthetic and bio-materials, the understanding of how biomimetically engineered systems self-assemble and function is equally important, as it gives insight in how non-living systems progressed to living organisms. Some of the most prominent examples of functional biomimics include polymersome based catalytic nanocompartments, multicompartment systems that mimic cellular organization and artificial organelles. In this thesis, the focus lies on understanding and applying the fundamental principles of biomimetic engineering by equipping colloidal nanoarchitectures (soft polymer nanoparticles and hollow sphere polymersomes) with functional biomolecules (transmembrane proteins and enzymes).&#13;\nFirst, the most important questions are addressed – why do polymer nanoarchitectures present ideal building blocks for creating novel biomimics, how do biomimics self-assemble in solution, which methods are most frequently used for their characterization, and where the applications of biomimics are in technology and medicine. Both colloidal and 2D supported/free standing polymeric nanoarchitectures structures are discussed in order to familiarize the reader with the wide range of nanoarchitectures that can be formed by polymers, however the focus primarily rests on biomimetic design of colloidal nanoarchitectures, as their colloidal nature favours them as therapeutic agents that can act on the cellular level.&#13;\nTo develop a pH responsive protein delivery agent, a biomimetic approach is applied in equipping self-assembled poly(ethylene glycol)-b-poly(methylcaprolactone)-b-poly(2- (N,Ndiethylamino)ethyl methacrylate) (PEG-b-PMCL-b-PDMAEMA) polymer nanoparticles with a therapeutic enzyme, acid sphingomyelinase. Due to the electrostatic interactions between the negatively charged enzyme and the positively charged PDMAEMA groups present in the nanoparticle corona, the biomimetically engineered nanoparticles display a distinct protein localization on their corona and a pH dependent release behavior of the attached protein. The application of the self-assembled system as a very efficient delivery agent for catalytically active biomolecules is demonstrated in human epithelial HeLa cells.&#13;\nNext, a more complex nanoscale biomimic - a pH triggered catalytic nanocompartment - is built by biomimetically engineering the nanoarchitecture of poly(2-methyl-2-oxazoline)- block-polydimethylsiloxane-block-poly(2-methyl-2-oxazoline) (PMOXA-b-PDMS-b- PMOXA) polymersomes. Aqueous cavities of polymersomes are loaded with horseradish peroxidase while a chemically modified Outer membrane protein F (OmpF) is reconstituted in polymersome membranes. The chemical modification of OmpF transforms the otherwise unspecific pore into a selective and pH responsive pore, through which molecules can only diffuse once the attached molecular cap blocking the pore is cleaved. Hence, once the modified OmpF is functionally reconstituted in polymersome membranes it allows the developed biomimic to present an on demand catalytic activity. As a first proof of concept of a pH responsive catalytic nanocompartment the system demonstrates that a spatial control of a reaction inside a nanocompartment can be achieved and supports the further development of complex reaction spaces that can act in an analogous manner to cellular compartments, where in situ reactions are modulated by a plethora of responsive proteins.&#13;\nFinally, biomimetically engineered polymersomes are designed for an in vitro and in vivo application as artificial organelles. In order to mimic processes taking place in lipid membranes of cellular organelles, polymersome membranes are equipped with a genetically and chemically modified OmpF. The structural modifications done at the rim of the OmpF pore, limit the OmpF permeability to small molecular weight molecules, but make it capable of responding to the presence of small signaling molecules. When the modified OmpF is reconstituted in membranes of enzyme loaded polymersomes it prevents the enzyme to access enzymatic substrates. However, the presence of of glutathione, which for example is found in abundant concentrations in the cytoplasm, readily cleaves the chemical modification of OmpF and opens the pore, thereby allowing the encapsulated enzyme to catalyze a reaction. The responsiveness of the self-assembled system to glutathione, abundantly present in the cytoplasm, makes the developed biomimic a suitable candidate for intracellular functionality as an artificial organelle. To demonstrate this, we not only show that the system is functional in the cellular microenvironment of human epithelial HeLa cells but also that it is robust enough to function in vivo in Zebrafish embryos.

Bacterial microcompartments (BMC) are complex macromolecular assemblies that participate in varied chemical processes in about one fourth of bacterial species. BMC-encapsulated enzymatic activities are segregated from other cell contents by means of semipermeable shells, justifying why BMC are viewed as prototype nano-reactors for biotechnological applications. Herein, we undertook a comparative study of bending propensities of BMC hexamers (BMC-H), the most abundant shell constituents. Published data show that some BMC-H, like β-carboxysomal CcmK, tend to assemble flat whereas other BMC-H often build curved objects. Inspection of available crystal structures presenting BMC-H in tiled arrangements permitted us to identify two major assembly modes with a striking connection with experimental trends. All-atom molecular dynamics (MD) supported that BMC-H bending is triggered robustly only from the arrangement adopted in crystals by BMC-H that experimentally form curved objects, leading to very similar arrangements to those found in structures of recomposed BMC shells. Simulations on triplets of planar-behaving hexamers, which were previously reconfigured to comply with such organization, confirmed that bending propensity is mostly defined by the precise lateral positioning of hexamers, rather than by BMC-H identity. Finally, an interfacial lysine was pinpointed as the most decisive residue in controlling PduA spontaneous curvature. Globally, results presented herein should contribute to improve our understanding of the variable mechanisms of biogenesis characterized for BMC, and of possible strategies to regulate BMC size and shape.

Deletion of major shell proteins of ethanolamine utilization microcompartment reduces intrinsic antibiotic resistance, biofilm, and intracellular survival of Salmonella Typhimurium.