Biology subjects

Tullman-Ercek, D.

Publications and source records attributed to Tullman-Ercek, D..

At least 19 recordsLinked to original sources

bioRxiv2026

Glycerol alone effects 1,3-propanediol production via the aerobic propanediol utilization pathway in Salmonella enterica

Crude glycerol is an underutilized waste stream. Viable routes for converting it to 1,3-propanediol (1,3-PDO) can conserve important resources and add value to its supply chain. Biological methods are appealing because they can circumvent expensive preprocessing steps while operating under mild conditions. Here, we show that the propanediol utilization pathway of Salmonella enterica serovar Typhimurium LT2 can be used to convert glycerol, including unprocessed crude glycerol, into 1,3-PDO under aerobic conditions in minimal media. Additionally, we demonstrate that high concentrations of expensive cofactors are not necessary to achieve optimal production titers. This study lays the groundwork for continual iteration on this pathway for bioprocess development. Key pointsO_LIS. enterica can produce 1,3-propanediol from crude glycerol alone C_LIO_LIGlycerol-to-1,3-propanediol conversion is dependent on expression of the propanediol utilization (Pdu) pathway C_LIO_LISub-saturating concentrations of exogenous vitamin B12 can boost cell growth and 1,3-propanediol yield C_LI

microbiology↗

bioRxiv2026

Engineering the Self-assembly of Bacterial Microcompartment Shell Proteins via Charged Mutations

AbstractProtein self-assembly is a fundamental biological process of great importance for the design and synthesis of biomaterials. Developing the ability to precisely manipulate protein assembly would greatly expand both our understanding of the process and our biotechnological capabilities. Within bacteria, proteins that self-organize to form bacterial microcompartments (MCPs) offer an excellent model system for studying protein self-assembly and advancing biomaterial design capabilities. MCPs consist of irregular polyhedral shells that encase an enzyme core, functioning as enzymatic nanoreactors. In isolation, the abundant shell proteins of the 1,2-propanediol utilization (Pdu) MCP, PduA and PduJ, have a high propensity to self-assemble into tubular structures, analogous in form to carbon nanotubes. Here, we design and characterize tubular structures formed by hexameric PduA and PduJ proteins. We demonstrate that altering hexamer charge offers a systematic strategy for modulating the higherorder assembly of PduA and PduJ across multiple contexts by integrating molecular dynamics simulations with heterologous overexpression, cell-free, and in vivo Salmonella enterica serovar Typhimurium LT2 experiments. First, using molecular simulation, we find that tube chirality and radius play critical roles in determining structural stability and flexibility. Next, overexpression and cell-free experiments show that increasing the overall negative charge of assembling subunits consistently promotes self-assembly into tubular structures. We find that this holds true in the native MCP system, as these same mutations promote the formation of tubular MCP structures in S. enterica LT2. Our results collectively reveal that both electrostatic interactions and fields generated by charges on proteins can be leveraged to control protein-based nanostructures.

biochemistry↗

bioRxiv2025

Determinants of Protein-Level Parameters Governing MS2 VLP Reassembly

MS2 virus-like particles (VLPs) are widely used as protein nanocages for cargo encapsulation, yet in vitro disassembly-reassembly protocols remain poorly standardized, and reassembly yields are reported inconsistently. As a result, the same experiments reported in literature produce widely divergent yields, limiting reproducibility and cross-study comparability. Here, we introduce a cargo-specific, quantitative framework for standardized MS2 VLP reassembly yield determination. We evaluate commonly used disassembly and post-disassembly processing methods and identify practical trade-offs between protein recovery, accessibility, and reproducibility. Reassembly yield is quantified using size exclusion chromatography calibrated against purified VLP standards, enabling robust, cargo-specific yield measurement. Using this framework, we apply a full factorial design of experiments to quantify the individual and combined effects of coat protein concentration, ionic strength, buffer pH, and molecular crowding on reassembly yield. The resulting statistical model explains more than 99% of the explainable variance and its linear fit to the experimental data indicates that optimal reassembly conditions extend beyond those tested to date. Protein concentration and ionic strength dominate reassembly yield, whereas pH and osmolyte concentration contribute more modestly within the tested ranges. Finally, we propose practical guidelines for standardized MS2 VLP disassembly, reassembly, and yield reporting, defining a transferable operating envelope for MS2 VLP reconstruction. While demonstrated here using a single nucleic acid cargo (tr-DNA), the framework is readily extensible to alternative cargos and coat protein variants.

biophysics↗

bioRxiv2025

A high throughput flow cytometry assay for quantifying type 3 secretion system assembly in Salmonella

Recombinant protein production in bacteria is limited by costly cell lysis and multi-step purification. To bypass these limitations, leveraging native bacterial secretion systems to secrete proteins offers a promising alternative. The type 3 secretion system (T3SS) of Salmonella enterica serovar Typhimurium can secrete proteins at hundreds of milligrams per liter. While promising, higher titers from this and other natural systems are needed to be relevant commercially. A major engineering target to enhance protein secretion is the T3SS secretion apparatus. However, efforts to quantify its assembly are hindered by bottlenecks in scalability and complexity. Assembly involves [~]20 structural proteins forming with precise stoichiometry under dynamic regulation. Membrane- and periplasmic-embedded components are difficult to probe without costly, time-intensive methods. To fully realize the T3SS as a tool for scalable protein production, new tools are needed for rapid and accurate characterization of the assembled apparatus. Here, we establish a high throughput flow cytometry method in S. Typhimurium by quantifying the abundance of the needle-bound tip protein SipD as a proxy for T3SS apparatus assembly. We then adapted a super resolution microscopy method, known as Structured Illumination Microscopy (SR-SIM), to visualize the presence of SipD and validate the flow cytometry results. Applying this approach, we revealed how overexpression of key T3SS regulators hilA and hilD impact assembly, expanded the assay with a secretion-compatible fluorescent reporter to link assembly with secretion, and uncovered how a PrgI variant impacts apparatus architecture. Together, these tools enable rapid insights into T3SS assembly and advance heterologous secretion platform development. ImportanceProducing proteins in bacteria often requires expensive and complex steps. This study focuses on S. Typhimurium, a bacterium that naturally secretes proteins using a specialized system. We developed a fast and scalable method to measure assembly of the secretion machinery, ultimately accelerating the process of optimizing this system for bacterial protein production. Further, this method provides new mechanistic insights into how the changes to the system impact the ability for the secretion machinery to form. We expect the platform could be adopted to other systems displayed on the bacterial cell surface, including T3SS homologs as well as a wide variety of multi-component membrane transporters.

synthetic biology↗

bioRxiv2025

Evaluation of Bacterial Microcompartment Cofactor Recycling and Permeability with a Model Guided In Vitro Assay

Biomanufacturing is a promising strategy for sustainable chemical production. However, challenges such as cofactor competition and low pathway flux prevent competitive titers. Some bacteria address these challenges by encapsulating metabolic pathways in bacterial microcompartments (MCPs), many of which contain dedicated cofactor recycling enzymes. We sought to determine how pathway cofactor recycling and intermediate sequestration in MCPs benefit pathway performance using an in vitro assay and kinetic model of the 1,2-propanediol utilization (Pdu) system. Guided by model simulations, we performed experimental design to characterize permeability, a key and difficult-to-measure property of MCPs. Using our model and measurements of metabolite concentrations over time, we estimate MCP permeability values in the range of 10-5 cm/s. We also demonstrated that NAD+/NADH recycling in the Pdu MCP benefits increased pathway flux. This study integrates experiments and systems modeling to advance our understanding of why pathways are encapsulated and to inform bioengineering applications.

biochemistry↗

bioRxiv2025

Self-assembling protein materials with genetically programmable morphology and size

Materials are challenging to synthetically program down to the atom level. Nature, however, excels at creating hierarchical materials from nanoscale building blocks, a feat that remains a major challenge in synthetic systems. A deeper understanding of the molecular rules governing self-assembly would unlock the potential for designing genetically programmable materials with atomic precision. Hexameric bacterial microcompartment (BMC-H) proteins offer a powerful model system for exploring this question. These sequence-defined proteins naturally assemble into complex architectures and can be expressed biologically, making them ideal candidates for studying how minor sequence variations influence supramolecular structure. In this work, we leverage cell-free protein synthesis (CFPS) alongside immunostaining and super-resolution microscopy to investigate the self-assembly behavior of two BMC-H proteins, PduA and PduJ. We find that both proteins form micro-to millimeter scale structures when expressed in vitro. Further, we demonstrate how single point mutation changes lead PduA and PduJ to form significantly different supramolecular structures when produced using CFPS. These studies support the future exploration of self-assembling proteins as programmable scaffolds in broad materials applications. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=147 SRC="FIGDIR/small/666636v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@3376faorg.highwire.dtl.DTLVardef@c81730org.highwire.dtl.DTLVardef@6a6a5borg.highwire.dtl.DTLVardef@6cb4ff_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2025

Distinguishing Protein and Gene Delivery Enables Characterization and Bioengineering of Extracellular Vesicle-Adeno-Associated Virus Vectors

Adeno-associated virus (AAV) gene therapies have achieved some clinical success, with multiple products reaching regulatory approval. Encapsulation of AAV vectors within engineered extracellular vesicles (EVs) is an emerging strategy which could help overcome challenges including pre-existing anti-capsid immunity and the need for controlling targeting and tropism. To guide the development of EV-AAV technologies, we developed an assay for quantifying and controlling for the contribution of pseudotransduction to evaluations of EV-AAV-mediated transduction. We developed an AAV vector that switches its transgene output from one reporter to another when acted upon by Cre recombinase expressed in a recipient cell. Using this platform, we investigated EV-AAV transduction as a function of various engineered EV surface modifications. For actively endocytic cells (HEK293FTs), modifications that enhance EV uptake and membrane fusion influence pseudotransduction but not true transduction. Conversely, in less endocytic Jurkat T cells, modifications enhancing EV uptake enhanced both pseudotransduction and true transduction. These conclusions held across two AAV serotypes. Our results provide new insight into prior reports and suggest that effects of enhancing uptake and membrane fusion of EV-AAV vectors are recipient cell type-specific. The methods developed here unambiguously dissect EV-AAV transduction mechanisms and can guide future bioengineering of EV-AAV vectors.

bioengineering↗

bioRxiv2025

Synthetic Rewiring of Virus-Like Particles via Circular Permutation Enables Modular Peptide Display and Protein Encapsulation

Virus-like particles (VLPs) are self-assembling nanoparticles derived from viruses with potential as scaffolds for myriad applications. They are also excellent testbeds for engineering protein superstructures. Engineers often employ techniques such as amino acid substitutions and insertions/deletions. Yet evolution also utilizes circular permutation, a powerful natural strategy that has not been fully explored in engineering self-assembling protein nanoparticles. Here, we demonstrate this technique using the MS2 VLP as a model self-assembling, proteinaceous nanoparticle. We constructed, for the first time, a comprehensive circular permutation library of the fused MS2 coat protein dimer construct. The strategy revealed new terminal locations, validated via cryo-electron microscopy, that enabled C-terminal peptide tagging and led to a stable protein encapsulation strategy via covalent bonding - a feature the native coat protein does not permit. Our systematic study demonstrates the power of circular permutation for engineering new features as well as quantitatively and systematically exploring VLP structural determinants.

synthetic biology↗

bioRxiv2025

A High Throughput Assay for Measuring Secreted Protein Based on a de novo Fluorescent Reporter Reveals Regulatory and Structural Insights in Salmonella Type Three Secretion System

Intracellular protein production in bacteria is limited by the need for lysis and costly purification. A promising alternative is to engineer the host organism for protein secretion. While the Salmonella enterica serovar Typhimurium (S. Typhimurium) Type 3 Secretion System (T3SS) has been utilized for protein secretion, its study and eventual applicability for recombinant protein production is constrained by the lack of high-throughput assays to quantitatively measure secretion titer. Developing such assays is challenging, as proteins must remain unfolded for secretion, limiting the use of several common reporter proteins. In this work, we develop a high-throughput secretion assay using mini-Fluorescent Activating Protein (mFAP). mFAP forms a chromophore only upon addition of an exogenous substrate, allowing secretion and subsequent fluorescence detection. We demonstrate mFAP secretion via the T3SS with an N-terminal secretion tag and show that the fluorescent signal in the secreted fraction is rapid and linear over three orders of magnitude. Using this assay, we screen S. Typhimurium strains with secretion-enhancing mutations, identifying a constitutively active strain and reveal temporally controlled secretion dynamics. We also show that this assay may be applicable to other secretion systems, providing a universal tool for tracking heterologous protein secretion.

synthetic biology↗

bioRxiv2024

Encapsulation of Select Violacein Pathway Enzymes in the 1,2-Propanediol Utilization Bacterial Microcompartment to Divert Pathway Flux

A continual goal in metabolic engineering is directing pathway flux to desired products and avoiding loss of pathway intermediates to competing pathways. Encapsulation of the pathway is a possible solution, as it creates a diffusion barrier between pathway intermediates and competing enzymes. It is hypothesized that bacteria use organelles known as bacterial microcompartments - proteinaceous shells encapsulating a metabolic pathway - for this purpose. We aim to determine to what degree this hypothesized benefit is conferred to encapsulated pathways. To this end, we used bacterial microcompartments to encapsulate select enzymes from the violacein pathway, which is composed of five enzymes that produce violacein as the main product and deoxyviolacein as a side product. Importantly, we studied the pathway in a cell-free context, allowing us to hold constant the concentration of unencapsulated and encapsulated enzymes and increase our control over reaction conditions. The VioE enzyme is a branch point in that it makes the precursor for both violacein and deoxyviolacein, the VioC enzyme is required for production of deoxyviolacein, and the VioD enzyme is required for violacein production. When we encapsulated VioE and VioC and left VioD unencapsulated, the product profile shifted toward deoxyviolacein and away from violacein compared to when VioC and VioD were both unencapsulated. This work provides the first fully quantitative evidence that microcompartment-based encapsulation can be used to divert pathway flux to the encapsulated pathway. It provides insight into why certain pathways are encapsulated natively and could be leveraged for metabolic engineering applications.

synthetic biology↗

bioRxiv2024

Signal sequences target enzymes and structural proteins to bacterial microcompartments and are critical for microcompartment formation

Spatial organization of pathway enzymes has emerged as a promising tool to address several challenges in metabolic engineering, such as flux imbalances and off-target product formation. Bacterial microcompartments (MCPs) are a spatial organization strategy used natively by many bacteria to encapsulate metabolic pathways that produce toxic, volatile intermediates. Several recent studies have focused on engineering MCPs to encapsulate heterologous pathways of interest, but how this engineering affects MCP assembly and function is poorly understood. In this study, we investigated the role of signal sequences, short domains that target proteins to the MCP core, in the assembly of 1,2-propanediol utilization (Pdu) MCPs. We characterized two novel Pdu signal sequences on the structural proteins PduM and PduB, which constitutes the first report of metabolosome signal sequences on structural proteins rather than enzymes. We then explored the role of enzymatic and structural Pdu signal sequences on MCP assembly by deleting their encoding sequences from the genome alone and in combination. Deleting enzymatic signal sequences decreased MCP formation, but this defect could be recovered in some cases by overexpressing genes encoding the knocked-out signal sequence fused to a heterologous protein. By contrast, deleting structural signal sequences caused similar defects to knocking out the genes encoding the full length PduM and PduB proteins. Our results contribute to a growing understanding of how MCPs form and function in bacteria and provide strategies to mitigate assembly disruption when encapsulating heterologous pathways in MCPs.

synthetic biology↗

bioRxiv2024

Characterization and Engineering of the Type 3 Secretion System Needle Monomer from Salmonella Through the Construction and Screening of a Comprehensive Mutagenesis Library

Protein production strategies in bacteria are often limited due to the need for cell lysis and complicated purification schemes. To avoid these challenges, researchers have developed bacterial strains capable of secreting heterologous protein products outside the cell, but secretion titers often remain too low for commercial applicability. Improved understanding of the link between secretion system structure and its secretory abilities can help overcome the barrier to engineering higher secretion titers. Here we investigated this link with the PrgI protein, the monomer of the secretory channel of the Type 3 Secretion System (T3SS) of Salmonella enterica. Despite detailed knowledge of the PrgI needles assembly and structure, little is known about how its structure influences its secretory capabilities. To study this, we recently constructed a comprehensive codon mutagenesis library of the PrgI protein utilizing a novel one pot recombineering approach. We then screened this library for functional T3SS assembly and secretion titer by measuring the secretion of alkaline phosphatase using a high-throughput activity assay. This allowed us to construct a first-of-its-kind secretion fitness landscape (SFL) to characterize the PrgI needles mutability at each position as well as the mutations which lead to enhanced T3SS secretion. We discovered new design rules for building a functional T3SS as well as identified hypersecreting mutants. This work can be used to increase understanding of the T3SSs assembly and identify further targets for engineering. This work also provides a blueprint for future efforts to engineer other complex protein assemblies through the construction of fitness landscapes. ImportanceProtein secretion offers a simplified alternative method for protein purification from bacterial hosts. However, the current state-of-the-art methods for protein secretion in bacteria are still hindered by low yields relative to traditional protein purification strategies. Engineers are now seeking strategies to enhance protein secretion titers from bacterial hosts, often through genetic manipulations. In this study, we demonstrate that protein engineering strategies focused on altering the secretion apparatus can be a fruitful avenue toward this goal. Specifically, this study focuses on how changes to the PrgI needle protein from the type 3 secretion system from Salmonella enterica can impact secretion titer. We demonstrate that this complex is amenable to comprehensive mutagenesis studies and that this can yield both PrgI variants with increased secretory capabilities and insight into the normal functioning of the type 3 secretion system.

bioengineering↗

bioRxiv2022

Systematic Engineering of Virus-Like Particles to Identify Self-Assembly Rules for Shifting Particle Size

Virus-like particles (VLPs) are promising scaffolds for biomaterials as well as diagnostic and therapeutic applications. However, there are some key challenges to be solved, such as the ability to engineer alternate sizes for varied use cases. To this end, we created a library of MS2 VLP variants at two key residues in the coat protein which have been implicated as important to controlling VLP size and geometry. By adapting a method for systematic mutagenesis coupled with size-based selections and high-throughput sequencing as a readout, we developed a quantitative assessment of two residues in MS2 coat protein that govern the size shift in MS2 VLPs. We then applied the strategy to the equivalent residues in Q{beta} VLPs, an MS2 homolog, and demonstrate that the analogous pair of residues are also able to impact VLP size and shape. These results underscore the power of fitness landscapes in identifying critical features for assembly.

bioengineering↗

bioRxiv2022

Enzymatically-active bacterial microcompartments follow substrate gradients and are protected from aggregation in a cell-free system

The ability to dynamically control organelle movement and position is essential for cellular function. Yet the underlying mechanisms driving this organization have not been fully resolved. Here, we draw from recent experimental observations and theoretical models of enzyme chemotaxis to demonstrate the chemotaxis of a bacterial organelle, the 1,2 propanediol (1,2-PD) utilization bacterial microcompartment (MCP) from Salmonella enterica. Upon encapsulating MCPs in a cell-like, biomimetic compartment, we observed the directed movement of MCPs along an external gradient of substrate. Our analysis shows that MCPs not only chemotax towards their substrate but also that enzymatic activity and substrate turnover protect them against large-scale aggregation. Our results provide a first experimental demonstration of organelle chemotaxis in a synthetic cellular system and support a recent theoretical model of chemotaxis. Together this work reveals a potentially significant driver of organelle organization while contributing to the construction of synthetic cell-like materials.

synthetic biology↗

bioRxiv2022

Construction of a constitutively active type III secretion system for heterologous protein secretion

Proteins comprise a multibillion-dollar industry in enzymes and therapeutics, but bacterial protein production can be costly and inefficient. Proteins of interest (POIs) must be extracted from lysed cells, purified, and resolubilized. The Salmonella pathogenicity island 1 type III secretion system (T3SS) is a protein secretion complex in Salmonella that has been engineered to secrete heterologous proteins and addresses the problems associated with bacterial protein production. However, the current best practices method of T3SS pathway activation for secretion is not ideal for industrial scaleup. Previously, the T3SS was activated by plasmid-based overexpression of the T3SS transcriptional regulator, hilA, which requires the addition of a small molecule inducer to the culture media and adds significant cost to the production media. Plasmid-based expression is also subject to instability in large-scale fermentation. Here, we show that we can constitutively activate the T3SS by modulating the upstream transcriptional regulator, hilD, either through knocking out hilE, a repressor of HilD, or by adding transcriptional fusions to hilD. Finally, we combine the two most promising genomic modifications to build a constitutively active T3SS capable of secreting a range of heterologous proteins at titers comparable to those reported with synthetic induction of hilA. These improvements further our goal of making an industrially competitive protein production strain that reduces the challenges associated with plasmid induction and maintenance. ImportanceProteins are used in our everyday lives as therapeutics (insulin), industrial enzymes (laundry detergent), and bio-based materials (spider silk). Current industrial protein production in bacteria is costly because it requires purification of the target protein from the other proteins inside the cell. We solve this problem by engineering the Salmonella pathogenicity island 1 type III secretion system (T3SS) to export the target protein into the cell growth media. This makes the protein purification process more efficient and cheaper. However, this system currently requires an expensive inducer reagent to activate it which significantly increases the cost of the production media. We show here the creation of a constitutively active T3SS, meaning the T3SS pathway is always on. In doing so, we successfully created a Salmonella strain that eliminates the need for the inducer reagent and exports proteins at levels comparable to the inducer-activated system, ultimately reducing the cost of T3SS protein production.

synthetic biology↗

bioRxiv2021

Comprehensive Fitness Landscape of a Multi-Geometry Protein Capsid Informs Machine Learning Models of Assembly

Virus-like particles (VLPs) are non-infections viral-derived nanomaterials poised for biotechnological applications due to their well-defined, modular self-assembling architecture. Although progress has been made in understanding the complex effects that mutations may have on VLPs, nuanced understanding of the influence particle mutability has on quaternary structure has yet to be achieved. Here, we generate and compare the apparent fitness landscapes of two capsid geometries (T=3 and T=1 icosahedral) of the bacteriophage MS2 VLP. We find significant shifts in mutability at the symmetry interfaces of the T=1 capsid when compared to the wildtype T=3 assembly. Furthermore, we use the generated landscapes to benchmark the performance of in silico mutational scanning tools in capturing the effect of missense mutation on complex particle assembly. Finding that predicted stability effects correlated relatively poorly with assembly phenotype, we used a combination of de novo features in tandem with in silico results to train machine learning algorithms for the classification of variant effects on assembly. Our findings not only reveal ways that assembly geometry affects the mutable landscape of a self-assembled particle, but also establish a template for the generation of predictive mutational models of self-assembled capsids using minimal empirical training data.

bioengineering↗

bioRxiv2021

Vertex protein PduN tunes encapsulated pathway performance by dictating bacterial metabolosome morphology

Engineering subcellular organization in microbes shows great promise in addressing bottlenecks in metabolic engineering efforts; however, rules guiding selection of an organization strategy or platform are lacking. Here, we study compartment morphology as a factor in mediating encapsulated pathway performance. Using the 1,2-propanediol utilization microcompartment (Pdu MCP) system from Salmonella enterica serovar Typhimurium LT2, we find that we can shift the morphology of this protein nanoreactor from polyhedral to tubular by removing vertex protein PduN. Analysis of the metabolic function between these Pdu microtubes (MTs) shows that they provide a diffusional barrier capable of shielding the cytosol from a toxic pathway intermediate, similar to native MCPs. However, kinetic modeling suggests that the different surface area to volume ratios of MCP and MT structures alters encapsulated pathway performance. Finally, we report a microscopy-based assay that permits rapid assessment of Pdu MT formation to enable future engineering efforts on these structures.

bioengineering↗

bioRxiv2021

Linking the Salmonella enterica 1,2-propanediol utilization bacterial microcompartment shell to the enzymatic core via the shell protein PduB

Bacterial microcompartments (MCPs) are protein-based organelles that house the enzymatic machinery for metabolism of niche carbon sources, allowing enteric pathogens to outcompete native microbiota during host colonization. While much progress has been made toward understanding MCP biogenesis, questions still remain regarding the mechanism by which core MCP enzymes are enveloped within the MCP protein shell. Here we explore the hypothesis that the shell protein PduB is responsible for linking the shell of the 1,2-propanediol utilization (Pdu) MCP from Salmonella enterica serovar Typhimurium LT2 to its enzymatic core. Using fluorescent reporters, we demonstrate that all members of the Pdu enzymatic core are encapsulated in Pdu MCPs. We also demonstrate that PduB is the sole protein responsible for linking the entire Pdu enzyme core to the MCP shell. Using MCP purifications, transmission electron microscopy, and fluorescence microscopy we find that shell assembly can be decoupled from the enzymatic core, as apparently empty MCPs are formed in Salmonella strains lacking PduB. Mutagenesis studies also reveal that PduB is incorporated into the Pdu MCP shell via a conserved, lysine-mediated hydrogen bonding mechanism. Finally, growth assays and systems-level pathway modeling reveal that unencapsulated pathway performance is strongly impacted by enzyme concentration, highlighting the importance of minimizing polar effects when conducting these functional assays. Together, these results provide insight into the mechanism of enzyme encapsulation within Pdu MCPs and demonstrate that the process of enzyme encapsulation and shell assembly are separate processes in this system, a finding that will aid future efforts to understand MCP biogenesis. ImportanceMCPs are unique, genetically encoded organelles used by many bacteria to survive in resource-limited environments. There is significant interest in understanding the biogenesis and function of these organelles, both as potential antibiotic targets in enteric pathogens and also as useful tools for overcoming metabolic engineering bottlenecks. However, the mechanism by which these organelles are formed natively is still not completely understood. Here we provide evidence of a potential mechanism inS. enterica by which a single protein, PduB, links the MCP shell and metabolic core. This finding is critical for those seeking to disrupt MCPs during pathogenic infections or for those seeking to harness MCPs as nanobioreactors in industrial settings.

synthetic biology↗