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 needle's 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 needle's 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 T3SS's 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. Importance: Protein 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.

Kinetic Growth of Multicomponent Microcompartment Shells.

An important goal of systems and synthetic biology is to produce high value chemical species in large quantities. Microcompartments, which are protein nanoshells encapsulating catalytic enzyme cargo, could potentially function as tunable nanobioreactors inside and outside cells to generate these high value species. Modifying the morphology of microcompartments through genetic engineering of shell proteins is one viable strategy to tune cofactor and metabolite access to encapsulated enzymes. However, this is a difficult task without understanding how changing interactions between the many different types of shell proteins and enzymes affect microcompartment assembly and shape. Here, we use multiscale molecular dynamics and experimental data to describe assembly pathways available to microcompartments composed of multiple types of shell proteins with varied interactions. As the average interaction between the enzyme cargo and the multiple types of shell proteins is weakened, the shell assembly pathway transitions from (i) nucleating on the enzyme cargo to (ii) nucleating in the bulk and then binding the cargo as it grows to (iii) an empty shell. Atomistic simulations and experiments using the 1,2-propanediol utilization microcompartment system demonstrate that shell protein interactions are highly varied and consistent with our multicomponent, coarse-grained model. Furthermore, our results suggest that intrinsic bending angles control the size of these microcompartments. Overall, our simulations and experiments provide guidance to control microcomparmtent size and assembly by modulating the interactions between shell proteins.

Theoretical and Practical Aspects of Multienzyme Organization and Encapsulation.

The advent of biotechnology has enabled metabolic engineers to assemble heterologous pathways in cells to produce a variety of products of industrial relevance, often in a sustainable way. However, many pathways face challenges of low product yield. These pathways often suffer from issues that are difficult to optimize, such as low pathway flux and off-target pathway consumption of intermediates. These issues are exacerbated by the need to balance pathway flux with the health of the cell, particularly when a toxic intermediate builds up. Nature faces similar challenges and has evolved spatial organization strategies to increase metabolic pathway flux and efficiency. Inspired by these strategies, bioengineers have developed clever strategies to mimic spatial organization in nature. This review explores the use of spatial organization strategies, including protein scaffolding and protein encapsulation inside of proteinaceous shells, toward overcoming bottlenecks in metabolic engineering efforts.

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 and inclusion bodies, purified, and resolubilized, which adds significant time and cost to the protein-manufacturing process. The Salmonella pathogenicity island 1 (SPI-1) type III secretion system (T3SS) has been engineered to address these problems by secreting soluble, active proteins directly into the culture media, reducing the number of purification steps. However, the current best practices method of T3SS pathway activation 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 (IPTG) to the culture media. IPTG adds significant cost to production and plasmid-based expression is subject to instability in large-scale fermentation. Here, we modulate the upstream transcriptional regulator, hilD, to activate the T3SS via three distinct methods. In doing so, we develop a toolbox of T3SS activation methods and construct constitutively active T3SS strains capable of secreting a range of heterologous proteins at titers comparable to plasmid-based hilA overexpression. We also explore how each activation method in our toolbox impacts the SPI-1 regulatory cascade and discover an epistatic relationship between T3SS regulators, hilE and the hilD 3' untranslated region (hilD 3'UTR). Together, these findings further our goal of making an industrially competitive protein production strain that reduces the challenges associated with plasmid induction and maintenance. KEY POINTS: • Characterized 3 new type III secretion system (T3SS) activation methods for heterologous protein secretion, including 2 constitutive activation methods. • Eliminated the need for a second plasmid and a small molecule inducer to activate the system, making it more suitable for industrial production. • Discovered new regulatory insights into the SPI-1 T3SS, including an epistatic relationship between regulators hilE and the hilD 3' untranslated region.

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 Qß VLPs, an MS2 homolog, and demonstrate that the analogous pair of residues are also able to impact Qß VLP size and shape. These results underscore the power of fitness landscapes in identifying critical features for assembly.

Quantitative high-throughput measurement of bulk mechanical properties using commonly available equipment.

Machine learning approaches have introduced an urgent need for large datasets of materials properties. However, for mechanical properties, current high-throughput measurement methods typically require complex robotic instrumentation, with enormous capital costs that are inaccessible to most experimentalists. A quantitative high-throughput method using only common lab equipment and consumables with simple fabrication steps is long desired. Here, we present such a technique that can measure bulk mechanical properties in soft materials with a common laboratory centrifuge, multiwell plates, and microparticles. By applying a homogeneous force on the particles embedded inside samples in the multiwell plate using centrifugation, we show that our technique measures the fracture stress of gels with similar accuracy to a rheometer. However, our method has a throughput on the order of 103 samples per run and is generalizable to virtually all soft material systems. We hope that our method can expedite materials discovery and potentially inspire the future development of additional high-throughput characterization methods.

Virus-like particles for drug delivery: a review of methods and applications.

Virus-like particles (VLPs) are self-assembling protein nanoparticles that have great promise as vectors for drug delivery. VLPs are derived from viruses but retain none of their infection or replication capabilities. These protein particles have defined surface chemistries, uniform sizes, and stability properties that make them attractive starting points for drug-delivery scaffolds. Here, we review recent advances in tailoring VLPs for drug-delivery applications, including VLP platform engineering approaches as well as methods for cargo loading, activation, and release. Finally, we highlight several successes using VLPs for drug delivery in model systems.

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.

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 critical 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 reveal that PduB is incorporated into the Pdu MCP shell via a conserved, lysine-mediated hydrogen bonding mechanism. Finally, growth assays and system-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. IMPORTANCE MCPs 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 in S. 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.

Functional enzyme-polymer complexes.

SignificanceThe use of biological enzyme catalysts could have huge ramifications for chemical industries. However, these enzymes are often inactive in nonbiological conditions, such as high temperatures, present in industrial settings. Here, we show that the enzyme PETase (polyethylene terephthalate [PET]), with potential application in plastic recycling, is stabilized at elevated temperature through complexation with random copolymers. We demonstrate this through simulations and experiments on different types of substrates. Our simulations also provide strategies for designing more enzymatically active complexes by altering polymer composition and enzyme charge distribution.

Mussel Adhesive-Inspired Proteomimetic Polymer.

Herein, a synthetic polymer proteomimetic is described that reconstitutes the key structural elements and function of mussel adhesive protein. The proteomimetic was prepared via graft-through ring-opening metathesis polymerization of a norbornenyl-peptide monomer. The peptide was derived from the natural underwater glue produced by marine mussels that is composed of a highly repetitive 10 amino acid tandem repeat sequence. The hypothesis was that recapitulation of the repeating unit in this manner would provide a facile route to a nature-inspired adhesive. To this end, the material, in which the arrangement of peptide units was as side chains on a brush polymer rather than in a linear fashion as in the natural protein, was examined and compared to the native protein. Mechanical measurements of adhesion forces between solid surfaces revealed improved adhesion properties over the natural protein, making this strategy attractive for diverse applications. One such application is demonstrated, using the polymers as a surface adhesive for the immobilization of live cells.

Density-based binning of gene clusters to infer function or evolutionary history using GeneGrouper.

MOTIVATION: Identifying variant forms of gene clusters of interest in phylogenetically proximate and distant taxa can help to infer their evolutionary histories and functions. Conserved gene clusters may differ by only a few genes, but these small differences can in turn induce substantial phenotypes, such as by the formation of pseudogenes or insertions interrupting regulation. Particularly as microbial genomes and metagenomic assemblies become increasingly abundant, unsupervised grouping of similar, but not necessarily identical, gene clusters into consistent bins can provide a population-level understanding of their gene content variation and functional homology. RESULTS: We developed GeneGrouper, a command-line tool that uses a density-based clustering method to group gene clusters into bins. GeneGrouper demonstrated high recall and precision in benchmarks for the detection of the 23-gene Salmonella enterica LT2 Pdu gene cluster and four-gene Pseudomonas aeruginosa PAO1 Mex gene cluster among 435 genomes spanning mixed taxa. In a subsequent application investigating the diversity and impact of gene-complete and -incomplete LT2 Pdu gene clusters in 1130 S.enterica genomes, GeneGrouper identified a novel, frequently occurring pduN pseudogene. When investigated in vivo, introduction of the pduN pseudogene negatively impacted microcompartment formation. We next demonstrated the versatility of GeneGrouper by clustering distant homologous gene clusters and variable gene clusters found in integrative and conjugative elements. AVAILABILITY AND IMPLEMENTATION: GeneGrouper software and code are publicly available at https://pypi.org/project/GeneGrouper/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

High-Throughput Screening Test for Adhesion in Soft Materials Using Centrifugation.

High-throughput screening of mechanical properties can transform materials science research by both aiding in materials discovery and developing predictive models. However, only a few such assays have been reported, requiring custom or expensive equipment, while the mounting demand for enormous data sets of materials properties for predictive models is unfulfilled by the current characterization throughput. We address this problem by developing a high-throughput colorimetric adhesion screening method using a common laboratory centrifuge, multiwell plates, and microparticles. The technique uses centrifugation to apply a homogeneous mechanical detachment force across individual formulations in a multiwell plate. We also develop a high-throughput sample deposition method to prepare films with uniform thickness in each well, minimizing well-to-well variability. After establishing excellent agreement with the well-known probe tack adhesion test, we demonstrate the consistency of our method by performing the test on a multiwell plate with two different formulations in an easily discernible pattern. The throughput is limited only by the number of wells in the plates, easily reaching 103 samples/run. With its simplicity, low cost, and large dynamic range, this high-throughput method has the potential to change the landscape of adhesive material characterization.

Bacterial microcompartments: tiny organelles with big potential.

Organization of metabolic processes within the space of a cell is critical for the survival of many organisms. In bacteria, spatial organization is achieved via proteinaceous organelles called bacterial microcompartments, which encapsulate pathway enzymes, substrates, and co-factors to drive the safe and efficient metabolism of niche carbon sources. Microcompartments are self-assembled from shell proteins that encapsulate a core comprising various enzymes. This review discusses how recent advances in understanding microcompartment structure and assembly have informed engineering efforts to repurpose compartments and compartment-based structures for non-native functions. These advances, both in understanding of the native structure and function of compartments, as well as in the engineering of new functions, will pave the way for the use of these structures in bacterial cell factories.

Computational and Experimental Approaches to Controlling Bacterial Microcompartment Assembly.

Bacterial microcompartments compartmentalize the enzymes that aid chemical and energy production in many bacterial species. They are postulated to help bacteria survive in hostile environments. Metabolic engineers are interested in repurposing these organelles for non-native functions. Here, we use computational, theoretical, and experimental approaches to determine mechanisms that effectively control microcompartment self-assembly. We find, via multiscale modeling and mutagenesis studies, the interactions responsible for the binding of hexamer-forming proteins in a model system, the propanediol utilization bacterial microcompartments from Salmonella enterica serovar Typhimurium LT2. We determine how the changes in the microcompartment hexamer protein preferred angles and interaction strengths can modify the assembled morphologies. We demonstrate that such altered strengths and angles are achieved via amino acid mutations. A thermodynamic model provides guidelines to design microcompartments of various morphologies. These findings yield insight in controlled protein assembly and provide principles for assembling microcompartments for biochemical or energy applications as nanoreactors.

Dynamic Control of Gene Expression with Riboregulated Switchable Feedback Promoters.

One major challenge in synthetic biology is the deleterious impacts of cellular stress caused by expression of heterologous pathways, sensors, and circuits. Feedback control and dynamic regulation are broadly proposed strategies to mitigate this cellular stress by optimizing gene expression levels temporally and in response to biological cues. While a variety of approaches for feedback implementation exist, they are often complex and cannot be easily manipulated. Here, we report a strategy that uses RNA transcriptional regulators to integrate additional layers of control over the output of natural and engineered feedback responsive circuits. Called riboregulated switchable feedback promoters (rSFPs), these gene expression cassettes can be modularly activated using multiple mechanisms, from manual induction to autonomous quorum sensing, allowing control over the timing, magnitude, and autonomy of expression. We develop rSFPs in Escherichia coli to regulate multiple feedback networks and apply them to control the output of two metabolic pathways. We envision that rSFPs will become a valuable tool for flexible and dynamic control of gene expression in metabolic engineering, biological therapeutic production, and many other applications.

An optimized growth medium for increased recombinant protein secretion titer via the type III secretion system.

BACKGROUND: Protein secretion in bacteria is an attractive strategy for heterologous protein production because it retains the high titers and tractability of bacterial hosts while simplifying downstream processing. Traditional intracellular production strategies require cell lysis and separation of the protein product from the chemically similar cellular contents, often a multi-step process that can include an expensive refolding step. The type III secretion system of Salmonella enterica Typhimurium transports proteins from the cytoplasm to the extracellular environment in a single step and is thus a promising solution for protein secretion in bacteria. Product titer is sensitive to extracellular environmental conditions, however, and T3SS regulation is integrated with essential cellular functions. Instead of attempting to untangle a complex web of regulatory input, we took an "outside-in" approach to elucidate the effect of growth medium components on secretion titer. RESULTS: We dissected the individual and combined effects of carbon sources, buffers, and salts in a rich nutrient base on secretion titer. Carbon sources alone decreased secretion titer, secretion titer increased with salt concentration, and the combination of a carbon source, buffer, and high salt concentration had a synergistic effect on secretion titer. Transcriptional activity measured by flow cytometry showed that medium composition affected secretion system activity, and prolonged secretion system activation correlated strongly with increased secretion titer. We found that an optimal combination of glycerol, phosphate, and sodium chloride provided at least a fourfold increase in secretion titer for a variety of proteins. Further, the increase in secretion titer provided by the optimized medium was additive with strain enhancements. CONCLUSIONS: We leveraged the sensitivity of the type III secretion system to the extracellular environment to increase heterologous protein secretion titer. Our results suggest that maximizing secretion titer via the type III secretion system is not as simple as maximizing secreted protein expression-one must also optimize secretion system activity. This work advances the type III secretion system as a platform for heterologous protein secretion in bacteria and will form a basis for future engineering efforts.

Self-assembling Shell Proteins PduA and PduJ have Essential and Redundant Roles in Bacterial Microcompartment Assembly.

Protein self-assembly is a common and essential biological phenomenon, and bacterial microcompartments present a promising model system to study this process. Bacterial microcompartments are large, protein-based organelles which natively carry out processes important for carbon fixation in cyanobacteria and the survival of enteric bacteria. These structures are increasingly popular with biological engineers due to their potential utility as nanobioreactors or drug delivery vehicles. However, the limited understanding of the assembly mechanism of these bacterial microcompartments hinders efforts to repurpose them for non-native functions. Here, we comprehensively investigate proteins involved in the assembly of the 1,2-propanediol utilization bacterial microcompartment from Salmonella enterica serovar Typhimurium LT2, one of the most widely studied microcompartment systems. We first demonstrate that two shell proteins, PduA and PduJ, have a high propensity for self-assembly upon overexpression, and we provide a novel method for self-assembly quantification. Using genomic knock-outs and knock-ins, we systematically show that these two proteins play an essential and redundant role in bacterial microcompartment assembly that cannot be compensated by other shell proteins. At least one of the two proteins PduA and PduJ must be present for the bacterial microcompartment shell to assemble. We also demonstrate that assembly-deficient variants of these proteins are unable to rescue microcompartment formation, highlighting the importance of this assembly property. Our work provides insight into the assembly mechanism of these bacterial organelles and will aid downstream engineering efforts.

Engineering a Virus-like Particle to Display Peptide Insertions Using an Apparent Fitness Landscape.

Peptide insertions in the primary sequence of proteins expand functionality by introducing new binding sequences, chemical handles, or membrane disrupting motifs. With these properties, proteins can be engineered as scaffolds for vaccines or targeted drug delivery vehicles. Virus-like particles (VLPs) are promising platforms for these applications since they are genetically simple, mimic viral structure for cell uptake, and can deliver multiple copies of a therapeutic agent to a given cell. Peptide insertions in the coat protein of VLPs can increase VLP uptake in cells by increasing cell binding, but it is difficult to predict how an insertion affects monomer folding and higher order assembly. To this end, we have engineered the MS2 VLP using a high-throughput technique, called Systematic Mutagenesis and Assembled Particle Selection (SyMAPS). In this work, we applied SyMAPS to investigate a highly mutable loop in the MS2 coat protein to display 9,261 non-native tripeptide insertions. This library generates a discrete map of three amino acid insertions permitted at this location, validates the FG loop as a valuable position for peptide insertion, and illuminates how properties such as charge, flexibility, and hydrogen bonding can interact to preserve or disrupt capsid assembly. Taken together, the results highlight the potential to engineer VLPs in a systematic manner, paving the way to exploring the applications of peptide insertions in biomedically relevant settings.