Protein Libraries Research Articles

A functional helix shuffled variant of the B domain of Staphylococcus aureus.

The B domain of protein A is a biotechnologically important three-helix bundle protein. It binds the Fc fragment of antibodies with helix 1/2 and the Fab region with helix 2/3. Here we designed a helix shuffled variant by changing the connectivity of the helices, in order to redesign the helix bundle, yielding altered helix-loop-helix properties. The new loops that generate the new connectivity were created in several protein libraries, and Fc binding variants were selected for a detailed biochemical characterization. We were able to create variants with Fc binding affinity at the same level as the wild type B but with significantly reduced thermal stability. The NMR structure proved that the overall three-dimensional structure was maintained not only in the helix shuffled variant but also points to some potential local differences to wild-type B, which could be the reason for the reduced thermal stability. Therefore, protein A is an example of an optimized structure being more important for stability than for function. Using the helix shuffled variant as a ligand on an affinity column facilitates a robust and straightforward purification of antibodies, but allows for a milder elution at less extreme pH. Therefore, the helix shuffled variant is a suitable ligand to purify more pH-sensitive antibodies.

Expanding the scope of copper artificial metalloenzymes: A potential fluorinase?

Biocatalysts for fluorination are rare, and thus of great interest for artificial enzyme design. Biohybrid catalysts including Cu-based DNAzymes and dinucleotide catalysts can catalyse enantioselective electrophilic fluorination of β-ketoesters. Here we report the investigation of Cu-based artificial metalloenzymes as catalysts for electrophilic fluorination reactions. A library of artificial copper proteins was prepared by bioconjugation of bidentate and tridentate nitrogen ligands to cysteine variants of the Sterol Carrier Protein 2 L (SCP-2 L) and subsequent addition of Cu(II) salts. The resulting copper proteins were screened for activity for the fluorination of β-ketoesters using Selectfluor. Under aqueous acidic conditions it was observed that the designed catalysts did not outcompete the uncatalysed background reaction. This work highlights that careful consideration of substrate reactivity and background reactions is needed when considering potential reactions for artificial metalloenzyme catalysis.

Biophysical mapping of TREM2-ligand interactions reveals shared surfaces for engagement of multiple Alzheimer’s disease ligands

TREM2 is a signaling receptor expressed on microglia that has emerged as an important drug target for Alzheimer’s disease and other neurodegenerative diseases. While a number of TREM2 ligands have been identified, little is known regarding the structural details of how they engage. To better understand this, we created a protein library of 28 different TREM2 variants that could be used to map interactions with various ligands using biolayer interferometry. The variants are located in previously identified putative binding surfaces on TREM2 called the hydrophobic site, basic site, and site 2. We found that mutations to the hydrophobic site ablated binding to apoE4 and TDP-43. Competition binding experiments indicated that apoE4 and oAβ42 share overlapping binding sites on TREM2. In contrast, binding to C1q was disrupted most strongly by mutations to the basic site, including R46, with some mutations to the hydrophobic site also attenuating binding, thus suggesting a broader mediation of binding across the two sites. Supporting this, competition experiments indicated that C1q binding could be blocked by both apoE and oAβ42. TREM2 binding to IL-34 was mediated by the basic site at a surface centering on R76. Competition binding experiments validated the unique site for IL-34, showing little to no competition with either oAβ42 or apoE4. However, competition experiments between C1q and IL34 suggest that the ligands compete for binding at the basic site. Altogether, our results suggest that TREM2 utilizes the hydrophobic site (consisting of CDR1, CDR2, and CDR3) as a common site to engage multiple ligands, and uses distinct basic sites to engage others. Our findings imply that pharmaceutical strategies targeting these surfaces might be effective to modulate TREM2 functions.

High-throughput protein binder discovery by rapid in vivo selection.

Proteins that selectively bind to a target of interest are foundational components of research pipelines 1,2 , diagnostics 3 , and therapeutics 4 . Current immunization-based 5,6 , display- based 7-14 , and computational approaches 15-17,18 for discovering binders are laborious and time- consuming - taking months or more, suffer from high false positives - necessitating extensive secondary screening, and have a high failure rate, especially for disordered proteins and other challenging target classes. Here we establish Phage-Assisted Non-Continuous Selection of Protein Binders (PANCS-binders), an in vivo selection platform that links the life cycle of M13 phage to target protein binding though customized proximity-dependent split RNA polymerase biosensors, allowing for complete and comprehensive high-throughput screening of billion-plus member protein variant libraries with high signal-to-noise. We showcase the utility of PANCS- Binders by screening multiple protein libraries each against a panel of 95 separate therapeutically relevant targets, thereby individually assessing over 10 11 protein-protein interaction pairs, completed in two days. These selections yielded large, high-quality datasets and hundreds of novel binders, which we showed can be affinity matured or directly used in mammalian cells to inhibit or degrade targets. PANCS-Binders dramatically accelerates and simplifies the binder discovery process, the democratization of which will help unlock new creative potential in proteome-targeting with engineered binder-based biotechnologies.

Discovery of Carbonic Anhydrase 9 as a Novel CLEC2 Ligand in a Cellular Interactome Screen.

Membrane proteins, especially extracellular domains, are key therapeutic targets due to their role in cell communication and associations. Yet, their functions and interactions often remain unclear. This study presents a general method to discover interactions of membrane proteins with immune cells and subsequently to deorphanize their respective receptors. We developed a comprehensive recombinant protein library of extracellular domains of human transmembrane proteins and proteins found in the ER-Golgi-lysosomal systems. Using this library, we conducted a flow-cytometric screen that identified several cell surface binding events, including an interaction between carbonic anhydrase 9 (CAH9/CA9/CAIX) and CD14high cells. Further analysis revealed this interaction was indirect and mediated via platelets bound to the monocytes. CA9, best known for its diverse roles in cancer, is a promising therapeutic target. We utilized our library to develop an AlphaLISA high-throughput screening assay, identifying CLEC2 as one robust CA9 binding partner. A five-amino-acid sequence (EDLPT) in CA9, identical to a CLEC2 binding domain in Podoplanin (PDPN), was found to be essential for this interaction. Like PDPN, CA9-induced CLEC2 signaling is mediated via Syk. A Hodgkin's lymphoma cell line (HDLM-2) endogenously expressing CA9 can activate Syk-dependent CLEC2 signaling, providing enticing evidence for a novel function of CA9 in hematological cancers. In conclusion, we identified numerous interactions with monocytes and platelets and validated one, CA9, as an endogenous CLEC2 ligand. We provide a new list of other putative CA9 interaction partners and uncovered CA9-induced CLEC2 activation, providing new insights for CA9-based therapeutic strategies.

Order-Disorder Balance in Silk-Elastin-like Polypeptides Determines Their Self-Assembly into Hydrogel Networks.

The biofabrication of recombinant structural proteins with a range of mechanical or structural features usually relies on the generation of protein libraries displaying variations in terms of amino acid composition, block structure, molecular weight, or physical/chemical cross-linking sites. This approach, while highly successful in generating a wealth of knowledge regarding the links between design features and material properties, has some inherent limitations related to its low throughput. This slows down the pace of the development of de novo recombinant structural proteins. Here, we propose an approach to tune the viscoelastic properties of temperature-responsive hydrogels made of silk-elastin-like polypeptides (SELPs) without modifying their sequence. To do so, we subject purified SELPs to two different postprocessing methods─water annealing or EtOH annealing─that alter the topology of highly disordered SELP networks via the formation of ordered intermolecular β-sheet physical cross-links. Combining different analytical techniques, we connect the order/disorder balance in SELPs with their gelling behavior. Furthermore, we show that introducing a functional block (in this case, a biomineralizing peptide) in the sequence of SELPs can disrupt its self-assembly and that such disruption can only be overcome by EtOH annealing. Our results suggest that postprocessing of as-purified SELPs might be a simple approach to tune the self-assembly of SELPs into biomaterials with bespoke viscoelastic properties beyond the traditional approach of developing SELP libraries via genetic engineering.

Expanding the landscape of antibody discovery.

Library:library screening technologies hold substantial promise for paired antibody:antigen discovery, but challenges have persisted. In this issue of Cell Reports Methods, Wagner etal. introduce a method that combines antibody-ribosome-mRNA complexes, antigen cell surface display, and single-cell RNA sequencing to successfully screen diverse antibody gene libraries against a library of viral receptor proteins.

Deep Proteome Coverage of Microglia Using a Streamlined Data-Independent Acquisition-Based Proteomic Workflow: Method Consideration for a Phenotypically Diverse Cell Type.

As the primary innate immune cells of the brain, microglia play a key role in various homeostatic and disease-related processes. To carry out their numerous functions, microglia adopt a wide range of phenotypic states. The proteomic landscape represents a more accurate molecular representation of these phenotypes; however, microglia present unique challenges for proteomic analysis. This study implemented a streamlined liquid- and gas-phase fractionation method with data-dependent acquisition (DDA) and parallel accumulation-serial fragmentation (PASEF) analysis on a TIMS-TOF instrument to compile a comprehensive protein library obtained from adult-derived, immortalized mouse microglia with low starting material (10 µg). The empirical library consisted of 9140 microglial proteins and was utilized to identify an average of 7264 proteins/run from single-shot, data-independent acquisition (DIA)-based analysis microglial cell lysate digest (200 ng). Additionally, a predicted library facilitated the identification of 7519 average proteins/run from the same DIA data, revealing complementary coverage compared with the empirical library and collectively increasing coverage to approximately 8000 proteins. Importantly, several microglia-relevant pathways were uniquely identified with the empirical library approach. Overall, we report a simplified, reproducible approach to address the proteome complexity of microglia using low sample input and show the importance of library optimization for this phenotypically diverse cell type.

Cavity-Based Discovery of New Fatty Acid Photodecarboxylases.

Light-dependent fatty acid photodecarboxylases (FAPs) hold significant potential for biotechnology, due to their capability to produce alka(e)nes directly from the corresponding (un)saturated natural fatty acids requiring light as the only reagent. This study expands the family of FAPs through cavity-based enzyme discovery methods. Thirty enzyme candidates with potential photodecarboxylation activity were identified by matching the cavities of four related template structures against the Protein Data Bank's flavoproteins, a library of proteins identified via the Foldseek Search Server, and homology models of sequences resulting from BLAST. Subsequent docking experiments narrowed this library to ten promising enzymes, which were expressed and assessed in vitro, identifying four photodecarboxylases. Out of these enzymes, the GMC oxidoreductase from Coccomyxa sp. Obi (CoFAP) was characterized in detail, which revealed high activity in the decarboxylation reactions of palmitic acid and octanoic acid and a broad pH tolerance (pH 6.5-9.5).

Rapid and Specific Detection of Bacillus cereus Using Phage Protein-Based Lateral Flow Assays.

Rapid and precise diagnostic techniques are essential for identifying foodborne pathogens, including Bacillus cereus (B. cereus), which poses significant challenges to food safety. Traditional detection methods are limited by long incubation times and high costs. In this context, gold nanoparticle (AuNP)-based lateral flow assays (LFAs) are emerging as valuable tools for rapid screening. However, the use of antibodies in LFAs faces challenges, including complex production processes, ethical concerns, or variability. Here, we address these challenges by proposing an innovative approach using bacteriophage-derived proteins for pathogen detection on LFAs. We used the engineered endolysin cell-wall-binding domain (CBD) and distal tail proteins (Dit) from bacteriophages that specifically target B. cereus. The protein-binding properties, essential for the formation of efficient capture and detection biointerfaces in LFAs, were extensively characterized from the microstructural to the LFA device level. Machine-learning models leverage knowledge of the protein sequence to predict advantageous protein orientations on the nitrocellulose membrane and AuNPs. The study of the biointerface binding quantified the degree of attachment of AuNPs to bacteria, providing, for the first time, a microscopic model of the number of AuNPs binding to bacteria. It highlighted the binding of up to one hundred 40 nm AuNPs per bacterium in conditions mimicking LFAs. Eventually, phage proteins were demonstrated as efficient bioreceptors in a straightforward LFA prototype combining the two proteins, providing a rapid colorimetric response within 15 min upon the detection of 105 B. cereus cells. Recombinantly produced phage binding proteins present an opportunity to generate a customizable library of proteins with precise binding capabilities, offering a cost-effective and ethical alternative to antibodies. This study enhances our understanding of phage protein biointerfaces, laying the groundwork for their utilization as efficient bioreceptors in LFAs and rapid point-of-care diagnostic assays, thus potentially strengthening public health measures.

Controllable Autolytic Leaky E. coli Platform for the Recovery of Intracellular Proteins.

Escherichia coli is a commonly used platform for the production of heterologous proteins. Extraction and purification of intracellularly expressed recombinant proteins rely on efficient cell disruption. To facilitate downstream processing, controlled autolytic cells have been designed that lyse automatically to release intracellular proteins when triggered with an internal or external signal. In the cases when a weak promoter has to be adopted to control autolysis, cell lysis and product release progress slowly even in the presence of surfactants or other adjuvants. In this study, we report an improved autolytic E. coli strain controlled by a weak promoter with higher efficiency without the use of any facilitating chemical. Cell lysis was initiated upon arabinose-induced expression of T4 lysozyme with N-terminal fusion of amphipathic cell-penetrating peptides via a flexible peptide linker. Furthermore, genes involved in membrane permeability were individually deleted and screened for leaky phenotypes. Deletion of lpp (encoding Braun's lipoprotein) combined with the autolytic system caused 96% cell lysis in 4 h of induction and released 84% or 67% of mCherry or a super large Cas13a fusion protein (160.8 kDa), respectively, in 10 h of induction. This autolytic leaky strain shows great promise for protein recovery and library screening.

Combinatorial effector targeting (COMET) for transcriptional modulation and locus-specific biochemistry.

Understanding how human gene expression is coordinately regulated by functional units of proteins across the genome remains a major biological goal. Here, we present COMET, a high-throughput screening platform for combinatorial effector targeting for the identification of transcriptional modulators. We generate libraries of combinatorial dCas9-based fusion proteins, containing two to six effector domains, allowing us to systematically investigate more than 110,000 combinations of effector proteins at endogenous human loci for their influence on transcription. Importantly, we keep full proteins or domains intact, maintaining catalytic cores and surfaces for protein-protein interactions. We observe more than 5800 significant hits that modulate transcription, we demonstrate cell type specific transcriptional modulation, and we further investigate epistatic relationships between our effector combinations. We validate unexpected combinations as synergistic or buffering, emphasizing COMET as both a method for transcriptional effector discovery, and as a functional genomics tool for identifying novel domain interactions and directing locus-specific biochemistry.

Human IL-22 receptor-targeted small protein antagonist suppress murine DSS-induced colitis

BackgroundHuman interleukin-22 (IL-22) is known as a “dual function” cytokine that acts as a master regulator to maintain homeostasis, structural integrity of the intestinal epithelial barrier, and shielding against bacterial pathogens. On the other hand, the overexpression of IL-22 is associated with hyper-proliferation and recruitment of pathologic effector cells, leading to tissue damage and chronic inflammation in specific diseases including inflammatory bowel disease (IBD). To study a role of IL-22-mediated signaling axis during intestinal inflammation, we generated a set of small protein blockers of IL-22R1 and verified their inhibitory potential on murine model of colitis.MethodsWe used directed evolution of proteins to identify binders of human IL-22 receptor alpha (IL-22R1), designated as ABR ligands. This approach combines the assembly of a highly complex combinatorial protein library derived from small albumin-binding domain scaffold and selection of promising protein variants using ribosome display followed by large-scale ELISA screening. The binding affinity and specificity of ABR variants were analyzed on transfected HEK293T cells by flow cytometry and LigandTracer. Inhibitory function was further verified by competition ELISA, HEK-Blue IL-22 reporter cells, and murine dextran sulfate sodium (DSS)-induced colitis.ResultsWe demonstrate that ABR specifically recognizes transgenic IL-22R1 expressed on HEK293T cells and IL-22R1 on TNFα/IFNγ-activated HaCaT cells. Moreover, some ABR binders compete with the IL-22 cytokine and function as IL-22R1 antagonists in HEK-Blue IL22 reporter cells. In a murine model of DSS-induced acute intestinal inflammation, daily intraperitoneal administration of the best IL-22R1 antagonist, ABR167, suppressed the development of clinical and histological markers of colitis including prevention of mucosal inflammation and architecture deterioration. In addition, ABR167 reduces the DSS-induced increase in mRNA transcript levels of inflammatory cytokines such as IL-1β, IL-6, IL-10, and IL-17A.ConclusionsWe developed small anti-human IL-22R1 blockers with antagonistic properties that ascertain a substantial role of IL-22-mediated signaling in the development of intestinal inflammation. The developed ABR blockers can be useful as a molecular clue for further IBD drug development.

Biophysical mapping of TREM2-ligand interactions reveals shared surfaces for engagement of multiple Alzheimer's disease ligands.

TREM2 is a signaling receptor expressed on microglia that has emerged as an important drug target for Alzheimer's disease and other neurodegenerative diseases. While a number of TREM2 ligands have been identified, little is known regarding the structural details of how they engage. To better understand this, we created a protein library of 28 different TREM2 variants that could be used to map interactions with various ligands using biolayer interferometry. The variants are located in previously identified putative binding surfaces on TREM2 called the hydrophobic site, basic site, and site 2. We found that mutations to the hydrophobic site ablated binding to apoE4 and TDP-43. Competition binding experiments indicated that apoE4 and oAβ42 share overlapping binding sites on TREM2. In contrast, binding to C1q was disrupted most strongly by mutations to the basic site, including R46, with some mutations to the hydrophobic site also attenuating binding, thus suggesting a broader mediation of binding across the two sites. Supporting this, competition experiments indicated that C1q binding could be blocked by both apoE and oAβ42. TREM2 binding to IL-34 was mediated by the basic site at a surface centering on R76. Competition binding experiments validated the unique site for IL-34, showing little to no competition with either oAβ42 or apoE4. However, competition experiments between C1q and IL34 suggest that the ligands compete for binding at the basic site. Altogether, our results suggest that TREM2 utilizes the hydrophobic site (consisting of CDR1, CDR2, and CDR3) as a common site to engage multiple ligands, and uses distinct basic sites to engage others. Our findings imply that pharmaceutical strategies targeting these surfaces might be effective to modulate TREM2 functions.

Enriching productive mutational paths accelerates enzyme evolution

Darwinian evolution has given rise to all the enzymes that enable life on Earth. Mimicking natural selection, scientists have learned to tailor these biocatalysts through recursive cycles of mutation, selection and amplification, often relying on screening large protein libraries to productively modulate the complex interplay between protein structure, dynamics and function. Here we show that by removing destabilizing mutations at the library design stage and taking advantage of recent advances in gene synthesis, we can accelerate the evolution of a computationally designed enzyme. In only five rounds of evolution, we generated a Kemp eliminase—an enzymatic model system for proton transfer from carbon—that accelerates the proton abstraction step >108-fold over the uncatalyzed reaction. Recombining the resulting variant with a previously evolved Kemp eliminase HG3.17, which exhibits similar activity but differs by 29 substitutions, allowed us to chart the topography of the designer enzyme’s fitness landscape, highlighting that a given protein scaffold can accommodate several, equally viable solutions to a specific catalytic problem.

Placing a Full Protein Library Under Pressure

Placing a Full Protein Library Under Pressure

Safe and effective in vivo delivery of DNA and RNA using proteolipid vehicles

Genetic medicines show promise for treating various diseases, yet clinical success has been limited by tolerability, scalability, and immunogenicity issues of current delivery platforms. To overcome these, we developed a proteolipid vehicle (PLV) by combining features from viral and non-viral approaches. PLVs incorporate fusion-associated small transmembrane (FAST) proteins isolated from fusogenic orthoreoviruses into a well-tolerated lipid formulation, using scalable microfluidic mixing. Screening a FAST protein library, we identified a chimeric FAST protein with enhanced membrane fusion activity that improved gene expression from an optimized lipid formulation. Systemically administered FAST-PLVs showed broad biodistribution and effective mRNA and DNA delivery in mouse and non-human primate models. FAST-PLVs show low immunogenicity and maintain activity upon repeat dosing. Systemic administration of follistatin DNA gene therapy with FAST-PLVs raised circulating follistatin levels and significantly increased muscle mass and grip strength. These results demonstrate the promising potential of FAST-PLVs for redosable gene therapies and genetic medicines.

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 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.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.

A batch screening technique for the calculation of chromatographic separability

This paper employs a high-throughput parallel batch (microtiter plate) adsorption screen with sequential salt step increases to rapidly generate protein elution profiles for multiple resins at different pHs using a protein library. The chromatographic set used in this work includes single mode, multimodal anion-exchange (MMA), and multimodal cation-exchange (MMC) resins. The protein library consists of proteins with isoelectric points ranging from 5.1 to 11.4 with varying hydrophobicities as determined by their retention on hydrophobic interaction chromatography. The batch sequential experiments are carried out using one protein at a time with a wide set of resins at multiple pH conditions, thus enabling simple microtiter plate detection. A mathematical formulation is then used to determine the first moment of the distributions from each chromatogram (sequential step elution) generated in the parallel batch experiments. Batch data first moments (expressed in salt concentration) are then compared to results obtained from column linear salt gradient elution, and the techniques are shown to be consistent. In addition, first moment data are used to calculate one-resin separability scores, which are a measure of a resin's ability, at a specified pH, to separate the entire set of proteins in the library from one another. Again, the results from the batch and column experiments are shown to be comparable. The first moment data sets were then employed to calculate the two-resin separability scores, which are a measure of the ability of two resins to synergistically separate the entire set of proteins in the library. Importantly, these results based on the two-resin separability performances derived from the batch and column experiments were again shown to be consistent. This approach for rapidly screening large numbers of chromatographic resins and mobile phase conditions for their elution behavior may prove useful for enabling the rapid discovery of new chromatographic ligands and resins.

Development of a Recombineering System for the Acetogen Eubacterium limosum with Cas9 Counterselection for Markerless Genome Engineering.

Eubacterium limosum is a Clostridial acetogen that efficiently utilizes a wide range of single-carbon substrates and contributes to metabolism of health-associated compounds in the human gut microbiota. These traits have led to interest in developing it as a platform for sustainable CO2-based biofuel production to combat carbon emissions, and for exploring the importance of the microbiota in human health. However, synthetic biology and metabolic engineering in E. limosum have been hindered by the inability to rapidly make precise genomic modifications. Here, we screened a diverse library of recombinase proteins to develop a highly efficient oligonucleotide-based recombineering system based on the viral recombinase RecT. Following optimization, the system is capable of catalyzing ssDNA recombination at an efficiency of up to 2%. Addition of a Cas9 counterselection system eliminated unrecombined cells, with up to 100% of viable cells encoding the desired mutation, enabling creation of genomic point mutations in a scarless and markerless manner. We deployed this system to create a clean knockout of the extracellular polymeric substance (EPS) gene cluster, generating a strain incapable of biofilm formation. This approach is rapid and simple, not requiring laborious homology arm cloning, and can readily be retargeted to almost any genomic locus. This work overcomes a major bottleneck in E. limosum genetic engineering by enabling precise genomic modifications, and provides both a roadmap and associated recombinase plasmid library for developing similar systems in other Clostridia of interest.