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.

Characterization and engineering of the type 3 secretion system needle monomer from 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.

Deconstructing synthetic biology across scales: a conceptual approach for training synthetic biologists.

Synthetic biology allows us to reuse, repurpose, and reconfigure biological systems to address society's most pressing challenges. Developing biotechnologies in this way requires integrating concepts across disciplines, posing challenges to educating students with diverse expertise. We created a framework for synthetic biology training that deconstructs biotechnologies across scales-molecular, circuit/network, cell/cell-free systems, biological communities, and societal-giving students a holistic toolkit to integrate cross-disciplinary concepts towards responsible innovation of successful biotechnologies.

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

Unlabelled: 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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Apparent size and morphology of bacterial microcompartments varies with technique.

Bacterial microcompartments (MCPs) are protein-based organelles that encapsulate metabolic pathways. Metabolic engineers have recently sought to repurpose MCPs to encapsulate heterologous pathways to increase flux through pathways of interest. As MCP engineering becomes more common, standardized methods for analyzing changes to MCPs and interpreting results across studies will become increasingly important.

Multiplexed mass spectrometry of individual ions improves measurement of proteoforms and their complexes.

An Orbitrap-based ion analysis procedure determines the direct charge for numerous individual protein ions to generate true mass spectra. This individual ion mass spectrometry (IMS) method for charge detection enables the characterization of highly complicated mixtures of proteoforms and their complexes in both denatured and native modes of operation, revealing information not obtainable by typical measurements of ensembles of ions.