Single-component synthetic bacterial microcompartments

Abstract

The present invention relates to single-component synthetic bacterial microcompartments, their uses, and their methods of assembly.

Description

[0001] SINGLE-COMPONENT SYNTHETIC BACTERIAL MICROCOMPARTMENTS FIELD

[0002] The present invention relates to single-component synthetic bacterial microcompartments, their uses, and their methods of assembly.

[0003] STATEMENT OF RELATED APPLICATIONS

[0004] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 584,563, filed September 22, 2023, the entire contents of which are incorporated herein by reference for all purposes.

[0005] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0006] This invention was made with government support under 1941966 awarded by the National Science Foundation. The government has certain rights in the invention.

[0007] SEQUENCE LISTING

[0008] The text of the computer readable sequence listing filed herewith, titled “UM-42105- 601_SQL”, created September 19, 2024, having a file size of 2,057 bytes, is hereby incorporated by reference in its entirety.

[0009] BACKGROUND

[0010] Bacterial microcompartments (BMCs) are a diverse and widespread class of organelles that have selectively permeable protein shells that can confine and increase enzymatic activities, as well as improve the efficacy of specific reactions in bacteria. In native BMCs, these selectively permeable protein shells are self-assembled using multiple homologs of protein oligomers to create complex icosahedral BMC shells. BMC shells lack the ability to selfassemble in vitro.

[0011] SUMMARY

[0012] Provided herein are compositions and methods related to a single-component synthetic bacterial microcompartment.

[0013] For example, the present disclosure provides a microcompartment comprising a single shell protein subunit, CcmK2. In some embodiments, the microcompartment encapsulates an agent. In some embodiments, the agent is a nanomaterial and / or an enzyme. In some embodiments, the nanomaterial is an organic molecule, protein (e.g., enzyme), inorganic molecule, or combinations thereof, In some embodiments, the enzyme is ribulose- 1,5- bisphosphate carboxylase / oxygenase (RuBisCo). In some embodiments, the microcompartment has a diameter of from 90 to 600 nm.

[0014] In some embodiments, the microcompartment is in a solution, in vitro. In some embodiments, the microcompartment is in a cell. In some embodiments, the cell is in culture or otherwise isolated or removed from a natural environment. In some embodiments, the cell is a bacterial cell, gram-negative bacterium, Escherichia coli (E. coli), cyanobacteria, plant cell or an animal cell. In some embodiments, the microcompartment is in an organism, plant, animal, mammal or human. In some embodiments, the organism is obtained from a lake, stream, river, or any combination thereof. In some embodiments, the microcompartment is in a storage buffer.

[0015] Also, provided herein are systems expressing one or more components of the microcompartment, the systems comprising an expression vector encoding CcmK2. In some embodiments, the expression vector further encodes at least one purification tag operably coupled to CcmK2. In some embodiments, the vector further encodes at least detection tag operably coupled to CcmK2.

[0016] Further provided herein is a method of producing the microcompartment comprising: assembling the microcompartment with CcmK2 peptides (e.g., by expression a plurality of CcmK2 peptides under microcompartment forming conditions). In some embodiments, the microcompartment is assembled in vitro. In some embodiments, one or more factors or agents is encapsulated in the microcompartment during formation.

[0017] Further provided here are methods for conducting a reaction (e.g., a biological reaction) in a microcompartment. For example, in some embodiments, the methods comprise conducting an enzymatic reaction in a microcompartment. In some embodiments, the enzymatic reaction is a carbon fixing enzymatic reaction. In some embodiments, the enzymatic reaction comprises fixing carbon by encapsulation of RuBisCo in the microcompartment.

[0018] BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIG. 1 shows a representative image of native CcmK2 shells under in vitro conditions self-assembled into uniformly sized shells.

[0020] FIG. 2 shows a representative image of CcmK2 shells assembled under different pH conditions. FIG. 3 shows a representative image of CcmK2 shell size assembly as KC1 concentrations arc increased.

[0021] FIG. 4 shows a bar graph of the propensity of CcmK2 assembly to decrease as the assembly buffer pH is increased. White bars indicate the proportion of soluble CcmK2, while black bars indicate the proportion of CcmK2 assembled into shells.

[0022] FIG. 5 shows a violin plot of increased CcmK2 shell sizes (diameter) as the assembly buffer pH levels are increased.

[0023] FIG. 6 shows a representative image of CcmK2 shells remaining intact under increased pH conditions.

[0024] FIG. 7 shows a bar graph of CcmK2 shell size assembly as KC1 concentrations are increased. White bars indicate the proportion of soluble CcmK2, while black bars indicate the proportion of CcmK2 assembled into shells.

[0025] FIG. 8 shows a violin plot of different KC1 concentrations having no significant effect on the CcmK2 shell sizes.

[0026] FIG. 9 shows a representative image of CcmK2 shells remaining intact under increased salt concentrations.

[0027] FIG. 10 shows that CcmK2 assembles into monodisperse microscale compartments. A. Cartoon representation of the P-carboxysome shell from S. elongatus. B. CcmK2 is an obligate hexamer with six-fold symmetry. D. DIC images of particles assembled at different concentrations of CcmK2. E. Diameters of particles assembled at different concentrations of CcmK2.

[0028] FIG. 11 shows that CcmK2 particles radially assemble. A. Fluorescence intensity plots of AF647 (pink) and AF488 (green) of cross sections of particles imaged in A. Intensity values were normalized so the maximum value = 1.0. n=50. B. Cartoon schematic of assembly of layered particles. C. Final diameter of particles assembled at initial concentrations of CcmK2, or after adding a second layer of CcmK2 to assembled particles.

[0029] FIG. 12 shows that CcmK2 assembly is influenced by salt concentration and pH. A. Schematic of pelleting assay to quantify CcmK2 assembly. B. DIC images of CcmK2 compartments assembled at various KC1 concentrations. C. Diameter of CcmK2 compartments across KC1 concentrations. D. Proportion of CcmK2 in the pellet and supernatant fraction after the pelleting assay in A. E. DIC images of CcmK2 compartments assembled at various pH values. F. Diameter of CcmK2 compartments across pH values. G. Proportion of CcmK2 in the pellet and supernatant fraction after the pelleting assay in A.

[0030] FIG.13 shows that CcmK2 particles show pH-dependent reversible assembly. A, E. Schematic of disassembly assay in B comparing samples assembled at the indicated condition (top) or assembled and then buffer exchanged into the indicated condition (bottom). B, F. DIC images of CcmK2 particles assembled at (top) or buffer exchanged (bottom). The initial pH before buffer exchange is indicated with a red box. C. Circular dichroism (CD) traces of CcmK2 hexamers, particles, and dissolved particles. D. DIC and fluorescence microscopy images of CcmK2 particles assembled at pH 6.2 (left), buffer exchanged into pH 7.2 (middle), and incubated after 0.5 uL of IN HC1 was added to the sample (right). E. DIC images of CcmK2 compartments treated as described in C. The initial concentration before dilution is indicated with a red box. G. Schematic of assay in H comparing samples assembled at the indicated protein concentration (top) or diluted to the indicated concentration (bottom). H. DIC images of CcmK2 particles assembled de novo or diluted to the indicated concentration. The initial protein concentration before dilution is indicated with a red box.

[0031] FIG. 14 shows that CcmK2 compartments are stable. A. Pearson’s correlation coefficient (PCC) of AF488 and AF647. B. PCC of AF488 and AF647.

[0032] FIG. 15 shows that histidine residues are important in CcmK2 compartment assembly. A. Each CcmK2 monomer has three histidine residues (cyan). Right: zoomed display of three histidine residues. B. DIC images of wildtype and mutant CcmK2 variants after incubation in assembly buffer at the indicated pH. C. Proportion of wildtype and mutant CcmK2 variants that pelleted or remained in the supernatant.

[0033] FIG. 16 shows that CcmK2 particles assemble on a minutes-timescale. A. Schematic of time-resolved dynamic light scattering (DLS) of CcmK2 assembly. B. Population wide intensity of CcmK2 sample. C. Change in the percentage of the population corresponding to the CcmK2 hexamer over time. D. Schematic of assembly of CcmK2 compartments in flow cells. E. Representative timelapse of an assembling CcmK2 compartment. F. Average sum intensity of regions of interest (ROIs) over time.

[0034] FIG. 17 shows an exemplary mechanism of CcmK2 particle assembly, (i) When the pH drops < 6.4, the imidazole ring on His75 of CcmK2 is protonated. The His75 -protonated CcmK2 hexamer is the substrate for the subsequent steps of particle assembly, (ii) CcmK2 hexamers assemble into nanotubes via side-side hexamer contacts, (iii) CcmK2 nanotubes cluster by interacting via facc-facc hcxamcr contacts, (iv, v) CcmK2 nanotubcs continue to cluster radially outwards.

[0035] DEFINITIONS

[0036] To facilitate an understanding of the present technology, a number of terms and phrases are defined below. Additional definitions are set forth throughout the detailed description.

[0037] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0038] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0039] Unless otherwise defined herein, scientific, and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear; in the event, however of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0040] The term “agent” is used in its broadest sense. Agents include chemical and biological agents. Chemical agents may be but are not limited to, any chemical element or compound, on their own or mixed, as it occurs in the natural state or as produced, used, or released. Chemical agents may be obtained from any source including animals, plants, and microorganisms and encompass fluids, solids, tissues, and gases. Biological agents may be but are not limited to, any molecules or materials obtained from bacteria, viruses, fungi, or other microorganisms (e.g., proteins, nucleic acids, lipids, metabolites, small molecules, etc.). Agents may be obtained from any source including animals, plants, and microorganisms and encompass fluids, solids, tissues, and gases. In some embodiments, the agent is from an environmental sample. Environmental samples include, but arc not limited to, those obtained from lakes, streams, rivers, or any combination thereof.

[0041] As used herein, a “microcompartment” is a structure comprising oligomer proteins that may include, for example, shell proteins from a- and P-carboxysomes, found in but not limited to, a- and P-cyanobacteria, respectively. The polymers or oligomers may be heterogenous or homogenous in composition and may be isolated from naturally occurring sources or may be artificially or synthetically produced.

[0042] As used herein, a “nanomaterial” is a class of material(s) with, but not limited to, at least one external dimension which may range in size from 1 to 1000 nm in diameter (e.g., about 1 nm, about 10 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 110 nm, about 120 nm, about 130 nm, about 140 nm, about 150 nm, about 160 nm, about 170 nm, about 180 nm, about 190 nm, about 200 nm, about 210 nm, about 220 nm, about 230 nm, about 240 nm, about 250 nm, about 260 nm, about 270 nm, about 280 nm, about 290 nm, about 300 nm, about 310 nm, about 320 nm, about 330 nm, about 340 nm. about 350 nm, about 360 nm, about 370 nm, about 380 nm, about 390 nm, about 400 nm, about 410 nm, about 420 nm, about 430 nm, about 440 nm, about 450 nm, about 460 nm, about 470 nm, about 480 nm, about 490 nm, about 500 nm, about 510 nm, about 520 nm, about 5530 nm, about 540 nm, about 550 nm, about 560 nm, about 570 nm, about 580 nm, about 590 nm or about 600 nm, about 620 nm, about 630 nm, about 640 nm, about 650 nm, about 660 nm, about 670 nm, about 680 nm, about 690 nm, about 700 nm, about 710 nm, about 720 nm, about 730 nm, about 740 nm. about 750 nm, about 760 nm, about 770 nm, about 780 nm, about 790 nm, about 800 nm, about 810 nm, about 820 nm, about 830 nm, about 840 nm, about 850 nm, about 860 nm, about 870 nm, about 880 nm, about 890 nm, about 900 nm.). The nanomaterial may be derived from nature or artificially made.

[0043] Preferred methods and materials are described below, although methods and materials similar’ or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting. DETAILED DESCRIPTION

[0044] Disclosed herein arc synthetic single-component bacterial microcompartments. In some embodiments, the microcompartments encapsulate one or more agents (e.g., for use in conducting, studying, or regulating enzymatic and metabolic reactions).

[0045] Bacterial microcompartments (BMCs) are organelles that segregate essential and sensitive metabolic pathways from the surrounding metabolic environment within an organism. Naturally occurring BMCs comprise a modular protein shell which surrounds an enzymatically active core, with the shell functioning as a semipermeable membrane for substrates and products that vary functionally amongst distinct BMCs. A key function of BMCs is their ability to selfassemble both their shell and enzymatic actions e.g., cargo from multiple protein oligomers to create complex protein structures. Specialized proteins are utilized to facilitate cargo aggregation as well as small encapsulation peptides that specifically associate proteins to the lumen of the shell. BMCs contribute to the metabolic versatility of bacteria, providing a competitive advantage in specific environmental niches. The present knowledge of native BMCs has resulted in an increase in the understanding of their role in microbial metabolism but also a burgeoning interest of their capabilities in a variety of applications including the field of synthetic biology. However, creating a stable BMC that can exist outside an organism with functioning enzymatic reactions occurring simultaneously has remained elusive within the biological sciences field.

[0046] The compositions and methods herein provide single component microcompartments that can encapsulate any agents of interest. In some embodiments, the shells are generated in vitro, providing a new approach to encapsulation and use of agents. This method offers multiple advantages over native BMCs as it allows for (1) the simplified study of or use BMCs and (2) a BMC system capable of perpetuating nanofactories and molecular scaffolds both in vitro and within more complex structures such as cells.

[0047] As described herein, reaction conditions may be adjusted to control the size and other properties of the shells. For example, regulation of shell size is controllable via selection of pH during the assembly process. Size selectability broadens the range of uses, including the size, concentration, and complexity of cargo contained in the shell and the diversity of environments in which shells may be employed. The microcompartments also provide optimal systems for reductionistic investigations of protein interactions on both the interior and exterior sides of BMC shells due to their simple and controllable make-up. CcmK2 proteins

[0048] The synthetic bacterial microcompartments comprise an assembly of CcmK2 proteins. The CcmK2 proteins may be obtained from any organism and may be wild-type or variant proteins, including natural variants. In some embodiments, the CcmK2 protein is from a cyanobacteria. In some embodiments, the CcmK2 protein has a sequence comprising the sequences of any of UniProtKB Accession Numbers Q03511 (MPIAVGMIETLGFPAVVEAADAMVKAARVTLVGYEKIGSGRVTVIVRGDVSEVQASVS AGLDSAKRVAGGEVLSHHIIARPHENLEYVLPIRYTEAVEQFRM (SEQ ID NO:1)), A0A0C1N5L4, A0A0C2M8I8, A0A0C2Q5E0, A0A0D6ACS2, A0A0D6L1Q9, A0A0H3JZB4, A0A0M3V6T6, A0A0P8CDZ6, A0A0S3TQS4, A0A139WV47, A0A1C0UX57, A0A1E2WRJ4, A0A1E5QXS7, A0A1Q4RAR4, A0A1U7GV96, A0A1U7HWB0, A0A1Y0RG45, A0A1Z4G808, A0A1Z4H7Z1, A0A1Z4I2M9, A0A1Z4IT55, A0A1Z4IXU7, A0A1Z4L585, A0A1Z4N9D9, A0A1Z4Q5A5, A0A1Z4S6M8, A0A1Z4T977, A0A1Z4UV52, A0A235I6K6, A0A235J489, A0A252CW73, A0A252CYI2, A0A252DEY2, A0A2D0HB63, A0A2H2XNG7, A0A2I8A1E6, A0A2K8STU8, A0A2K8WNK1, A0A2L2NQR4, A0A2N6K2F1, A0A2N6KEY1, A0A2N6MLE7, A0A2P8QNE4, A0A2R5FSU0, A0A2S6CU20, A0A350Y1W9, A0A352AND3, A0A355DU61, A0A367PV81, A0A367RD01, A0A367RIH4, AOA3G6X1F8, A0A3M1LAC1, A0A3S1AUR3, A0A401IDE9, A0A433VP62, A0A433W4M1, A0A479ZS82, A0A522XGF6, A0A5P8W4B4, A0A6G9SF45, A0A6N0LW66, A0A6N2DJ18, A0A6P0Q9D6, A0A841WPQ8, A0A844MYD4, A0A856MB42, A0A8I6XQH9, A0A8J7DL76, A0A8J7HW33, A0A8J7I648, A0A8K1MUT4, A0A926YSB8, A0A926ZRK5, A0A927A366, A0A930T1N0, A0A930TEV5, A0A930XHD1, A0A938ZK90, A0A938ZTG1, A0A941GMH0, A0A951PTQ6, A0A968J3N2, A0A968NRY5, A0A968YNL7, A0A968Z8P8, A0A969TJK1, A0A969TQP7, A0A969V679, A0A975TD60, A0A978SWY7, A0A978T8C2, A0A978U46I, A0A9D7VFR6, A0A9D9K9B3, A0A9E6RWT9, B8HX53, K9QWL8, K9U5P9, K9YIB5, K9ZJ01, L8LWP2, A0A3M9Z4C2, A0A8J7KDI3, and A0A968LRK6, or sequences having at least 80% identity thereto (e.g., at least 90%, at least 95%, at least 98%, at least 99%) or having one or more conservative or non-conservative amino acid substitution therein (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10).

[0049] The ability to synthesize CcmK2 proteins for assembly in vitro allows significant flexibility in employing modified or variant CcmK2 proteins. For example, in some embodiments, the CcmK2 proteins are expressed as fusion proteins. Fusion partners include, but arc not limited to, purification tags, detection tags, and display peptides. Purification tags facilitate purification of expressed CcmK2 proteins and, in some embodiments, are removed prior to microcompartment assembly. Detection tags, such as tag comprising a fluorophore, facilitate detection of CcmK2 proteins and assembled microcompartments. Display peptides allow for the presentation of desired peptide sequence on the outer or inner surface of assembled microcompartments. Such display peptides find use, for example, to regulate binding of molecules to a surface of a microcompartment. In some embodiments, proteins are post- translationally modified. For example, in some embodiments, proteins may be modified with a carbohydrate, lipid, binding moiety, linker, polyethylene glycol (PEGylated), nucleic acid molecule, or the like. In some embodiments, the modifications are employed to reduce antigenicity of the system. Variant CcmK2 proteins may include deletions (e.g., truncations), insertions, and / or one or more conservative or non-conservative amino acids substitutions relative to a wild-type CcmK2 protein. A “conservative” amino acid substitution refers to the substitution of an amino acid in a peptide or polypeptide with another amino acid having similar chemical properties, such as size or charge. For purposes of the present disclosure, each of the following eight groups contains amino acids that are conservative substitutions for one another: 1) Alanine (A) and Glycine (G); 2) Aspartic acid (D) and Glutamic acid (E); 3) Asparagine (N) and Glutamine (Q); 4) Arginine (R) and Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), and Valine (V); 6) Phenylalanine (F), Tyrosine (Y), and Tryptophan (W); 7) Serine (S) and Threonine (T); and 8) Cysteine (C) and Methionine (M). Naturally occurring residues may be divided into classes based on common side chain properties, for example: polar positive (histidine (H), lysine (K), and arginine (R)); polar negative (aspartic acid (D), glutamic acid (E)); polar neutral (serine (S), threonine (T), asparagine (N), glutamine (Q)); non-polar aliphatic (alanine (A), valine (V), leucine (L), isoleucine (I), methionine (M)); non-polar aromatic (phenylalanine (F), tyrosine (Y), tryptophan (W)); proline and glycine; and cysteine. As used herein, a “semi-conservative” amino acid substitution refers to the substitution of an amino acid in a peptide or polypeptide with another amino acid within the same class. In some embodiments, unless otherwise specified, a conservative or semi-conservative amino acid substitution may also encompass non-naturally occurring amino acid residues that have similar chemical properties to the natural residue. These non-natural residues are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include, but arc not limited to, pcptidomimctics (c.g., chemically modified peptides, peptoids (side chains arc appended to the nitrogen atom of the peptide backbone, rather than to the a-carbons), P-peptides (amino group bonded to the 0 carbon rather than the a carbon), etc.) and other reversed or inverted forms of amino acid moieties. Embodiments herein may, in some embodiments, be limited to natural amino acids, non-natural amino acids, and / or amino acid analogs. Nonconservative substitutions involve the exchange of a member of one class for a member from another class.

[0050] Assembly

[0051] The present disclosure provides methods for producing or assembling or modulating or expressing or sizing a single component synthetic bacterial microcompartment.

[0052] In some embodiments, one or more CcmK2 proteins is cloned into one or more expression vectors. The expression vectors are introduced into a host cell and CcmK2 proteins are expressed in the host cell. CcmK2 proteins are purified. In some embodiments, purified proteins are concentrated. Purified proteins are added to an assembly buffer to form microcompartments. In some embodiments, one or more cargo agents are included in the reaction mixture to generate microcompartments encapsulating the cargo agents. The pH of the assembly buffer is selected to allow for assembly and to regulate the size and / or other properties of the microcompartment. Experiments conducted during the development of the invention surprisingly found that acidic pH conditions allowed CcmK2 proteins to assemble into microcompartments .

[0053] Any suitable expression vector or host cell may be used in the assembly methods. In some embodiments, the expression vector is a plasmid, virus, or nucleic acid fragment. In some embodiments the expression vector includes a regulatory sequence comprising a promoter(s). In some embodiments, the promoter is a constitutive, regulatable, or inducible, cell-type specific tissue-specific, or species-specific promoter. In addition to the sequence sufficient to direct transcription, an expression vector can also include sequences of other regulatory elements that are involved in modulating transcription (e.g., enhancers, Kozak sequences, etc.). In addition, any promoter / regulatory sequences useful for driving constitutive expression of a protein may be used, but are not limited to, for example constitutive promoters (Pcpc, PpsbA, Prbc), heterologous / inducible promoters (Plac, Ptrc, Ptet, PR), modified promoters (Pcpc (e.g. PcpcB, Pcpc560, Pcpt, PCP)), lac operons (T7, T3, Sp6), synthetic and hybrid promoters (J23-scrics, Pconll, etc.) and native, inducible promoters (metal-inducible promoters, environmental sensor- or metabolic state-dependent promoters and cell type-specific promoters). In some embodiments, the expression vector encodes CcmK2 peptides in combination with targeting molecules, detectable labels, enzymes, stable isotopes, antibodies, sequence tags, spacers, capture sequences, and the like. In some embodiments, the expression vector is transformed or transfected into the cells. Suitable host organisms include, but are not limited to, bacteria such as E. coli, B. subtilis, S. cerevisiae, a-cyanobacteria (.S'. elongatus, Spirulina, Anabaena, Nostoc and Oscillatoria). plants, algae, fungi, or other eukaryotic organisms.

[0054] Expressed CcmK2 proteins can be purified by any suitable method. In some embodiments, CcmK2 proteins arc expressed with affinity tags and expressed proteins are purified using an affinity column that specifically finds the affinity tag. In some embodiments, affinity tags are removed prior to microcompartment assembly. In some embodiments, unmodified proteins are purified. In some embodiments, host cells are collected (e.g., by pelleting) and lysed (e.g., via mechanical disruption, liquid homogenization, sonication, freeze / thaw, grinding, chemically, etc.) to release proteins. Cells may be stored (e.g., flash frozen), if desired, prior to lysis or other further processing. In some embodiments, one or more purification buffers are added to cells during or after lysis. Buffers may include stabilizers, protease inhibitors, salts, surfactants, and the like. In some embodiments, the purification process comprises passing sample containing CcmK2 protein through a filter and / or size exclusion column. In some embodiments, purified, collected proteins are stored in a storage buffer.

[0055] In some embodiments, to produce a purified CcmK2 protein, the methods comprise, consist of, or consist essentially of: expressing a CcmK2 protein in a host cell, , purifying the host cell of a generate a purified component, resuspending the purified component in a lysis buffer to generate a lysate, centrifuging the lysate (e.g., for at least 30 minutes), clarifying the lysate onto a purification column, washing the purification column with a high salt buffer, preforming elutions on the purification column, desalting the purification column into the storage buffer, diluting the purification column into a storage buffer, filtering storage buffer through a syringe and passing the sample protein over a size exclusion column equilibrated in the storage buffer. In some embodiments, to produce a single component synthetic bacterial microcompartment from purified CcmK2 protein, the methods comprise, consist of, or consist essentially of: diluting purified CcmK2 protein in solution in a storage buffer into an assembly buffer, in vitro.

[0056] In some embodiments, the assembly buffer in which the purified CcmK2 protein is diluted has a pH of from 5.8 to 6.2. For example, the assembly buffer pH can be about 5.8, about 5.9, about 6.0, about 6.1 or about 6.2.

[0057] In some embodiments, the formed microcompartment may range in size from 90-600nm in diameter (e.g., about 90 nm, about 100 nm, about 110 nm, about 120 nm, about 130 nm, about 140 nm, about 150 nm, about 160 nm, about 170 nm, about 180 nm, about 190 nm, about 200 nm, about 210 nm, about 220 nm, about 230 nm, about 240 nm, about 250 nm, about 260 nm, about 270 nm, about 280 nm, about 290 nm, about 300 nm, about 310 nm, about 320 nm, about 330 nm, about 340 nm. about 350 nm, about 360 nm, about 370 nm, about 380 nm, about 390 nm, about 400 nm, about 410 nm, about 420 nm, about 430 nm, about 440 nm, about 450 nm, about 460 nm, about 470 nm, about 480 nm, about 490 nm, about 500 nm, about 510 nm, about 520 nm, about 530 nm, about 540 nm, about 550 nm, about 560 nm, about 570 nm, about 580 nm, about 590 nm or about 600 nm).

[0058] In some embodiments, formed microcompartments may be introduced into a variety of different environments. In some embodiments, microcompartments are maintained in vitro. In some embodiments, microcompartments are added to cells. In some embodiments, microcompartments are added to cell mixtures (e.g., in culture). In some embodiments, microcompartments are administered to an organism. In some embodiments, the microcompartments are provided in a liposome or other carrier. In some embodiments, microcompartments are included on or in a device (e.g., implanted device, sensor, artificial organ, etc.).

[0059] Cargo

[0060] The present disclosure provides a single component synthetic bacterial microcompartment comprising a single shell protein subunit, CcmK2. In some embodiments, one or more agents in included within or on the shell. Such agents are referred to herein as “cargo.” In some embodiments, agents include, but are not limited to proteins (e.g., fluorescent proteins, antigens, cytokines, macrophages, antioxidants antimicrobial proteins, signaling proteins, fibroins, albumins, gelatins, gliadines, legumins, 30Kcl9, lipoproteins and / or ferritin proteins), organic agents (c.g., polymeric micelles, dendrimers, carbons and / or polymeric micelles), inorganic agents (e.g., metal, non-metal elements in the forms of an oxides, hydroxides, chalcogenides and / or phosphate compounds), hybrid compounds (e.g., hydrogels (polymeric nanogels and / or macromolecular micelles), materials made of complex composition (e.g., metals, oxides, chalcogenides) and / or coordination polymer), enzymes (e.g., lipases, amylases, invertase, glucose oxidases and / or proteases), antioxidant nanozymes (e.g., Superoxide Dimutas (SOD) nanozymes, catalase mimetics nanozymes) and / or pro-oxidant nanozymes (e.g., peroxidase mimetic nanozymes) and the like. Any of a wide variety of enzymes or multi-enzyme systems may be included in the microcompartments. Examples, include, but are not limited to, oxidoreductases, transferases, hydrolases, lyases, isomerases, and ligases (e.g., a-amylase, catalase, laccase, peroxidase, lysozyme, and reporter enzymes alkaline phosphatase, and luciferase). The cargo may further comprise one or more agents (e.g., molecules) that enhance an activity or property of another cargo component. Such properties include, but are not limited to, stability, shell-life (storability), mobility, interactivity, etc.

[0061] In some embodiments, enzyme cofactors are included within the microcompartment or are introduced into the microcompartment (via pores) after assembly. Such cofactors include, but are not limited to prosthetic groups, coenzymes, and metal ions (e.g., ascorbate, biotin, cadmium, calcium, cobalt, cobamide, copper, flavoproteins, heme, iron, magnesium, manganese, molybdenum, nickel, potassium, pyridoxal phosphate, pyrroloquinoline quinone, pyruvate, siroheme, sulfur, thiamin diphosphate, thiamine, tungsten, zinc, etc.).

[0062] In some embodiments, microcompartment encapsulation composition may comprise a system for carbon fixation of RuBisCo. In some embodiments, microcompartment encapsulation composition may comprise a system for a biological reaction wherein the biological reaction is an enzymatic reaction. In some embodiments, the enzymatic reaction is a carbon fixing enzymatic reaction.

[0063] Uses

[0064] The synthetic microcompartments provided herein find use in a wide variety of research, diagnostic, industrial, agricultural, and clinical settings. For example, the microcompartments find use in any setting where it is desirable to enclose or isolate or segregate cargo. In some embodiments, the microcompartments provide an in vitro system for working with enzymes or other agents that are found naturally in endogenous BMCs. However, with the ability to synthesize monomers and assemble the microcompartments in vitro, a wide variety of additional agents may be employed on or in the microcompartments. The microcompartments provide an alternative system to enclose, isolate, or segregate enzymes, reactions, or other agents that are currently worked with in organelles, liposomes, exosomes, micelles, nanoparticles, wells, channels, microfluidic chambers, and the like.

[0065] Non-limiting examples of methods employing the microcompartments include: basic research, applied research, clinical research, drug screening, nanofactories, diagnostics, storage or stabilization of agents, isolation of agents (e.g., nanotoxic intermediates), synthetic biology, delivery of agents (e.g., in vitro, in vivo, environmentally, etc.), sustain / controlled release, and bioremediation.

[0066] EXAMPLES

[0067] The following are examples of the present invention and are not to be construed as limiting.

[0068] Example 1

[0069] MATERIALS AND METHODS

[0070] Sample Development

[0071] The His-SUM0-CcmK2 expression plasmid (pCMOl) was generated via Gibson Assembly using synthetized dsDNA for His-SUM0-CcmK2 that was inserted into a pETllb vector containing an ampicillin antibiotic resistance cassette. The His-SUMO-CcmK2[A68C] expression plasmid (pCM02) was generated from pCMOl via site-directed mutagenesis (Forward primer = CAAACGCGTGTGTGGCGGCGAAGTGCTGAGC, Reverse primer = GCGCTATCCAGACCCGCG). Constructs were verified by sanger sequencing. Cloning was performed in E. coli TOP 10 chemically competent cells.

[0072] Protein expression and purification

[0073] His-SUMO-CcmK2 and His-SUMO-CcmK2 (A68C) were expressed and purified in using expression plasmids that were transformed into E. coli- AE cells, and single transformants to inoculate in at least 10 mL overnight cultures. Cells were grown in LB + carbenicillin (100 pg / mL). For expression, one liter of culture was inoculated through a 1:100 dilution of the overnight culture and grown at 37 °C and 200 rpm shaking until an optical density of a sample measured at a wavelength of 600 nm of 0.6-0.8 was reached. The culture flasks were cooled in an ice bath for at least 5 min and protein expression was then induced with a final concentration of at least 1 mM IPTG. Cultures were grown at 16 °C for 20h, pelleted, flash-frozen in liquid nitrogen and stored at -80°C.The following buffers were used to purify the proteins: Buffer A (20 mM Tris-HCl [pH 7.8], 150 mM KC1, 10% glycerol, 20 mM imidazole, 5 mM BME), Buffer B (20 mM Tris-HCl [pH 7.8], 150 mM KC1, 10% glycerol, 1 M imidazole, 5 mM BME), Lysis Buffer (buffer A + 0.05 mg / mL lysozyme, protease inhibitor, 5 mM BME), and Storage Buffer (20 mM Tris-HCl [pH 7.8], 50 mM KC1, 10% glycerol, 2 mM DTT). Cell pellets were resuspended in 40 mL Lysis Buffer, and sonicated with cycles of at least 10 seconds on, 20 seconds off at 50% power for at least 6 minutes. The lysates were centrifuged at 13,000 x g at 4 °C for at least 30 min. The clarified lysates were decanted, syringe filtered (0.45 pm), and loaded onto a 1 mL HP His-TRAP (Cytiva) column equilibrated in buffer A. The column was washed with five column volumes of High Salt Buffer (20 mM Tris-HCl [pH 7.8], 1 M KC1, 10% glycerol, 20 mM imidazole), 5 column volumes of Buffer A, and then 5 column volumes of 5% Buffer B. Elution was performed using a 5-100% gradient of Buffer B via an AKTA Pure system (Cytiva). Peak fractions were pooled, and buffer exchanged into Storage Buffer using Zeba Spin Desalting Columns. Protein was diluted with Storage Buffer to < 3 mg / mL and incubated with the Ulpl protease at 30 °C overnight with gentle rocking. Samples were syringe filtered (0.2 pm) and passed over a size exclusion column (HiLoad 16 / 600 Superdex 200 pg; Cytiva) equilibrated in storage buffer. Peak fractions were pooled and concentrated to at most 1.5 mg / mL. Aliquots were flash-frozen and stored at -80 °C.

[0074] Fluorescent Labeling of CcmK2[A68C]

[0075] Prior to Ulpl treatment, His-SUMO-CcmK2[A68C] was fluorescently labeled with either Alexa Fluor 488 or 647 (Fisher Scientific). At least 10 mM of DTT was added to 0.5 mL aliquots of protein. Samples were then exposed to N2 gas for 2 min. To remove excess DTT, samples were buffer exchanged using Zeba Spin desalting columns equilibrated in Storage Buffer without DTT (20 mM Tris-HCl [pH 7.8], 50 mM KC1, 10% glycerol). Fluorescent dyes were added to samples in a 2: 1 molar ratio of dye to protein and incubated for at least 20 min at 25°C away from light. Reactions were quenched with 10 mM DTT. Protein samples were concentrated to at least < 500 pL through Amicon Ultra centrifugal filters. Labeled His-SUMO-CcmK2[A68C] were incubated with the Ulpl protease at 30 °C overnight with gentle rocking. Samples were syringe filtered (0.2 pm), passed over a size exclusion column (Superdex200 10 / 300GL; Cytiva) and equilibrated in Storage Buffer (20 mM Tris-HCl [pH 7.8], 50 mM KC1, 10% glycerol, 2 mM DTT). Peak fractions were pooled and concentrated, and flash-frozen aliquots were stored at -80 °C.

[0076] CcmK2 shell assembly

[0077] CcmK2 in storage buffer (20 mM Tris-HCl [pH 7.8], 50 mM KC1, 10% glycerol, 2 mM DTT) was concentrated to 1.0- 1.6 mg / mL, if needed. CcmK2 was diluted into assembly buffer (final concentrations of 20 mM KPi [pH 5.8-6.2] and 150 mM KC1) to a final concentration of 40 pM unless otherwise stated. For fluorescent shell assembly, Alexa Fluor 488- or 647-labeled CcmK2[A68C] was added in a 1:500 molar ratio of dark- to labeled-CcmK2. Reactions were incubated for at least 60 minutes prior to further experimentation.

[0078] CcmK2 shell imaging

[0079] For imaging, samples were transferred to 16 well CultureWells (Grace Biolabs) and blocked with 5% Pluronic F-127. Imaging was performed using a Nikon Ti2-E motorized inverted microscope (100 x objective and DIC analyzer cube) controlled by NIS Elements software with a Transmitted LED Lamp house and a Photometries Prime 95B Back-illuminated sCMOS Camera. Image analysis was performed using Fiji v 1.0.

[0080] Quantification of CcmK2 shell assembly

[0081] After the 60 min reaction incubation, samples were spun down at 16,000 x g for at least 10 min. The supernatant was removed and saved, and the pellet was resuspended in fresh sample buffer using the initial sample volume. 4x SDS-PAGE loading dye was added to each sample to a final concentration of lx, and samples were boiled at 95 °C for at least 15 min. Samples were ran on a 4-12% Bis-Tris NuPAGE gel (Invitrogen) at 100 V for at least 70 min. Gels were stained with InstantBlue Coomasie Stain (Abeam) for l-3h and destained in water overnight. The intensities of the bands were quantified using Fiji v 1.0 and resultant data graphed using GraphPad Prism 9.0.1 for macOS (GraphPad Software, San Diego, CA, www dot GraphPad dot com).

[0082] Example 2

[0083] CcmK2 self-assembles into microcompartments in vitro.

[0084] In vivo studies of native P-carboxysome assembly have demonstrated that enzymatic core assembly is a prerequisite to shell assembly. These assembly studies also found that the pentameric shell protein, CcmL is required for proper carboxysome morphology; without the pentameric protein, elongated “bar-like” carboxysomes assemble instead. Identified, under in vitro conditions, were native CcmK2 alone that self-assembled into uniformly sized shells (FIG 1). This led to the current models of p-carboxysome assembly as the first single BMC shell protein assembling into a microcompartment in vitro.

[0085] Example 3

[0086] CcmK2 self-assembly is sensitive to pH and salt concentrations.

[0087] Previous studies have shown that hexameric BMC shell proteins can assemble into flat sheets in vitro, and that this sheet assembly is sensitive to pH and salt concentrations. Characterization of the ability of CcmK2 shells to assemble under various environmental conditions, showed that shell assembly is sensitive to pH-dependent and salt concentrations (FIG 2 and FIG 3).

[0088] Example 4

[0089] CcmK2 shell size (diameter) during assembly is decreased as pH of assembly buffer is increased.

[0090] Shell assembly is highly pH-dependent, with the propensity of assembly decreasing as pH increases. Intriguingly, CcmK2 shell diameter increased as the pH of the assembly buffer increased (FIG 4 and FIG 5).

[0091] Example 5

[0092] CcmK2 shells remain stable under increased pH concentrations. CcmK2 shell size remained stable under increased pH concentrations. Shells assembled at pH 6.2 were buffer exchanged into higher pH conditions. Exchanged shells remained intact up to pH 7.0 (FIG 6).

[0093] Example 6

[0094] CcmK2 shell size (diameter) during assembly is stable under increased KC1 concentrations.

[0095] Unlike buffer pH, salt concentration did not affect the final diameter of the CcmK2 shells (FIG 7 and FIG 8).

[0096] Example 7

[0097] CcmK2 shell assembly is stable under increased KC1 concentrations.

[0098] CcmK2 shell size remained stable under increased salt concentration. Shells readily assembled between 50-500 mM KC1, as salt concentration increased (FIG 9).

[0099] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0100] Example 8

[0101] CcmK2 assembles into monodisperse and pH-reversible microparticles

[0102] METHODS

[0103] Construct Development. The His-SUMO-CcmK2 expression plasmid (pCMOl) was generated via Gibson Assembly (Gibson et al., 2009) using synthesized dsDNA for His-SUMO-CcmK2 that was inserted into a pET I lb vector containing an ampicillin antibiotic resistance cassette. All other constructs used in this study were generated via site-directed mutagenesis of pCMOl, except pCM14 which was generated via site-directed mutagenesis of pCM13 (see table 1 for primers). Plasmids were sequence verified upon construction. Cloning was performed in chemically competent E. coli Top 10 cells (Takara Bio). Table 1

[0104] Protein expression and purification. Wild-type and mutant valiants of CcmK2 were expressed with an N-terminal His-SUMO tag off a pETl lb vector in E. coli BL21-AE (Invitrogen). All cells were grown in LB + carbenicillin (100 pg / ml). One liter cultures used for expression were inoculated using overnight cultures at a 1:100 dilution. Cultures were grown to an OD600 of 0.6- 0.8 and cooled on ice prior to expression induction using final concentration of 1 mM isopropyl B-D-l -thiogalactopyranoside (IPTG). Cultures were grown at 16 °C for 20h, pelleted, flash- frozen in liquid nitrogen and stored at -80°C.

[0105] The following buffers were used to purify wild-type and mutant variants of CcmK2 : Buffer A (20 mM Tris-HCl [pH 7.8], 150 mM KC1, 10% glycerol, 20 mM imidazole, 5 mM BME), Lysis Buffer (buffer A + 0.05 mg / mL lysozyme, protease inhibitor, 5 mM BME), High Salt Buffer (20 mM Tris-HCl [pH 7.8], 1 M KC1, 10% glycerol, 20 mM imidazole), Buffer B (20 mM Tris-HCl [pH 7.8], 150 mM KC1, 10% glycerol, 1 M imidazole, 5 mM BME), and Storage Buffer (20 mM Tris-HCl [pH 7.8], 50 mM KC1, 10% glycerol, 2 mM DTT). Cell pellets were resuspended in 40 mL Lysis Buffer, and sonicated with cycles of 10 seconds on, 20 seconds off at 50% power for 6 min. The lysates were centrifuged at 19,000 ref at 4 °C for 30 min. The clarified lysates were decanted, syringe filtered (0.45 pm), and loaded onto a 1 mL HP His- TRAP (Cytiva) column equilibrated in Buffer A. The column was washed with five column volumes of High Salt Buffer, 5 column volumes of Buffer A, and 5 column volumes of 5% Buffer B. Elution was performed using a 5-100% gradient of Buffer B via an AKTA Pure system (Cytiva). Peak fractions were pooled, and buffer exchanged into Storage Buffer using Zeba Spin Desalting Columns. Protein was diluted with Storage Buffer to < 3 mg / mL and incubated with Ubiquitin-like specific protease 1 (Ulpl) at 30 °C for 24 h with gentle rocking. Samples were syringe filtered (0.2 pm) and passed over a size exclusion column (HiLoad 16 / 600 Superdex 200 pg; Cytiva) equilibrated in Storage Buffer. Peak fractions were pooled and concentrated to 1.8- 2.0 mg / mL. Aliquots were flash-frozen and stored at -80 °C.

[0106] Fluorescent Labeling of CcmK2[A68C]. Prior to Ulpl treatment, His-SUMO-CcmK2[A68C] was fluorescently labeled with either Alexa Fluor 488 or 647 (Fisher Scientific). 10 mM DTT was added to 0.5 mF aliquots of protein, and samples were exposed to N2 gas for 2 min. To remove excess DTT, samples were buffer exchanged using Zeba Spin desalting columns equilibrated in Storage Buffer without DTT (20 mM Tris-HCl [pH 7.8], 50 mM KC1, 10% glycerol). Fluorescent dye was added to samples in a 2:1 molar ratio of dye to protein, and samples were incubated for 20 min at 25 °C away from light. Reactions were quenched with 10 mM DTT. Protein samples were concentrated to < 500 pL using Amicon Ultra centrifugal filters. Labeled His-SUMO-CcmK2[A68C] was incubated with the Ulpl protease at 30 °C overnight with gentle rocking. Samples were syringe filtered (0.2 pm) and passed over a size exclusion column (Superdex200 10 / 300GL; Cytiva) equilibrated in Storage Buffer (20 mM Tris-HCl [pH 7.8], 50 mM KC1, 10% glycerol, 2 mM DTT). Peak fractions were pooled and concentrated, and flash-frozen aliquots were stored at -80 °C.

[0107] CcmK2 particle assembly. CcmK2 (in Storage Buffer) was diluted into an assembly buffer (final concentrations of 20 mM KPi [pH 6.2] and 150 mM KC1 unless otherwise stated) to a final concentration of 40 pM unless otherwise stated. For fluorescent particle assembly, Alexa Fluor 488- or 647-labeled CcmK2A68Cwas mixed in a 1:1000 molar ratio of labeled- to dark-CcmK2 prior to dilution into the assembly buffer. Reactions were incubated at room temperature for 60 minutes prior to further experimentation unless otherwise noted.

[0108] CcmK2 particle size measurements. Diameters of CcmK2 particles were analyzed in Fiji v 1.0 and resultant data was graphed using GraphPad Prism 10.3.0 for Windows (GraphPad Software, Boston, Massachusetts USA, www.graphpad.com).

[0109] Quantification of particle assembly via centrifugation. After the 60 min reaction incubation, samples were spun down at 16,000 ref for 10 min. The supernatant was removed and saved, and the pellet was resuspended in water using the initial sample volume. 4x SDS-PAGE loading dye was added to pellet and supernatant samples to a final concentration of lx, and samples were boiled at 95 °C for 15 min. Samples were run on an 4-12% Bis-Tris NuPAGE gel (Invitrogen) at 110 V for 70 min. Gels were stained with InstantBlue Coomasie Stain (Abeam) for 1-3 h, destained in water overnight, and imaged on a Fluorchem M system (ProteinSimple). The intensities of the bands were quantified using Fiji v 1.0 and resultant data graphed using GraphPad Prism 10.3.0 for Windows (GraphPad Software, San Diego, CA, www.graphpad.com).

[0110] Microscopy of CcmK2 particles

[0111] Differential Interference Contrast (DIC) and fluorescence microscopy. Samples were transferred to 16 well CultureWells (Grace Biolabs) blocked with 5% Pluronic F-127. All samples were incubated for 60 min at room temperature unless otherwise stated. Imaging was performed using a Nikon Ti2-E motorized inverted microscope (100 x DIC objective and DIC analyzer cube) controlled by NIS Elements software with a Transmitted LED Lamp house and a Photometries Prime 95B Back-illuminated sCMOS Camera. AF 488 signal was acquired using a ‘FITC’ filter set [excitation / emission 488 / 460]. AF 647 signal was acquired using a ‘Cy5’ filter set [excitation / emission 647 / 630]. Image analysis was performed using Fiji v 1.0.

[0112] Laser Scanning Confocal microscopy. Samples were imaged on a Zeiss LSM 980 Airyscan 2 system at room temperature using a 63x objective with immersion oil. Scan speed = 15. Z-stacks were taken in 0.15 pm increments. Samples were prepared for imaging as in fluorescence microscopy. Fluorescence recovery after photobleaching. FRAP measurements were performed after particle assembly in flow cells. All fluorescence imaging was performed using a Nikon Ti2-E motorized inverted microscope controlled by NIS Elements software with a SOLA 365 LED light source, a xlOO objective lens (Oil CFI60 Plan Apochromat Lambda Series for DIC), and a Photometries Prime 95B Back-illuminated sCMOS camera. AF 488 signal was acquired using a ‘FITC’ filter set [excitation, 470 / 40 nm (450-490 nm); emission, 525 / 50 nm (500-550 nm); dichroic mirror, 495 nm]. Bleaching was conducted with a 405-nm laser at 40% power (20 mW) with a 200-ps dwell time. Recovery was monitored with a time-lapse video with 5-s intervals for 5 min. Image analysis was done in Fiji v 1.0. Intensities from bleached regions of interest were background subtracted and normalized using an unbleached particle to account for any full field of view photobleaching. The values for each particle were then normalized such that a value of 1 was set to the pre -bleach intensity and a value of 0 was set to the intensity immediately postbleaching. Data were exported, further graphed, and analyzed using GraphPad Prism 10.3.0 for Windows.

[0113] Circular dichroism. Far-UV CD spectra were obtained using J- 1500 CD spectrometer (Jasco). For all protein samples analyzed, far-UV CD spectra were obtained using a J-1500 CD spectrometer (Jasco). All measurements were taken with 200 pl of protein at 0.25 mg / ml in 20 mM KPi. Measurements were taken using a quartz cell with a path length of 0.1 cm. The spectra were acquired from 260 to 190 nm with a 0.1 -nm interval, 50 nm / min scan speed, and at 25°C.

[0114] Flow cell. Y-inlet flow cell assembly was performed as previously described. Flow cells were passivated for 1-3 h (1 mg / mL BSA in 20 mM KPi pH 6.6, 150 mM KC1) and subsequently washed (20 mM KPi [pH 6.6], 150 mM KC1). Syringe 1 was loaded with assembly trigger buffer (20 mM KPi [pH 5.8], 150 mM KC1, 0.6 mg / mL ascorbic acid), and syringe 2 was loaded with 40 uM soluble CcmK2 in loading buffer (20 mM KPi [pH 6.6], 150 mM KC1). With the flow cell on the microscope, syringes were attached to each inlet with Tfzl 1 / 16X tubing, and samples were flowed in simultaneously at 50 pL / min for 2 min. Flow was stopped, and video acquisition taken in 5-s intervals for 30 min. Image analysis was done in Fiji v 1.0, and data were exported, further graphed, and analyzed using GraphPad Prism 10.3.0 for Windows. Dynamic Light Scattering. All sizing and polydispersity measurements were carried out on a Zetasizer Nano ZSP (Malvern) in 10 mm pathlength polystyrene cuvettes (Fisher Scientific) using 173° backscattering. For time-resolved DLS, 40 uM soluble CcmK2 (20 mM KPi [pH 6.6], 150 mM KC1) was added to an equal volume of assembly trigger buffer (20 mM KPi [pH 5.8], 150 mM KC1). Measurements were acquired every 2 min, with mixing via pipetting in between to prevent particle aggregation and sedimentation. Data were exported, graphed, and analyzed using GraphPad Prism 10.3.0 for Windows.

[0115] RESULTS AND DISCUSSION

[0116] CcmK2 self-assembles into monodisperse microparticles

[0117] The P-carboxysome from the P-cyanobacterium Synechococcus elongatus (S. elongatus) is a well-established model for the study of BMC assembly (Figure 10A). The major BMC-H protein of the P-carboxysome is CcmK2, making up -70% of the shell surface area (Figure 10B).1BMC-H shell proteins have been found to form alternative superstructures, such as flat sheets and nanotubes, making them ideal candidates for the development of protein-based tools and biomaterials.2 6Several groups have shown that purified CcmK2 can assemble into flat sheets in vztro.3,5-8These previous studies were all conducted on CcmK2 with an N- or C- terminal histidine tag, but both termini face the sides of CcmK2 associated with inter-hexamer interactions.3,9His-SUMO-tagged CcmK2 was purified, and upon cleavage of the tag, Differential Interference Contrast (DIC) microscopy showed that tagless CcmK2 formed spherical and highly monodisperse microparticles (Figure 10C) at or above 5 pM CcmK2 in standard buffer conditions (20 mM KPi pH 6.2, 150 mM KC1) (Figure 10D). Particle diameter increased with increasing CcmK2 protein concentration in solution, but monodispersity was maintained at a given protein concentration (Figure 10E).

[0118] This is the first time CcmK2, or any BMC shell protein, has been shown to assemble into single-component monodisperse microparticles. There are several modes of self-assembly that typify spherical protein suprastructures, including phase separated condensates and amyloidogenic aggregates called particulates and spherulites.10Alternatively, it is possible that CcmK2 particles represent a minimized carboxysome shell that use a mode of assembly similar to that of native carboxysomes; albeit previous studies found BMC-P proteins to be required for proper shell curvature.1 1 13The assembly parameters and structural characteristics of the aforementioned suprastructurcs arc well-documented and serve as a benchmark for the characterization of CcmK2 particles. The physicochemical properties and assembly parameters of CcmK2 particles was characterized to determine their molecular nature, starting with higher- resolution imaging of their ultrastructure.

[0119] CcmK2 microparticles are composed of a dense meshwork of nanotubes

[0120] The ultrastructure of CcmK2 particles was investigated by performing Airyscan super resolution confocal microscopy on particles assembled using fluorescent-labelled CcmK2. CcmK2 was labeled with AlexaFluor488 (CcmK2AF488) and AlexaFluor647 (CcmK2AF647). The dyes were maleimide-conjugated to CcmK2[A68C], which has a cysteine residue strategically inserted in a loop region facing the luminal side of the hexamer and distant from the hexamer- and tiling-interfaces of CcmK2. CcmK2AF488and CcmK2AF64formed particles similar to unlabeled CcmK2, validating that dye labeling does not interfere with assembly. The particles were found to be decorated equally with CcmK2AF488and CcmK2AF647throughout the particle volume. The data show that CcmK2 particles are absent of a hollow interior and do not represent a minimized carboxysome shell that assembles in a manner similar to that of native carboxysomes.

[0121] To decipher the surface and core features of particles, scanning electron microscopy (SEM) was used, which confirmed the particles were highly spherical and monodisperse. There were also examples of particles that seemingly fused during assembly. CryoSEM provided higher resolution images that unveiled a fibrous network at the particle surface. Some particles cracked open during sample preparation, exposing a dense core consistent with fluorescence data. The interiors displayed amorphous densities. CryoEM was thus used to visualize particle interiors. The particles were composed of a dense cluster of nanotubes that varied greatly in length, but maintained a fixed diameter of 20 nm. 3D reconstruction of the cryo-EM tilt series indicated that the nanotubes are twisted and contain a hollow core.

[0122] Negative-stain TEM was also performed, but intact CcmK2 particles were not observed, indicating that sample preparation may have destroyed the particles. However, small clusters of CcmK2 nanotubes of fixed diameter (19 ± 2 nm) and varied lengths (64 ± 22 nm) were identified. The nanotube diameter corresponds well with the clustered nanotubes observed in cryo-EMand with previously reported BMC-H nanotubes (20 nm).5Moreover, the surface pattern of the nanotubcs is consistent with CcmK2 hexamer tiling.

[0123] These data identify CcmK2 particles as a newly reported and non-canonical assembly of a BMC-H protein. CcmK2 hexamers tessellate as single layers on the facets of native carboxysome shells in vivo. In vitro, tagged variants of CcmK2 have been shown to selfassemble into flat sheets. Tagless CcmK2 hexamers can assemble into monodisperse microparticles comprised of a dense meshwork of nanotubes. This is the first report of any BMC- H protein assembling into highly monodisperse microparticles. This finding highlights the propensity of BMC shell proteins to assemble into off-pathway supramolecular structures.

[0124] CcmK2 particles radially assemble to sizes limited by substrate depletion

[0125] Particle diameter increased with higher solution concentrations of CcmK2 (see Figure 10D-E), indicating that particle growth is likely limited by substrate depletion. To determine the mode of particle growth and to confirm that particle sizing is governed by substrate depletion, a pulse-chase experiment whereby a second round of labelled CcmK2AF647was added to preassembled particles labelled with CcmK2AF488was performed. Increasing concentrations of CcmK2AF488were pre-incubated for one hour prior to the addition of an equimolar concentration of CcmK2AF647. One hour after the addition of CcmK2AF647, all particles were composed of both fluorescent variants of CcmK2. However, the distribution was not homogenous. As shown by cross-section analysis of CcmK2 particles assembled at “5 + 5 pM” and above, the CcmK2AF647signal was broader than that of CcmK2AF488(Figure 11A). Furthermore, at “10 + 10 pM” and above, the CcmK2AF647intensity plateaued, or decreased, in the particle core where the CcmK2AP488signal peaked. The findings show that CcmK2AI 64 / was preferentially added to the particle perimeter, and did not efficiently penetrate the pre-assembled cores (Figure 11BC). The resulting CcmK2Ar647‘halo’ around the CcmK2Ar488core also indicates that soluble hexamers do not dynamically exchange over the period of the experiment. Indeed, fluorescence recovery after photobleaching (FRAP) on CcmK2AF488indicted no notable fluorescence recovery was displayed, further demonstrating that the particles are stable assemblies.

[0126] The particle diameters observed in the pulse-chase experiments were similar to those when assembled in one-step at the same final concentration of CcmK2 (Figure 11C). The finding shows that the fluorescent-labelling of CcmK2 does not alter particle assembly and also confirms that particle sizing is limited by substrate depletion of soluble CcmK2 hexamers in solution. Furthermore, the data support that the addition of CcmK2 hcxamcrs to existing particles is significantly faster than de novo particle nucleation, as we did not observed any particles consisting of CcmK2AI 64 / alone. It was thus concluded that CcmK2 particles radially assemble from a nucleation core to sizes limited by substrate depletion.

[0127] CcmK2 particle assembly is influenced by salt concentration and pH.

[0128] Along with protein concentration, salt and pH have major effects on the assembly and final properties of protein condensates, particulates, spherulites, and BMC structures.4 10The assembly and morphology of CcmK2 particles across a range of salt concentrations and pH was thus investigated. A pelleting assay was developed to quantify the proportion of CcmK2 protein assembling into particles versus remaining in solution (Figure 12A). Soluble CcmK2 was added to a specified sample condition to be tested and incubated. Assembled CcmK2 particles were then pelleted via low-speed centrifugation. The supernatant and pellet contain the soluble and particle fractions of CcmK2, respectively. SDS-PAGE analysis was then performed on the supernatant and pellet fractions. CcmK2 band intensities at a given condition were quantified to determine the proportion of CcmK2 incorporated into particles. Samples were also imaged on the microscope to verify the pelleted fraction represents CcmK2 particles and not protein aggregation.

[0129] Increasing salt concentration showed a gradual effect on particle assembly (Figure 12B). From 50 to 400 mM KC1, particles assembled to similar diameters and monodispersity (Figure 12C). From 450 to 600 mM KC1, particle sizes decreased slightly, but remained monodisperse at each given condition. Particles did not assemble at or above 600 mM KC1 (Figure 12D). Unlike salt concentration that showed only a gradual effect on particle assembly (Figure 12D), a very narrow pH range dramatically influenced particle assembly and sizing (Figure I2E). CcmK2 particles only formed at pH < 6.4 (Figure 4F). Within this pH range supporting assembly, particles increased in size with increasing pH, but monodispersity remained constant for each condition (Figure 12G). The data show that pH can be used a simple method to control CcmK2 particle assembly and sizing in vitro. Taken together, the decrease in CcmK2 particles at higher salt implicates electrostatic interactions, and the decrease in particles as pH increases indicates that positively charged residues may be important in self-assembly process. CcmK2 particles show pH-dependent reversible assembly.

[0130] After discovering the strict pH dependence on CcmK2 particle assembly, the effect of pH on pre-assembled particles was next determined. CcmK2 particles were first assembled in our standard buffer conditions (20 mM KPi pH 6.2, 150 mM KC1), and then pelleted and resuspended in buffers with increasing pH (Figure 13A). As shown above, robust particle assembly only occurs at pH < 6.4 (Figure 13B, top). When these pre-formed particles were pelleted and resuspended into buffers at pH 6.6 or pH 7, the particles displayed decreased contrast in the DIC channel, showing a loss of protein density (Figure 13B, bottom). Above pH 7, the particles completely dissolved. Circular Dichroism (CD) analysis confirmed that the secondary structure of CcmK2 hexamers before particle assembly and after particle dissolution are identical (Figure 13C). This finding is important because it shows that particle assembly is not the result of CcmK2 unfolding into non-native amyloidogenic P-sheets, which is an irreversible form of aggregation and a hallmark of spherulites and particulates. The particles scattered too much light for an interpretable CD analysis of the “in-particle” CcmK2 structure (Figure 13C).

[0131] Overall, the CD data indicates that CcmK2 particles disassemble into native hexamers upon shifting to high pH. It was therefore hypothesized that particle assembly and disassembly could be cycled by pH shifts. Indeed, when preformed CcmK2AF647particles were disassembled by increasing the pH from 6.2 to 7.2, and then spiked in HC1 to once again drop the pH, particles reassembled (Figure 13D). The reassembled particles were smaller likely because the HC1 treatment dropped the pH significantly lower than the initial assembly buffer (pH 6.2). It was also tested if salt or protein concentration could provide switch-like regulation of particle disassembly like pH. As shown in Figure 12D, high KC1 concentrations (> 500 mM) prevented de novo particle assembly (Figure 13E, top). When pre-formed particles were pelleted and resuspended into high salt buffers, the particles remained assembled with no change in size or contrast, even at IM KC1 (Figure 13E, bottom). The effect of protein concentration was also tested. As shown in Figure 10D, de novo particle assembly requires a threshold concentration of soluble CcmK2 (Figure 3F, top). A serial dilution was performed on particles assembled at 40 p M CcmK2 (Figure 13F, bottom). These preformed particles remained assembled even after a 128-fold dilution down to ~ 0.3 pM CcmK2, which is ~ 10-fold lower than the minimum concentration required for de novo particle assembly. Together, the data show that de novo assembly of CcmK2 particles is highly dependent upon protein and salt concentration as well as pH. Following assembly, particles become refractory to changes in salt or protein concentration, but remain exquisitely sensitive to changes in pH.

[0132] CcmK2 particles display high stability pH reversible assembly make CcmK2 particles ideal candidates for biotechnological applications. Therefore, CcmK2 particles were subjected to a variety of biotechnologically relevant stressors to further test their stability, using fluorescent hexamer exchange as a measure of particle destabilization. When particle assembly was initiated by mixing CcmK2AF488and CcmK2AF647together, both fluorescent variants were evenly distributed across all particles, as indicated by a high Pearson’s Correlation Coefficient (PCC) between the CcmK2AF488and CcmK2AF647signals (PCC= 0.80 ± 0.01) (Figure 14A). However, when the two dye-labelled CcmK2 variants were independently assembled into particles and then mixed post-assembly, no CcmK2 exchange was observed even after several days / weeks / months of incubation at room temperature (PCC= 0.04 ± 0.01) (Figure 14A). The data show that the separately labelled CcmK2 particles are useful tools in the study of particle stability and CcmK2 hexamer exchange after exposure to stressors.

[0133] CcmK2 particles showed no hexamer exchange or changes in particle morphology after being subjected to centrifugation, sonication, or vortexing (Figure 14A). Particles assembled at room temperature were exposed to a temperature ramp to test thermal stability. Samples were then imaged at room temperature. Particles that were either cooled to 4 °C or kept at room temperature showed no significant exchange in PCC (Figure 14B). Particles incubated at 37 °C for 30 min retained the same size and morphology, but slightly increase is hexamer exchange (PCC =0.27 ± 0.03). At > 70°C, the PCC values were equivalent to when CcmK2Ar488and CcmK2AF647hexamers were mixed prior to assembly (Figure 14B), which indicates these particles dissolved and reassembled when brought to room temperature for imaging. Together, the data show that CcmK2 particles are highly stable under a number of physical stressors, which is an appealing feature for a potential tool in synthetic biology and biotechnological applications.

[0134] Protonation-state of a single histidine residue controls CcmK2 particle assembly The key residues governing the pH-reversible assembly of CcmK2 particles were next idcntifcd. CcmK2 particles robustly assembled at pH < 6.2, which is close to the pKa of histidine (~ 6) where its imidazole side chain can gain or lose a proton when the pH is above or below the pKa, respectively. The data strongly implicate histidine (His) residues as important in CcmK2 particle self-assembly. CcmK2 contains three His residues (H75, H76, H82) (Figure 15A); all are surface exposed and near the tiling interface of the hexamer. It was therefore hypothesized that one or more His residue(s) and their protonation state controls the pH-regulated assembly of CcmK2 particles.

[0135] To test if histidine protonation drives particle assembly, all three histidines were mutated to asparagines (Asn) (CcmK23xH^N). Mutating His to Asn removes charge, but maintains polarity. CcmK23xH^Nwas unable to form particles across the pH range that supported wildtype CcmK2 particle assembly (Figure 15B-C), demonstrating that one or more histidine was required for assembly. We next tested if only one of the histidines were necessary for particle assembly by making individual His-to-Asn substitutions (CcmK2H75N, CcmK2H76N, and CcmK2H82N). Intriguingly, only CcmK2H75Ncould not form particles (Figure 15B-C), showing that H75 is the critical residue for particle assembly.

[0136] CcmK2H76Nunexpectedly formed particles more readily than wild type (Figure 15B), as quantified by the greater amount of protein in the particle pellet fraction at higher pH (Figure 15C, dashed line). CcmK2H76Nparticles were also larger than wildtype at a given pH. The data indicate that the H76N substitution improves CcmK2 particle assembly. One possibility is that when both H76 and H75 are protonated, their close proximity causes an electrostatic repulsion that destabilizes particle assembly. Alternatively, there could be competition between H75 and H76 to form the required electrostatic associations, with only H75 interactions driving particle assembly. Particles formed with CcmK2H82Nwere not significantly different from wildtype, showing H82 does not play a significant role in assembly (Figure 15B-C).

[0137] Importantly, all His-to-Asn mutants of CcmK2 remained hexameric when soluble. Given that mutating H75 and H76 had effects on particle assembly, while mutating H82 did not, the data strongly implicates the convex, core-facing side of the CcmK2 hexamer as a key interaction surface for particle assembly (see Figure 15A). It is contemplated that H75 and H76, and surrounding residues, are potential targets for modulating particle assembly and morphology for future tool development. CcmK2 particles assembly kinetics

[0138] Assembly kinetics of CcmK2 particles were determined using Dynamic Light Scattering (DLS). The insoluble proportion of CcmK2 over time with increasing pH was quantitated. It was found that CcmK2 assembly at pH 5.8 and 6.0 was almost complete in under 5 minutes, while the majority of CcmK2 at pH > 6.2 remained soluble. By two hours, the proportion of insoluble CcmK2 at pH 6.2 plateaued around 0.80. By six hours, the proportion of insoluble CcmK2 at pH 6.4 plateaued around 0.60. CcmK2 remained soluble at pH > 6.6, even after 20 h.

[0139] These initial experiments showed that the kinetics of assembly may be slow enough to use DLS to monitor potential pathway intermediates and their sizes. At pH 7.2, CcmK2 existed as a single species around 6 nm, which agrees with the solved crystal structure of CcmK2 (diameter of -7 nm).14At pH 6.0, on the other hand, there was a single population at -600 nm in diameter, consistent with the sizing of CcmK2 particles as quantified from our DIC microscopy. Time- resolved DLS was used to track both the hexamer and particle populations of CcmK2 during assembly. Soluble CcmK2 at pH 6.6 was rapidly mixed into a cuvette containing assembly triggering buffer with a pH of 5.8 (Figure 16A). At early time points, two peaks were detected in the population- wide intensity; one at a diameter of ~7 nm, corresponding to soluble CcmK2 hexamers, and the other at 100-400 nm, which represented higher-order structures of CcmK2 (Figure 16B). Overtime, the CcmK2 hexamer population decreased in intensity and eventually disappeared, while the larger diameter peak increased in intensity and shifted further towards even larger diameters. Within 30 seconds, almost 20% of the CcmK2 hexamer population was already incorporated into higher order structures (Figure 16C). The data show CcmK2 particles gradually increasing in size over time, consistent with our microscopy data showing radial assembly.

[0140] The peak corresponding to the larger population continued to shift to a larger diameter after the peak corresponding to CcmK2 hexamers disappeared. The data support that CcmK2 hexamers form an intermediate structure first, and then those intermediate structures continue to assemble into the final particles. From the high-resolution imaging, these intermediate structures are likely CcmK2 nanotubes. There are no peaks corresponding to an intermediate size, but it is possible that tubes arc too elongated to have a radius of gyration faithfully measured by DLS. A microfluidics approach using a Y-inlet flow cell was used to visualize the de novo assembly of CcmK2 particles (Figure 16D). Soluble CcmK2AF488was flowed at pH 6.6 in one inlet, and an assembly-triggering buffer with a pH of 5.8 in the second inlet. The fast flow from both inlets created a laminar boundary that prevented mixing. Once the samples entered the flow cell, the flow was stopped, which breaks the laminar boundary and causes the two samples to mix. As the samples mixed, the pH equilibrated to ~6.2, which initiated particle assembly. Fluorescence microscopy was used to visualize assembly in real time (Figure 16E).

[0141] Assembly as an increase in focus size and increase in the total fluorescence intensity of the region of interest (ROI) equivalent to the final foci size overtime was tracked (Figure 16F). A non-linear increase in fluorescence intensity overtime was observed. There was an initial lag in the first few minutes of assembly, followed by a period of fast growth before the rate of fluorescence intensity decreased and plateaued. Figure 17 shows an exemplary mechanism of assembly of CcmK2 particles.

[0142] Example 8 references

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[0165] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

CLAIMSWc claim:

1. A single-component synthetic bacterial microcompartment shell comprising: a single shell protein subunit, CcmK2.

2. The composition of claim 1, further comprising an agent encapsulated in said singlecomponent synthetic bacterial microcompartment shell.

3. The composition of claim 2, wherein said agent is a nanomaterial.

4. The composition of claim 3, wherein said nanomaterial is an organic molecule.

5. The composition of claim 3, wherein said nanomaterial is a protein.

6. The composition of claim 3, wherein said nanomaterial is an inorganic molecule.

7. The composition of claim 3, wherein said nanomaterial is a hybrid compound molecule.

8. The composition of claim 5, wherein said protein is an enzyme.

9. The composition of claim 8, wherein said enzyme is ribulose-l,5-bisphosphate carboxylase / oxygenase (RuBisCo).

10. The composition of any of claims 1-9, wherein said single-component synthetic bacterial microcompartment shell has a diameter of 90 to 600 nm.

11. A composition comprising the single-component synthetic bacterial microcompartment of any of claims 1-10.

12. The composition of claim 11, wherein said single-component synthetic bacterial microcompartment shell is in a solution, in vitro.

13. The composition of claim 11, wherein said single-component synthetic bacterial microcompartment shell is in a cell.

14. The composition of claim 13, wherein said cell is a bacterial cell.

15. The composition of claim 1 , wherein said bacterial cell is a gram- negative bacterium.

16. The composition of claim 15, wherein said gram-negative bacterium is Escherichia coli (E. coli).

17. The composition of claim 15, wherein said gram-negative bacterium is a cyanobacteria.

18. The composition of claim 13, wherein said cell is a plant cell.

19. The composition of claim 13, wherein said cell is an animal cell.

20. The composition of claim 11, wherein said single-component synthetic bacterial microcompartment shell is in an organism.

21. The composition of claim 20, wherein said organism is a plant.

22. The composition of claim 20, wherein said organism is an animal.

23. The composition of claim 22, wherein said animal is a mammal.

24. The composition of claim 23, wherein said mammal is a human.

25. The composition of claim 20, wherein said organism is obtained from a lake, stream, river or any combination thereof.

26. A system for expressing components of the single-component synthetic bacterial microcompartment shell of claim 1, comprising an expression vector encoding CcmK2.

27. The system of claim 26, wherein said expression vector further encodes at least one purification tag operably coupled to CcmK2.

28. A composition comprising: the purified CcmK2 protein in a storage buffer.

29. The composition of claim 28, wherein the storage buffer contains at least one stabilization agent.30.The composition of claim 29, wherein the at least one stabilization agent comprises Tris-HCl and DTT.

31. The composition of claim 28, wherein the storage buffer contains at least one neutralizing agent.

32. The composition of claim 31, wherein the at least one neutralizing agent comprises KC1.

33. The composition of claim 28, wherein the storage buffer contains at least one preserving agent.

34. The composition of claim 33, wherein at least one preserving agent comprises glycerol.

35. A method of producing the single-component microscale shell comprising, assembling the single-component synthetic bacterial microcompartment shell of any of claims 1-24 with CcmK2.

36. A method of fixing carbon by encapsulation of RuBisCo in the single-component microscale shell of any of the claims 1-24.

37. Use of the single-component synthetic bacterial microcompartment shell of any of the claims 1-24.

38. Use of the single-component synthetic bacterial microcompartment shell of any of claims 1- 24 for encapsulating a biological reaction.

39. The use of claim 38, wherein the biological reaction is an enzymatic reaction.

40. The use of claim 39, wherein enzymatic reaction is a carbon fixing enzymatic reaction.

41. A method for assembling a single-component synthetic bacterial microcompartment shell in vitro comprising the steps of: diluting the composition of any of claims 27-34 into an assembly buffer.

42. The method of claim 41, wherein the assembly buffer comprises at least one neutralizing agent.

43. The method of claim 42, wherein at least one neutralizing agent comprises KPi and KC1.

44. The method of any of claims 41-43, further comprising the step of incubating for a period of at least 60 minutes.

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