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Enzyme nanoreactors are nanoscale compartments consisting of encapsulated enzymes and a selectively permeable barrier. Sequestration and colocalization of enzymes can increase catalytic activity, stability, and longevity, highly desirable features for many biotechnological and biomedical applications of enzyme catalysts. One promising strategy to construct enzyme nanoreactors is to repurpose protein nanocages found in nature. However, protein-based enzyme nanoreactors often exhibit decreased catalytic activity, partially caused by a mismatch of protein shell selectivity and the substrate requirements of encapsulated enzymes. No broadly applicable and modular protein-based nanoreactor platform is currently available. Here, we introduce a pore-engineered universal enzyme nanoreactor platform based on encapsulins-microbial self-assembling protein nanocompartments with programmable and selective enzyme packaging capabilities. We structurally characterize our protein shell designs via cryo-electron microscopy and highlight their polymorphic nature. Through fluorescence polarization assays, we show their improved molecular flux behavior and highlight their expanded substrate range via a number of proof-of-concept enzyme nanoreactor designs. This work lays the foundation for utilizing our encapsulin-based nanoreactor platform for diverse future biotechnological and biomedical applications.

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Prokaryotes organize intracellular compartments with protein-based organelles called encapsulins. Encapsulins with icosahedral symmetry can encapsulate specific cargo proteins mediated by targeting peptides or encapsulation-mediating domains. Encapsulins have been used in eukaryotic cells for bioengineering, vaccine development, and nanoparticle alignment. Their versatility makes them attractive for research; however, detailed structural information on encapsulins is crucial for further applied research. However, cargo proteins are randomly oriented inside the icosahedral encapsulins. The random orientation of cargo proteins presents a challenge for structural analysis that relies on averaging processes such as x-ray crystallography and cryo-electron microscopy (cryo-EM) single-particle imaging. Therefore, we aimed to accurately estimate the secondary structure content and elucidate the structure of cargo proteins inside the particle by measuring the circular dichroism (CD) spectra using vacuum ultraviolet circular dichroism (VUVCD) spectroscopy. Thus, the structure of the cargo protein inside encapsulin was evaluated. This approach could potentially set a standard for evaluating cargo proteins inside particles in future applied research on encapsulins.

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Rhodanese-like domains (RLDs) represent a widespread protein family canonically involved in sulfur transfer reactions between diverse donor and acceptor molecules. RLDs mediate these transsulfuration reactions via a transient persulfide intermediate, created by modifying a conserved cysteine residue in their active sites. RLDs are involved in various aspects of sulfur metabolism, including sulfide oxidation in mitochondria, iron-sulfur cluster biogenesis, and thio-cofactor biosynthesis. However, due to the inherent complexity of sulfur metabolism caused by the intrinsically high nucleophilicity and redox sensitivity of thiol-containing compounds, the physiological functions of many RLDs remain to be explored. Here, we focus on a single domain Acinetobacter baumannii RLD (Ab-RLD) associated with a desulfurase encapsulin which is able to store substantial amounts of sulfur inside its protein shell. We determine the 1.6 Å x-ray crystal structure of Ab-RLD, highlighting a homodimeric structure with a number of unusual features. We show through kinetic analysis that Ab-RLD exhibits thiosulfate sulfurtransferase activity with both cyanide and glutathione acceptors. Using native mass spectrometry and in vitro assays, we provide evidence that Ab-RLD can stably carry a persulfide and thiosulfate modification and may employ a ternary catalytic mechanism. Our results will inform future studies aimed at investigating the functional link between Ab-RLD and the desulfurase encapsulin.

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Protein nanoparticles are effective platforms for antigen presentation and targeting effector immune cells in vaccine development. Encapsulins are a class of protein-based microbial nanocompartments that self-assemble into icosahedral structures with external diameters ranging from 24 to 42 nm. Encapsulins from Myxococcus xanthus were designed to package bacterial RNA when produced in E. coli and were shown to have immunogenic and self-adjuvanting properties enhanced by this RNA. We genetically incorporated a 20-mer peptide derived from a mutant strain of the SARS-CoV-2 receptor binding domain (RBD) into the encapsulin protomeric coat protein for presentation on the exterior surface of the particle, inducing the formation of several nonicosahedral structures that were characterized by cryogenic electron microscopy. This immunogen elicited conformationally relevant humoral responses to the SARS-CoV-2 RBD. Immunological recognition was enhanced when the same peptide was presented in a heterologous prime/boost vaccination strategy using the engineered encapsulin and a previously reported variant of the PP7 virus-like particle, leading to the development of a selective antibody response against a SARS-CoV-2 RBD point mutant. While generating epitope-focused antibody responses is an interplay between inherent vaccine properties and B/T cells, here we demonstrate the use of orthogonal nanoparticles to fine-tune the control of epitope focusing.

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Vaccines are one of the most effective medical interventions, playing a pivotal role in treating infectious diseases. Although traditional vaccines comprise killed, inactivated, or live-attenuated pathogens that have resulted in protective immune responses, the negative consequences of their administration have been well appreciated. Modern vaccines have evolved to contain purified antigenic subunits, epitopes, or antigen-encoding mRNAs, rendering them relatively safe. However, reduced humoral and cellular responses pose major challenges to these subunit vaccines. Protein nanoparticle (PNP)-based vaccines have garnered substantial interest in recent years for their ability to present a repetitive array of antigens for improving immunogenicity and enhancing protective responses. Discovery and characterisation of naturally occurring PNPs from various living organisms such as bacteria, archaea, viruses, insects, and eukaryotes, as well as computationally designed structures and approaches to link antigens to the PNPs, have paved the way for unprecedented advances in the field of vaccine technology. In this review, we focus on some of the widely used naturally occurring and optimally designed PNPs for their suitability as promising vaccine platforms for displaying native-like antigens from human viral pathogens for protective immune responses. Such platforms hold great promise in combating emerging and re-emerging infectious viral diseases and enhancing vaccine efficacy and safety.

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Encapsulins are self-assembling nano-compartments that naturally occur in bacteria and archaea. These nano-compartments encapsulate cargo proteins that bind to the shell's interior through specific recognition sequences and perform various metabolic processes. Encapsulation enables organisms to perform chemical reactions without exposing the rest of the cell to potentially harmful substances while shielding cargo molecules from degradation and other adverse effects of the surrounding environment. One particular type of cargo protein, the ferritin-like protein (FLP), is the focus of this review. Encapsulated FLPs are members of the ferritin-like protein superfamily, and they play a crucial role in converting ferrous iron (Fe+2) to ferric iron (Fe+3), which is then stored inside the encapsulin in mineralized form. As such, FLPs regulate iron homeostasis and protect organisms against oxidative stress. Recent studies have demonstrated that FLPs have tremendous potential as biosensors and bioreactors because of their ability to catalyze the oxidation of ferrous iron with high specificity and efficiency. Moreover, they have been investigated as potential targets for therapeutic intervention in cancer drug development and bacterial pathogenesis. Further research will likely lead to new insights and applications for these remarkable proteins in biomedicine and biotechnology.

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Protein compartments offer definitive structures with a large potential design space that are of particular interest for green chemistry and therapeutic applications. One family of protein compartments, encapsulins, are simple prokaryotic nanocompartments that self-assemble from a single monomer into selectively permeable cages of between 18 and 42 nm. Over the past decade, encapsulins have been developed for a diverse application portfolio utilizing their defined cargo loading mechanisms and repetitive surface display. Although it has been demonstrated that encapsulation of non-native cargo proteins provides protection from protease activity, the thermal effects arising from enclosing cargo within encapsulins remain poorly understood. This study aimed to establish a methodology for loading a reporter protein into thermostable encapsulins to determine the resulting stability change of the cargo. Building on previous in vitro reassembly studies, we first investigated the effectiveness of in vitro reassembly and cargo-loading of two size classes of encapsulins Thermotoga maritima T = 1 and Myxococcus xanthus T = 3, using superfolder Green Fluorescent Protein. We show that the empty T. maritima capsid reassembles with higher yield than the M. xanthus capsid and that in vitro loading promotes the formation of the M. xanthus T = 3 capsid form over the T = 1 form, while overloading with cargo results in malformed T. maritima T = 1 encapsulins. For the stability study, a Förster resonance energy transfer (FRET)-probed industrially relevant enzyme cargo, transketolase, was then loaded into the T. maritima encapsulin. Our results show that site-specific orthogonal FRET labels can reveal changes in thermal unfolding of encapsulated cargo, suggesting that in vitro loading of transketolase into the T. maritima T = 1 encapsulin shell increases the thermal stability of the enzyme. This work supports the move toward fully harnessing structural, spatial, and functional control of in vitro assembled encapsulins with applications in cargo stabilization.

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Protein capsids are a widespread form of compartmentalization in nature. Icosahedral symmetry is ubiquitous in capsids derived from spherical viruses, as this geometry maximizes the internal volume that can be enclosed within. Despite the strong preference for icosahedral symmetry, we show that simple point mutations in a virus-like capsid can drive the assembly of unique symmetry-reduced structures. Starting with the encapsulin from Myxococcus xanthus, a 180-mer bacterial capsid that adopts the well-studied viral HK97 fold, we use mass photometry and native charge detection mass spectrometry to identify a triple histidine point mutant that forms smaller dimorphic assemblies. Using cryoelectron microscopy, we determine the structures of a precedented 60-mer icosahedral assembly and an unexpected 36-mer tetrahedron that features significant geometric rearrangements around a new interaction surface between capsid protomers. We subsequently find that the tetrahedral assembly can be generated by triple-point mutation to various amino acids and that even a single histidine point mutation is sufficient to form tetrahedra. These findings represent a unique example of tetrahedral geometry when surveying all characterized encapsulins, HK97-like capsids, or indeed any virus-derived capsids reported in the Protein Data Bank, revealing the surprising plasticity of capsid self-assembly that can be accessed through minimal changes in the protein sequence.

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Encapsulins are protein nanocompartments that regulate cellular metabolism in several bacteria and archaea. Myxococcus xanthus encapsulins protect the bacterial cells against oxidative stress by sequestering cytosolic iron. These encapsulins are formed by the shell protein EncA and three cargo proteins: EncB, EncC, and EncD. EncB and EncC form rotationally symmetric decamers with ferroxidase centers (FOCs) that oxidize Fe+2 to Fe+3 for iron storage in mineral form. However, the structure and function of the third cargo protein, EncD, have yet to be determined. Here, we report the x-ray crystal structure of EncD in complex with flavin mononucleotide. EncD forms an α-helical hairpin arranged as an antiparallel dimer, but unlike other flavin-binding proteins, it has no ß-sheet, showing that EncD and its homologs represent a unique class of bacterial flavin-binding proteins. The cryo-EM structure of EncA-EncD encapsulins confirms that EncD binds to the interior of the EncA shell via its C-terminal targeting peptide. With only 100 amino acids, the EncD α-helical dimer forms the smallest flavin-binding domain observed to date. Unlike EncB and EncC, EncD lacks a FOC, and our biochemical results show that EncD instead is a NAD(P)H-dependent ferric reductase, indicating that the M. xanthus encapsulins act as an integrated system for iron homeostasis. Overall, this work contributes to our understanding of bacterial metabolism and could lead to the development of technologies for iron biomineralization and the production of iron-containing materials for the treatment of various diseases associated with oxidative stress.

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Foot-and-mouth disease (FMD) is an acute zoonosis causes significant economic losses. Vaccines able to stimulate efficient protective immune responses are urgently needed. In this study, Escherichia coli-derived recombinant VP1 of serotype A and O FMD virus (FMDV) was conjugated to thermostable scaffold lumazine synthase (LS) or Quasibacillus thermotolerans encapsulin (QtEnc) using a robust plug-and-display SpyTag/SpyCatcher system to generate multimeric nanovaccines. These nanovaccines induced highly potent antibody responses in vaccinated mice. On day 14 after the first immunisation, antibody titres were approximately 100 times higher than those of monomer antigens. Both vaccines induced high and long-term IgG antibody production. Moreover, the QtEnc-VP1 nanovaccine induced higher antibody titres than the LS-VP1 nanovaccine. The nanovaccines also induced Th1-biased immune responses and higher levels of neutralising antibodies. These data indicated that FMDV nanovaccines generated by conjugating VP1 with a thermostable scaffold are highly immunogenic and ideal candidates for FMDV control in low-resource areas.

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The three most important commercial bacterial insecticides are all derived from subspecies of Bacillus thuringiensis (Bt). Specifically, Bt subsp. kurstaki (Btk) and Bt subsp. aizawai (Bta) are used to control larval lepidopteran pests. The third, Bt subsp. israelensis (Bti), is primarily used to control mosquito and blackfly larvae. All three subspecies produce a parasporal body (PB) during sporulation. The PB is composed of insecticidal proteins that damage the midgut epithelium, initiating a complex process that results in the death of the insect. Among these three subspecies of Bt, Bti is unique as it produces the most complex PB consisting of three compartments. Each compartment is bound by a multilaminar fibrous matrix (MFM). Two compartments contain one protein each, Cry11Aa1 and Cyt1Aa1, while the third contains two, Cry4Aa1/Cry4Ba1. Each compartment is packaged independently before coalescing into the mature spherical PB held together by additional layers of the MFM. This distinctive packaging process is unparalleled among known bacterial organelles, although the underlying molecular biology is yet to be determined. Here, we present structural and molecular evidence that the MFM has a hexagonal pattern to which Bti proteins Bt152 and Bt075 bind. Bt152 binds to a defined spot on the MFM during the development of each compartment, yet its function remains unknown. Bt075 appears to be derived from a bacteriophage major capsid protein (MCP), and though its sequence has markedly diverged, it shares striking 3-D structural similarity to the Escherichia coli phage HK97 Head 1 capsid protein. Both proteins are encoded on Bti's pBtoxis plasmid. Additionally, we have also identified a six-amino acid motif that appears to be part of a novel molecular process responsible for targeting the Cry and Cyt proteins to their cytoplasmic compartments. This paper describes several previously unknown features of the Bti organelle, representing a first step to understanding the biology of a unique process of sorting and packaging of proteins into PBs. The insights from this research suggest a potential for future applications in nanotechnology.

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Encapsulins are self-assembling protein nanocompartments able to selectively encapsulate dedicated cargo enzymes. Encapsulins are widespread across bacterial and archaeal phyla and are involved in oxidative stress resistance, iron storage, and sulfur metabolism. Encapsulin shells exhibit icosahedral geometry and consist of 60, 180, or 240 identical protein subunits. Cargo encapsulation is mediated by the specific interaction of targeting peptides or domains, found in all cargo proteins, with the interior surface of the encapsulin shell during shell self-assembly. Here, we report the 2.53 Å cryo-EM structure of a heterologously produced and highly cargo-loaded T3 encapsulin shell from Myxococcus xanthus and explore the systems' structural heterogeneity. We find that exceedingly high cargo loading results in the formation of substantial amounts of distorted and aberrant shells, likely caused by a combination of unfavorable steric clashes of cargo proteins and shell conformational changes. Based on our cryo-EM structure, we determine and analyze the targeting peptide-shell binding mode. We find that both ionic and hydrophobic interactions mediate targeting peptide binding. Our results will guide future attempts at rationally engineering encapsulins for biomedical and biotechnological applications.

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Phloroglucinol is an important chemical intermediate which has been tentatively produced by engineered bacteria. However, its biosynthesis in industry is limited due to its natural antibacterial activity. Our study firstly selected Yarrowia lipolytica as the chassis strain, which was verified to be tolerable to phloroglucinol. Then the gene of type III polyketone synthase PhlD (the key biosynthetic gene) was overexpressed to facilitate phloroglucinol production with a concentration of 107.4 mg/L. Furthermore, we introduced the prokaryotic nanocompartment to assist the intracellular catalytic activity. The results showed that the concentration of phloroglucinol was increased by about 2.5 times, indicating this multifunctional nanocompartment is orthogonal to the physiological activities of Y. lipolytica. Additionally, fermentations with xylose and lignocellulosic hydrolysates as the carbon source were performed with the engineered Y. lipolytica, resulting in a total concentration of 580.2 mg/L and 328.9 mg/L, respectively. These findings revealed the potential of Y. lipolytica in phloroglucinol production and provided an effective nanocompartment strategy to improve the catalytic activity of the enzyme for boosting phloroglucinol production. KEY POINTS: • The first time to select and use Y. lipolytica to produce phloroglucinol. • Successful construction of prokaryotic nanocompartment in Y. lipolytica to increase production of phloroglucinol. • Lignocellulose hydrolysate is used as a substrate in fermentation.

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Encapsulins are a class of protein nanocages that are found in bacteria, which are easy to produce and engineer in E. coli expression systems. The encapsulin from Thermotoga maritima (Tm) is well studied, its structure is available, and without modification it is barely taken up by cells, making it promising candidates for targeted drug delivery. In recent years, encapsulins are engineered and studied for potential use as drug delivery carriers, imaging agents, and as nanoreactors. Consequently, it is important to be able to modify the surface of these encapsulins, for example, by inserting a peptide sequence for targeting or other functions. Ideally, this is combined with high production yields and straightforward purification methods. In this chapter, we describe a method to genetically modify the surface of Tm and Brevibacterium linens (Bl) encapsulins, as model systems, to purify them and characterize the obtain nanocages.

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Rapid emergence of the SARS-CoV-2 variants has dampened the protective efficacy of existing authorized vaccines. Nanoparticle platforms offer a means to improve vaccine immunogenicity by presenting multiple copies of desired antigens in a repetitive manner which closely mimics natural infection. We have applied nanoparticle display combined with the SpyTag-SpyCatcher system to design encapsulin-mRBD, a nanoparticle vaccine displaying 180 copies of the monomeric SARS-CoV-2 spike receptor-binding domain (RBD). Here we show that encapsulin-mRBD is strongly antigenic and thermotolerant for long durations. After two immunizations, squalene-in-water emulsion (SWE)-adjuvanted encapsulin-mRBD in mice induces potent and comparable neutralizing antibody titers of 105 against wild-type (B.1), alpha, beta, and delta variants of concern. Sera also neutralizes the recent Omicron with appreciable neutralization titers, and significant neutralization is observed even after a single immunization.

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Charge transport across proteins can be surprisingly efficient over long distances-so-called long-range tunneling-but it is still unclear as to why and under which conditions (e.g., presence of co-factors, type of cargo) the long-range tunneling regime can be accessed. This paper describes molecular tunneling junctions based on an encapsulin (Enc), which is a large protein cage with a diameter of 24 nm that can be loaded with various types of (small) proteins, also referred to as "cargo". We demonstrate with dynamic light scattering, transmission electron microscopy, and atomic force microscopy that Enc, with and without cargo, can be made stable in solution and immobilized on metal electrodes without aggregation. We investigated the electronic properties of Enc in EGaIn-based tunnel junctions (EGaIn = eutectic alloy of Ga and In that is widely used to contact (bio)molecular monolayers) by measuring the current density for a large range of applied bias of ±2.5 V. The encapsulated cargo has an important effect on the electrical properties of the junctions. The measured current densities are higher for junctions with Enc loaded with redox-active cargo (ferritin-like protein) than those junctions without cargo or redox-inactive cargo (green fluorescent protein). These findings open the door to charge transport studies across complex biomolecular hierarchical structures.

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Targeted nanoparticles of different origins are considered as new-generation diagnostic and therapeutic tools. However, there are no targeted drug formulations within the composition of nanoparticles approved by the FDA for use in the clinic, which is associated with the insufficient effectiveness of the developed candidates, the difficulties of their biotechnological production, and inadequate batch-to-batch reproducibility. Targeted protein self-assembling nanoparticles circumvent this problem since proteins are encoded in DNA and the final protein product is produced in only one possible way. We believe that the combination of the endless biomedical potential of protein carriers as nanoparticles and the standardized protein purification protocols will make significant progress in "magic bullet" creation possible, bringing modern biomedicine to a new level. In this review, we are focused on the currently existing platforms for targeted self-assembling protein nanoparticles based on transferrin, lactoferrin, casein, lumazine synthase, albumin, ferritin, and encapsulin proteins, as well as on proteins from magnetosomes and virus-like particles. The applications of these self-assembling proteins for targeted delivery in vitro and in vivo are thoroughly discussed, including bioimaging applications and different therapeutic approaches, such as chemotherapy, gene delivery, and photodynamic and photothermal therapy. A critical assessment of these protein platforms' efficacy in biomedicine is provided and possible problems associated with their further development are described.

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The receptor-binding domain (RBD) of the SARS-CoV-2 spike is a primary target of neutralizing antibodies and a key component of licensed vaccines. Substantial mutations in RBD, however, enable current variants to escape immunogenicity generated by vaccination with the ancestral (WA1) strain. Here, we produce and assess self-assembling nanoparticles displaying RBDs from WA1 and BA.5 strains by using the SpyTag:SpyCatcher system for coupling. We observed both WA1- and BA.5-RBD nanoparticles to degrade substantially after a few days at 37 °C. Incorporation of nine RBD-stabilizing mutations, however, increased yield ~five-fold and stability such that more than 50% of either the WA1- or BA.5-RBD nanoparticle was retained after one week at 37 °C. Murine immunizations revealed that the stabilized RBD-nanoparticles induced ~100-fold higher autologous neutralization titers than the prefusion-stabilized (S2P) spike at a 2 µg dose. Even at a 25-fold lower dose where S2P-induced neutralization titers were below the detection limit, the stabilized BA.5-RBD nanoparticle induced homologous titers of 12,795 ID50 and heterologous titers against WA1 of 1767 ID50. Assessment against a panel of ß-coronavirus variants revealed both the stabilized BA.5-RBD nanoparticle and the stabilized WA1-BA.5-(mosaic)-RBD nanoparticle to elicit much higher neutralization breadth than the stabilized WA1-RBD nanoparticle. The extraordinary titer and high neutralization breadth elicited by stabilized RBD nanoparticles from strain BA.5 make them strong candidates for next-generation COVID-19 vaccines.

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Encapsulins, virus capsid-like bacterial nanocompartments have emerged as promising tools in medicine, imaging, and material sciences. Recent work has shown that these protein-bound icosahedral 'organelles' possess distinct properties that make them exceptionally usable for nanotechnology applications. A key factor contributing to their appeal is their ability to self-assemble, coupled with their capacity to encapsulate a wide range of cargos. Their genetic manipulability, stability, biocompatibility, and nano-size further enhance their utility, offering outstanding possibilities for practical biotechnology applications. In particular, their amenability to engineering has led to their extensive modification, including the packaging of non-native cargos and the utilization of the shell surface for displaying immunogenic or targeting proteins and peptides. This inherent versatility, combined with the ease of expressing encapsulins in heterologous hosts, promises to provide broad usability. Although mostly not yet commercialized, encapsulins have started to demonstrate their vast potential for biotechnology, from drug delivery to biofuel production and the synthesis of valuable inorganic materials. In this review, we will initially discuss the structure, function and diversity of encapsulins, which form the basis for these emerging applications, before reviewing ongoing practical uses and highlighting promising applications in medicine, engineering and environmental sciences.

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Protein nanocages have emerged as an important engineering platform for biotechnological and biomedical applications. Among naturally occurring protein cages, encapsulin nanocompartments have recently gained prominence due to their favorable physico-chemical properties, ease of shell modification, and highly efficient and selective intrinsic protein packaging capabilities. Here, we expand encapsulin function by designing and characterizing encapsulins for concurrent RNA and protein encapsulation in vivo. Our strategy is based on modifying encapsulin shells with nucleic acid-binding peptides without disrupting the native protein packaging mechanism. We show that our engineered encapsulins reliably self-assemble in vivo, are capable of efficient size-selective in vivo RNA packaging, can simultaneously load multiple functional RNAs, and can be used for concurrent in vivo packaging of RNA and protein. Our engineered encapsulation platform has potential for codelivery of therapeutic RNAs and proteins to elicit synergistic effects and as a modular tool for other biotechnological applications.

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