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Bacterial microcompartments: biomaterials for synthetic biology-based compartmentalization strategies Ashley Chessher, Rainer Breitling, and Eriko Takano ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.5b00059 • Publication Date (Web): 18 May 2015 Downloaded from http://pubs.acs.org on May 25, 2015

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ACS Biomaterials Science & Engineering

Bacterial microcompartments: biomaterials for synthetic biology-based compartmentalization strategies Ashley Chessher, Rainer Breitling, Eriko Takano* Manchester Synthetic Biology Research Centre SYNBIOCHEM, Manchester Institute of Biotechnology, The Faculty of Life Sciences, The University of Manchester, 131 Princess Street, Manchester, M1 7DN, United Kingdom Email [email protected] Keywords: Bacterial microcompartments, BMC, Synthetic Biology,

ABSTRACT Synthetic biology (SynBio) presents a new paradigm for how metabolic pathways can be designed, assembled and integrated within a cell. A key aim of SynBio is the development of orthogonal tools that facilitate the expression of heterologous genes and circuits in a non-native host (chassis). Compartmentalization represents one orthogonalization strategy, in particular for metabolic pathways, preventing unwanted protein–protein interactions and competition for resources with native pathways, while sequestering toxic intermediates and providing an appropriate environment to support metabolic channeling. A variety of biomaterials have been investigated for their ability to form intracellular compartments. Particularly versatile examples are bacterial microcompartments (BMCs), protein-based shells that sequester a multitude of metabolic reactions in their native host. These compartments provide a natural template for de novo compartmentalization and offer unprecedented opportunities for bioengineering using SynBio. Here we review BMCs as modular building blocks for a general compartmentalization

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methodology. We describe their role, structure and properties and discuss the prospects of using SynBio to assemble and engineer microcompartments. Finally, this review explores the future applications of synthetic BMCs and highlights key areas for further research on these unique structures.

INTRODUCTION Synthetic biology (SynBio) describes recent progress in our ability to enhance biological systems via genetic modification, by combining new experimental methodology and advanced engineering concepts1-3. Supporting these advances is an array of computational tools for the rational design, modelling and evaluation of biological systems, in conjunction with large-scale fabrication techniques for the construction and assembly of complex genetic constructs. Based on these core principles, research in SynBio has progressed4 from the design and construction of simple genetic devices and circuits5, 6, to more sophisticated engineering feats such as the manipulation of Saccharomyces cerevisiae for the production of artemisinic acid, a precursor for the antimalarial artemisinin7. Despite these notable successes, a number of challenges remain, including in the construction of orthogonal parts and modules. To enable an engineering approach to biology, a prerequisite of molecular and genetic building blocks is that they function largely in a modular fashion, insulated from the cellular context and without detrimental effect or interference (crosstalk) from the endogenous pathways/systems of a non-native host cell. Only the availability of such orthogonal parts will enable robust and predictable behavior, facilitating the design of new functional pathways or processes8.

COMPARTMENTALIZATION AS AN INSULATION STRATEGY A promising general strategy for insulating orthogonal modules from the cellular context uses the concept of spatial control via compartmentalization9,

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. Compartmentalization involves

physically insulating heterologous components from the cellular milieu, thereby minimizing interactions with the native system. Utilizing compartments not only limits metabolic crosstalk


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with competing host cell pathways, but prevents the release of toxic intermediates and increases reaction efficiency11-13. Critically, the use of compartmentalization also potentially supports metabolic channeling through the encapsulation of enzymes in close proximity with substrates and intermediates14. Indeed, stochastic reaction–diffusion modelling has suggested that compartmentalization might increase catalytic activity substantially compared to non-compartmentalized reactions, up to sixfold for the specific pathway analyzed15. Experimental evidence based on the bi-enzyme complex of tryptophan synthase, supports the concept of metabolic channeling with increased reaction efficiency of 1–2 orders of magnitude16. In natural systems, the co-localization of enzymes in close proximity has evolved in the form of subcellular compartments, such as peroxisomes and glycosomes, but also in the form of metabolons; supramolecular assemblies comprising metabolic enzymes, complexed with structural elements to perform sequential substrate-channeling reactions17. To date, numerous examples of metabolons have been documented in eukaryotes, plants and bacteria18. There has been a growing interest in using SynBio to engineer nano-biomaterials for synthetic compartmentalization strategies19, 20. Current efforts to develop synthetic compartments include the use of colloidosomes21; polymeric nano-structures22, peptides23 and an aqueous two-phase system (ATPS)24. As with many scientific endeavours, inspiration has been taken from nature, where many forms of membrane-based compartmentalization exist. However, attempts to construct synthetic nano-compartments have focused on applications outside of biological systems21. To engineer in vivo synthetic microcompartments, recent research has focused on prokaryotes where metabolons encapsulated in protein-based structures known as bacterial microcompartments (BMCs) are surprisingly widespread25.

BACTERIAL MICROCOMPARTMENTS: FUNCTION AND STRUCTURE Bacterial microcompartments (BMCs) are proteinaceous organelles; in general they are 80– 250nm in cross section, comprising 5,000 to 20,000 proteins that assemble to form a characteristic shell with polyhedral architecture25-27. The functional role of BMCs is to encapsulate metabolic pathways protecting the enzymes or sequestering toxic by-products, while


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regulating the transport of substrate and products between the microcompartment and cell cytoplasm (Figure 1). The best characterized examples of BMCs have been extensively reviewed25, 26, 28-30 and include the carboxysome of cyanobacteria and chemoautotrophs31, which encapsulates the enzymes ribulose-1,5-bisphosphate carboxylase–oxygenase32 and carbonic anhydrase33

for

carbon

fixation34.

Recent

attention

has

been

directed

towards

microcompartments that facilitate the utilization of 1,2-propanediol (Pdu), which accumulates following fermentation of the plant sugars rhamnose and fucose35 and for the utilization of ethanolamine (Eut), formed from the breakdown of phosphatidylethanolamine, a membrane lipid component of enterocytes36. Both microcompartments play a crucial role in providing a source of carbon, preventing cytotoxicity and improving carbon retention by sequestering acetaldehyde37, 38

.

A ubiquitous feature of the shells is their construction from members of a family of homologous BMC proteins, which possess a conserved ~80-100 residue BMC domain motif (Pfam00936) that forms an α/β fold with small helices flanking a four-stranded antiparallel sheet. Multiple shell paralogs have been identified among archetypal BMCs, such as the α/β-carboxysome (CcmK1/2/3/4 and CcoS1A/B/C respectively), Pdu (PduA-B-J-K-T-U) and Eut (EutL-M-K-S) microcompartments30. Individual shell proteins self-assemble in 2-dimensional layers approximately 20 Å in thickness as cyclic homohexamers ~70 Å in diameter; these homohexamers form the facets of the microcompartment. The arrangement of the cyclic homohexamers creates a narrow central pore for metabolite transport. Topological variations have been observed amongst BMC proteins, including tandem BMC domain shell proteins (CcsoS1D, EutL, PduT), which form trimers that self-assemble into pseudohexamers and are capable of undergoing conformational change27. While both single and tandem BMC domain proteins assemble to form the facets of the shell, a second conserved BMC domain protein (Pfam03319) found in the α/β-carboxysome (CsoS4A/B and CcmL), Pdu (PduN) and Eut (EutN) microcompartments, self-assembles into pentamers, forming the vertices where the shell facets converge, thereby sealing the microcompartment. The interaction between cyclohexamers in the shell facets and pentamers at the vertices gives rise to a regular icosahedral structure for the carboxysome, but structures exhibiting less pronounced geometric symmetry for both the Pdu and Eut microcompartments30. To date, the recombinant expression of shell proteins for the


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carboxysome (CcoS1A-B-C-S4A/B), 1,2-propanediol (PduA-B-J-K-N) and ethanolamine (EutSM-N-L-K) microcompartments has generated polyhedral structures in vivo39-41. The high degree of conservation among BMC domain proteins has facilitated comparative genome analyses for identifying additional BMC types in numerous bacterial species; this includes a radical glycyl B12-independent propanediol utilization-type (grp)42, an amino alcohol metabolism microcompartment exclusive to Mycobacterium species43, and a microcompartment in Planctomyces limnophilus for saccharide metabolism44. Interestingly, smaller BMC variants of approximately 24 nm termed encapsulins have also been observed in Thermotoga maritima andcomprise 60 copies of a 31-kDa monomer that forms an icosahedral shell45. These structures are believed to protect pH- and temperature-sensitive enzymes; ferritin-like protein (Flp) containing a ferroxidase active site that could oxidize and sequester reactive ferrous ions preventing free-radical formation, or a dye decolorizing peroxidase (Dyp) involved in protecting the cell from oxygen exposure45. Recombinant in vivo expression has since been demonstrated using the encapsulin of Brevibacterium linens46. More recently, the encapsulation of EGFP and luciferase using a 37 C-terminal amino acid sequence of DypB peroxidase to the Reencapsulin nanocompartment of Rhodococcus erythropolis N77147.With further advances in next generation sequencing more novel microcompartments are likely to be discovered, as the underlying design principle seems to be widespread amongst microbial species.

BACTERIAL MICROCOMPARTMENTS: ASSEMBLY, ENCAPSULATION AND TRANSPORT The precise mechanism coordinating microcompartment assembly remains elusive. One proposal suggests that compartment formation is initiated by a nucleus of pre-existing cargo (enzymes to be encapsulated), around which shell proteins orientate48. Contrary to this, experimental evidence has shown that internalized cargo may not always be necessary for microcompartment formation39, 41. Encapsulation of cargo into microcompartments probably depends on conserved N-terminal peptide sequences, which are absent in non-encapsulated homologues49. N-terminal sequences have also been associated with orientation of enzymes for metabolic channeling


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within microcompartments50, EutC

51

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. Targeting sequences derived from the N-terminal regions of

(MDQKQIEEIVRSVMASMGQ)41,

PduP

(NTSELETLIRTILSEQL)51,

and

PduV

(MKRLMFIGPSQCGKTSLTQSLRGEALHYKKTQAIEWSPMA)39, have been demonstrated to target heterologous proteins for encapsulation within microcompartments. Structural investigation of N-terminal targeting sequences revealed the absence of a conserved protein motif. However, a pattern of hydrophobic residues attached to a non-conserved potential linker sequence has been determined in the N-terminal regions of PduC-D-E, suggesting a helical element39. This structural arrangement reflects a common theme among signal sequences in protein translocation pathways52, which display hydrophobic sequences, but lack primary sequence homology53. Following the recent elucidation of the structure of PduP, the formation of alpha helices by N-terminal regions has been verified54.

A defining BMC feature is their capacity to control passage of metabolites and cofactors of varying size and charge in and out the microcompartment, while simultaneously sequestering toxic intermediates. The carboxysome must allow entry of ribulose-1,5-bisphosphate and the exit of glyceraldehyde-3-phosphate bicarbonate, which are both polar, while retaining non-polar CO255. Similarly, the ethanolamine and propanediol microcompartments must permit passage of relatively polar substrates (ethanolamine and 1,2-propanediol), while allowing the diffusion of ethanol and large molecules such as coenzyme A (CoA) (767 Da) and NADH (663 Da), yet retain the small polar intermediate acetaldehyde (44 Da). The selective permeability of BMCs has been attributed to small pores (4–7 Å), which are formed from the cyclic arrangement of single domain BMC proteins30. Residues that line the pore dictate electrostatic interactions and subsequently the transport or blockage of particular molecules. In the carboxysome, pores generated by CcmK2 and CcmK4 are ~7 Å, and ~4 Å in diameter, respectively, and possess an overall positive charge, presumably to facilitate the transport of negatively charged bicarbonate56. The crystal structure of PduA revealed a pore of ~11 Å, lined with hydrogen bond donors and acceptors57, which is in line with the earlier observations. Transport through pores formed from single domain proteins is thought to occur by passive diffusion. In contrast, the pore structures of tandem domain BMC proteins suggest an alternate transport mechanism. The crystal structures of tandem domain BMC proteins CsoS1D58, EutL59 and PduB60 indicate


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formation of trimers that assemble into a hexagonal tile structure, containing a large central pore (~7.5 Å to ~14 Å in diameter),. The comparatively large pore size suggests that tandem BMC shell proteins contribute to a ligand-gated transport mechanism for importing and exporting large cofactors and molecules. Upon binding of a molecule to specific residues on the pore surface, a conformational change is induced causing the pore to open. Studies of EutL have demonstrated a change in pore conformation following exposure to zinc ions61, while binding sites for Fe-S clusters have been identified in PduT57. This has been supported by the recent elucidation of the crystal structure of EutL bound to cobalamin62. A further variation has been observed in the CcmP shell protein of β-carboxysomes, which forms an individual subcompartment within the carboxysome63. The size restriction exhibited by pore structures of shell proteins has suggested a model whereby cofactors such as NADH and CoA are encapsulated upon assembly and are subsequently recycled within the compartment64. When designing heterologous pathways for encapsulation within synthetic microcompartments, it may therefore be necessary to include steps for regenerating pathway-associated cofactors.

BACTERIAL MICROCOMPARTMENTS: DESIGN AND ENGINEERING For synthetic compartmentalization to become a reality, a number of key challenges must be addressed, specifically the design of modular compartments with pre-designed pore sizes for assisting the reactions of encapsulated cargo. Advances in de novo DNA synthesis technologies65 and DNA assembly methods66 have facilitated the iterative design and construction of engineered synthetic microcompartments. Such tools provide the capacity for cloning heterologous shell proteins in new combinations to construct hybrid compartments, using the current diversity of shell proteins as the basis of a parts library. Recent bioinformatics tools such as the LoClass (Locus Classifier) algorithm have aided expansion of this library67. Despite the first report of a recombinant engineered BMC already 6 years ago68, a completely synthetic microcompartment has yet to be constructed. The capacity to assemble a synthetic compartment with pre-defined pore size to accommodate metabolites of varying size and charge remains a key objective in BMC design, if bespoke microcompartments are to be engineered. The role of key pore residues in dictating the


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permeability of the protein shell to metabolites is becoming increasingly evident. A recent study to characterize the effect of mutations in the pore region of the PduA shell protein demonstrated finely tuned specificity for transport of 1,2-propanediol (substrate) and retention of aldehyde (pathway intermediate). Furthermore, it was revealed that pore size and polarity could be modulated to accommodate co-factors and other metabolites69. Further progress towards custom microcompartments has been demonstrated with the construction of chimeric compartments using the carboxysome of Synechococcus elongatus PCC 794263. Firstly, hybrid compartments were assembled by replacing a shell protein from the β-carboxysome (CcmK4) with the αcarboxysome homologue (CsoS1), which has a different pore structure. Secondly, the pore properties of a α-carboxysome shell protein were transplanted to a β-carboxysome shell paralog, by mutating key residues responsible for a conserved motif that flanks the pore, thereby altering the electrostatic properties of the pore and presumably the permeability of the microcompartment63. These advances open the prospect of assembling bespoke compartments from increasingly modularized building blocks, using native shell proteins as a ‘template’ for constructing synthetic libraries. The recent adoption of genome-editing techniques within SynBio, such as the CRISPR-Cas9 system70, provides a strategy that could be repurposed in developing libraries for pore and compartment size engineering. Precise mutations could be introduced into the coding sequence of shell proteins to study the effects on solute passage and to elucidate transport mechanisms, allowing the assembly of compartments with tailor-made permeability properties to match the biosynthetic functions of an engineered cargo, such as a biosynthetic pathway. Chimeric microcompartments with mutated pore residues could then be functionally assessed using cell free transcription–translation systems (TX-TL), which would allow rapid prototyping of synthetic compartments71. The ensuing generation of shell protein libraries could also be utilized for selection of compartment size through hybrid subunits. A crucial factor in the assembly of chimeric microcompartments is the stoichiometric ratio of shell protein expression. SynBio has seen a substantial acceleration in the characterization of genetic parts, including the quantitative assessment of extensive libraries of regulatory elements72. These elements could be applied to fine-tune the expression of individual shell proteins to achieve the appropriate expression stoichiometry and therefore microcompartment


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assembly. The utilization of lac-based promoters that utilize small effectors such as IPTG, have been shown to affect the global transcriptome in E.coli during growth73 and potentially increase contextual complexity. The recent introduction of optogenetic methods to provide temporal control as part of a “just-in-time” response74 enables programmed gene expression while buffering against biological noise to increase the predictability of circuit behavior, thereby overcoming contextual dependencies associated with traditional vector systems. SynBio may also facilitate the development of N-terminal signal sequence libraries, based on documented examples; PduP50, PduD75, CcmN76, EutC41, and PduV39. The small number of targeting sequences currently available may however be a limiting factor for encapsulating large pathways. The tagging of different cargo with the same N-terminal sequence may promote irregular spatial assembly. Indeed, the use of the same PduP targeting sequence to target two different enzymes was found to compromise microcompartment stability54. A potential solution has been highlighted from the sequence analysis of PduP, which revealed three key binding residues that show two conservative replacements in the N-terminal sequence of PduD, suggesting these residues may mediate binding to different shell proteins51. Furthermore, sequence redundancy is displayed within N-terminal sequences, where residues of between 10 and 70 amino acids could be utilized for encapsulation of individual cargo50, 54. In addition, the finding that PduC lacks an N-terminal sequence, but is still targeted to recombinant Pdu microcompartments, suggests the existence of alternate targeting mechanisms that could also be exploited. Using knowledge of characterized N-terminal sequences, a library of characterized targeting sequences could be designed with considerable variation to alter binding affinities and control the orientation of cargo within engineered microcompartments. Further research is needed to elucidate whether different signaling sequences compete for the same interior binding site77. A critical parameter of microcompartment design yet to be addressed concerns the maximum number of enzymes that can be accommodated within the interior lumen of a synthetic microcompartment. The encapsulated pathways of characterized microcompartments are relatively small; consisting of carbonic anhydrase and RuBisCO of the carboxysome, a multi subunit diol dehydratase (PduCDE) and propionaldehyde dehydrogenase (PduP) (and possibly PduL and PduQ) in the Pdu microcompartment, while the Eut microcompartment contains the


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multisubunit

coenzyme-B12-dependent

ethanolamine

ammonia

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lyase

(EutABC),

phosphotransacetylase (EutD), acetaldehyde dehydrogenase (EutE), and alcohol dehydrogenase (EutG). Although it is certain that multiple copies of each enzyme are encapsulated, quantitative estimates regarding the total copy number of enzymes per compartment are limited. Quantitative data limited to the carboxysome suggests 214-296 RuBisCO complexes per carboxysome78, while the theoretical number of encapsulated enzymes in the Pdu microcompartment is thought to be approximately 700077. Little is known about the functional capacity of other compartment types. This limits the ability of synthetic biologists to engineer systems with the optimal number of enzymes to be incorporated; this becomes of paramount importance once large pathways, such as those involved with polyketide and non-ribosomal peptides synthesis79, are intended as cargo, as the space constraints will determine the feasibility of the encapsulation. The range of possible cargo sizes could be considerably extended by combining subunits to create chimeric microcompartments of varying size, including variants with increased interior volume. Our limited understanding of how functional pathways are targeted and assembled notwithstanding, the feasibility of encapsulating a non-native functional pathway within a recombinant microcompartment has recently been demonstrated in E.coli. An ethanol bioreactor consisting of pyruvate decarboxylase and alcohol dehydrogenase from Zymomonas mobilis was successfully targeted to the Pdu microcompartment of Citrobacter freundii using the N-terminal targeting peptide of PduP54.

APPLICATIONS AND FUTURE DEVELOPMENTS Applications for synthetic BMCs have been envisaged in a plug-and-play strategy for the expression of refactored antibiotic biosynthetic gene clusters. As part of this strategy, screening and production strains would express secondary metabolites within microcompartments to limit their toxicity and increase metabolic flux12. Synthetic microcompartments could also be harnessed in agriculture for enhancing CO2 fixation in crops80, but also the commercial synthesis of biofuels13. Indeed, the ethanolamine and propanediol microcompartments provide ideal candidates, since they have already evolved to sequester volatile intermediates.


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It has been proposed that engineering synthetic microcompartments with tissue-specific targeting peptides may enable them to act as drug delivery vehicles. Microcompartments could then be directed to precise cellular locations to release encapsulated drugs (either orally or parenterally) by either pH dependent assembly or through protein degradation through ubiquitylation81. Further applications include the use of BMCs as biotemplates for the assembly of nanowires82, and as important components in the development of cell-free systems83. Although the compartmentalization of metabolic pathways has been the most obvious early application, synthetic microcompartments are by no means limited to this purpose. Protein-based compartments have been observed in eukaryotes, where large ribonucleic particles (vaults) are thought to play a role in intracellular signaling and cell regulation84 Thus, elements of transcription and translation may also be subject to compartmentalization. Indeed, this would enable isolated microcompartments to be used for in vitro protein evolution, a powerful tool in SynBio for optimizing protein activity and fine-tuning synthetic regulatory circuits85. Although directed evolution has been performed in emulsified compartments for a long time86, proteinbased compartments would overcome difficulties commonly arising in the use of emulsions, specifically the denaturing effect of the matrix and the recovery of products87. A further advantage of using BMCs in preference to other compartmentalization strategies for industrial scale synthesis is conveyed by the proteinaceous nature of BMCs. The shell proteins forming the microcompartment are amenable to the incorporation of N-terminal His-tags to facilitate purification using immobilized metal affinity chromatography88. Despite evidence that a single His-tagged shell protein enables co-purification of other shell proteins39, 89, 90, the use of His-tagging to recover intact microcompartments has yet to be attempted. Future developments may entail more sophisticated compartmentalization strategies, potentially incorporating synthetic scaffolding, which has previously been used to achieve correct spatial orientation and enzyme stoichiometries91. This combined approach would involve engineering a synthetic scaffolding protein that expresses both N-terminal peptide tags to direct its encapsulation, and interaction domains for cognate ligands on enzyme targets. Alternatively, there is evidence to suggest that microcompartments are spatially regulated within the cell by cytoskeletal elements92, which could be harnessed to assemble an inter-compartment scaffold. In the long run, such a system could be radically engineered to enable the spatial arrangement of


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individual microcompartments, each encapsulating an alternate series of reactions. This would enable a sequential series of reactions with products from one microcompartment diffusing or being actively transported directly to an adjacent microcompartment, in effect providing a scaled and modularized version of metabolic channeling. The development of new tools for characterizing and modifying the structure and pore attributes of engineered BMCs is also an avenue for continued research. The recent development of a flow cytometry assay to characterize protein encapsulation, using GFP tagged with a mutant library of the signal sequence PduP1-18,93, promises rapid progress in this area.

CONCLUSION BMCs are a promising novel biomaterial that can be utilized to achieve spatial control over cellular processes by promoting metabolic channeling, directing transcription and translation, as delivery vehicles, and intracellular storage spaces. These novel structures have the potential to contain and insulate a wide range of heterologous pathways and represent a key component in SynBio for developing orthogonal systems in bacteria. To achieve the anticipated goals of unlimited, fully synthetic compartmentalization, further research is required to elucidate the mechanisms underlying pore selectivity, encapsulation and spatial regulation of BMCs within the cell cytoplasm. By far, the most controversial challenge faced in synthetic microcompartments concerns the design of pore structures that enable the diffusion of a particular substrate into the BMC and co-factor recycling, yet prevent the release of the desired product enabling it to be concentrated. For complex pathways, this goal may be beyond our current ability to engineer, limiting synthetic microcompartments to encapsulating short pathways or even individual metabolic reactions, analogous to the functions of native BMCs. Further advances in SynBio will likely yield more tools that facilitate the identification of new parts, design and assembly of de novo microcompartments to overcome such limitations, while accelerating the design–build–test cycle.


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Figure 1. Compared to the native situation (A), compartmentalization (B) increases the proximity of enzymes and substrates in support of metabolic channeling and enhanced pathway efficiency. The semi-permeability of the protein shell prevents the release of toxic intermediates, while insulating the enclosed reactions from competing endogenous pathways of the host cell that might otherwise consume key intermediates.


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AUTHOR INFORMATION Corresponding Author Eriko Takano Manchester Synthetic Biology Research Centre SYNBIOCHEM, Manchester Institute of Biotechnology, The Faculty of Life Sciences, The University of Manchester, UK, 131 Princess Street, Manchester, M1 7DN, UK; Email [email protected] Author Contributions The manuscript was written through contributions of all authors. Funding Sources AC has been funded by BBSRC (BB/L027593/1) as part of the ERA-IB Terpenosome Consortium: Engineered compartments for monoterpenoid production using synthetic biology. Notes The authors declare no competing financial interest. ABBREVIATIONS SynBio, Synthetic Biology; BMC, Bacterial Microcompartment;


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For Table of Contents Use Only

Bacterial microcompartments: biomaterials for synthetic biology-based compartmentalization strategies Ashley Chessher, Rainer Breitling, Eriko Takano*


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81x44mm (300 x 300 DPI)


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Bacterial Microcompartments - American Chemical Society

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