Search bioRxiv⌕ Search

Biology subjects

Tullman-Ercek, D.

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

26 records · Page 2Linked to original sources

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

MotivationIdentifying gene clusters of interest in phylogenetically proximate and distant taxa can help to infer phenotypes of interest. Conserved gene clusters may differ by only a few genes, which can be biologically meaningful, such as the formation of pseudogenes or insertions interrupting regulation. These qualities may allow for unsupervised clustering of similar gene clusters into bins that provide a population-level understanding of the genetic variation in similar gene clusters. ResultsWe 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 in 435 genomes containing 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 replicated in vivo, disruption of pduN with a frameshift mutation negatively impacted microcompartment formation. We next demonstrated the versatility of GeneGrouper by clustering both distant homologous gene clusters and variable gene clusters found in integrative and conjugative elements. AvailabilityGeneGrouper software and code are publicly available at https://github.com/agmcfarland/GeneGrouper.

bioinformatics↗

Computational and experimental approaches to controlling bacterial microcompartment assembly

Bacterial microcompartments compartmentalize the enzymes that aid chemical and energy production in many bacterial species. These protein organelles are found in various bacterial phyla and are postulated to help many of these organisms survive in hostile environments such as the gut of their hosts. 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. As a model system, we find via multiscale modeling and mutagenesis studies, the interactions responsible for the binding of hexamer-forming proteins propanediol utilization bacterial microcompartments from Salmonella and establish conditions that form various morphologies. We determine how the changes in the microcompartment hexamer protein preferred angles and interaction strengths can modify the assembled morphologies including the naturally occurring polyhedral microcompartment shape, as well as other extended shapes or quasi-closed shapes. We demonstrate experimentally that such altered strengths and angles are achieved via amino acid mutations. A thermodynamic model that agrees with the coarse-grained simulations provides guidelines to design microcompartments. These findings yield insight in controlled protein assembly and provide principles for assembling microcompartments for biochemical or energy applications as nanoreactors.

biophysics↗

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.

microbiology↗

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 systematic manner, paving the way to exploring the applications of peptide insertions in biomedically relevant settings.

biochemistry↗

Apparent size and morphology of bacterial microcompartments varies with technique

Bacterial microcompartments (MCPs) are protein-based organelles which 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. In this study, we demonstrate that different imaging techniques yield variations in the apparent size of purified MCPs from Salmonella enterica serovar Typhimurium LT2, likely due to variations in sample preparation methods. We provide guidelines for preparing samples for MCP imaging and outline expected variations in apparent size and morphology between methods. With this report we aim to establish an aid for comparing results across studies.

synthetic biology↗

Dynamic control of pathway expression with riboregulated switchable feedback promoters

Dynamic pathway regulation has emerged as a promising strategy in metabolic engineering for improved system productivity and yield, and continues to grow in sophistication. Bacterial stress-response promoters allow dynamic gene regulation using the hosts natural transcriptional networks, but lack the flexibility to control the expression timing and overall magnitude of pathway genes. Here, we report a strategy that uses RNA transcriptional regulators to introduce another layer of control over the output of natural stress-response promoters. This new class of gene expression cassette, called a riboregulated switchable feedback promoter (rSFP), can be modularly activated using a variety of mechanisms, from manual induction to quorum sensing. We develop and apply rSFPs to regulate a toxic cytochrome P450 enzyme in the context of a Taxol precursor biosynthesis pathway and show this leads to 2.4x fold higher titers than from the best reported strain. We envision that rSFPs will become a valuable tool for flexible and dynamic control of gene expression in metabolic engineering, protein and biologic production, and many other applications.

synthetic biology↗

Spatially organizing biochemistry: choosing a strategy to translate synthetic biology to the factory

Natural biochemical systems are ubiquitously organized both in space and time. Engineering the spatial organization of biochemistry has emerged as a key theme of synthetic biology, with numerous technologies promising improved biosynthetic pathway performance. One strategy, however, may produce disparate results for different biosynthetic pathways. We propose a spatially resolved kinetic model to explore this fundamental design choice in systems and synthetic biology. We predict that two example biosynthetic pathways have distinct optimal organization strategies that vary based on pathway-dependent and cell-extrinsic factors. Moreover, we outline this design space in general as a function of kinetic and biophysical properties, as well as culture conditions. Our results suggest that organizing biosynthesis has the potential to substantially improve performance, but that choosing the appropriate strategy is key. The flexible mathematical framework we propose can be adapted to diverse biosynthetic pathways, and lays a foundation to rationally choose organization strategies for biosynthesis.

synthetic biology↗

Engineered shell proteins confer improved encapsulated pathway behavior in a bacterial microcompartment

Bacterial microcompartments are a class of proteinaceous organelles comprising a characteristic protein shell enclosing a set of enzymes. Compartmentalization can prevent escape of volatile or toxic intermediates, prevent off-pathway reactions, and create private cofactor pools. Encapsulation in synthetic microcompartment organelles will enhance the function of heterologous pathways, but to do so, it is critical to understand how to control diffusion in and out of the microcompartment organelle. To this end, we explored how small differences in the shell protein structure result in changes in the diffusion of metabolites through the shell. We found that the ethanolamine utilization (Eut) protein EutM properly incorporates into the 1,2-propanediol utilization (Pdu) microcompartment, altering native metabolite accumulation and the resulting growth on 1,2-propanediol as the sole carbon source. Further, we identified a single pore-lining residue mutation that confers the same phenotype as substitution of the full EutM protein, indicating that small molecule diffusion through the shell is the cause of growth enhancement. Finally, we show that the hydropathy index and charge of pore amino acids are important indicators to predict how pore mutations will affect growth on 1,2- propanediol, likely by controlling diffusion of one or more metabolites. This study highlights the success of two strategies to engineer microcompartment control over metabolite transport: altering the existing shell protein pore via mutation of the pore-lining residues, and generating chimeras using shell proteins with the desired pores.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=99 SRC=\"FIGDIR/small/106716_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (26K):\norg.highwire.dtl.DTLVardef@1f69a12org.highwire.dtl.DTLVardef@fd5560org.highwire.dtl.DTLVardef@1f01d6aorg.highwire.dtl.DTLVardef@11b442c_HPS_FORMAT_FIGEXP M_FIG TOC Abstract Graphic C_FIG

synthetic biology↗