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At least 199 records · Page 11Linked to original sources

bioRxiv2023

ADEVO: Proof-of-concept of Adenovirus Directed EVOlution by random peptide display on the fiber knob

Directed evolution of viral vectors involves the generation of randomized libraries followed by artificial selection of improved variants. Directed evolution only yielded limited results in adenovirus vector (AdV) development until now, mainly due to insufficient complexities of randomized libraries. Clinical applications of AdVs as gene therapy or oncolytic vectors are still hampered by the predetermined tropism of natural types. To overcome this challenge, we hypothesized that the technology of randomized peptide insertions on the capsid surface can be incorporated into the AdV bioengineering toolbox for vector retargeting. Here we developed Adenovirus Directed EVOlution (ADEVO) protocols based on fiber knob peptide display. As a proof-of-concept, HAdV-C5-derived libraries were constructed following three distinct protocols and selected on A549-DCAR cells that lack the HAdV-C5 primary receptor, with the goal of identifying variants able to infect and lyse these tumor cells more efficiently. All protocols enabled the construction of high complexity libraries with up to 9.6x10^5 unique variants, an approximate 100-fold improvement compared to previously published AdV libraries. After selection, the most enriched variants did not display enhanced infectivity but rather more efficient replication and cell lysis. This warrants investigations into potential unsuspected involvement of the fiber protein in adenovirus replication. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=145 SRC="FIGDIR/small/567388v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@187f7baorg.highwire.dtl.DTLVardef@1990555org.highwire.dtl.DTLVardef@12d3c60org.highwire.dtl.DTLVardef@d703da_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2024

Targeting glioblastoma tumor hyaluronan to enhance therapeutic interventions that regulate metabolic cell properties

Despite extensive advances in cancer research, glioblastoma (GBM) still remains a very locally invasive and thus challenging tumor to treat, with a poor median survival. Tumor cells remodel their microenvironment and utilize extracellular matrix to promote invasion and therapeutic resistance. We aim here to determine how GBM cells exploit hyaluronan (HA) to maintain proliferation using ligand-receptor dependent and ligand-receptor independent signaling. We use tissue engineering approaches to recreate the three-dimensional tumor microenvironment in vitro, then analyze shifts in metabolism, hyaluronan secretion, HA molecular weight distribution, as well as hyaluronan synthetic enzymes (HAS) and hyaluronidases (HYAL) activity in an array of patient derived xenograft GBM cells. We reveal that endogenous HA plays a role in mitochondrial respiration and cell proliferation in a tumor subtype dependent manner. We propose a tumor specific combination treatment of HYAL and HAS inhibitors to disrupt the HA stabilizing role in GBM cells. Taken together, these data shed light on the dual metabolic and ligand - dependent signaling roles of hyaluronan in glioblastoma. SignificanceThe control of aberrant hyaluronan metabolism in the tumor microenvironment can improve the efficacy of current treatments. Bioengineered preclinical models demonstrate potential to predict, stratify and accelerate the development of cancer treatments.

bioengineering↗

bioRxiv2024

DNA microbeads for spatio-temporally controlled morphogen release within organoids

Organoids have proven to be powerful in vitro model systems that mimic features of the corresponding tissue in vivo. However, across tissue types and species, organoids still often fail to reach full maturity and function, because biochemical cues cannot be provided from within the organoid to guide their development. The establishment of such tools has been identified as a major goal of the field. Here, we introduce DNA microbeads as a novel tool for implementing spatio-temporally controlled morphogen gradients inside of organoids at any point in their life cycle. The DNA microbeads are formed in a simple one-pot process, they can be stored for a year and their viscoelastic behavior and surface modification is tunable to mimic the corresponding tissue. Employing medaka retinal organoids and early embryos, we show that DNA microbeads can be integrated into embryos and organoids by microinjection and erased in a non-invasive manner with light. Coupling a recombinant surrogate Wnt to the DNA microbeads we demonstrate the spatio-temporally controlled release of the morphogen from the microinjection site, which leads to the formation of retinal pigmented epithelium while maintaining neuroretinal ganglion cells. We were thus able to bioengineer retinal organoids to more closely mirror the cell type diversity of in vivo retinas. The DNA microbead technology can easily be adapted to other organoid applications for improved tissue mimicry.

bioengineering↗

bioRxiv2024

Empowering high-throughput high-content analysis of 3D tumor models: Open-source software for automated non-confocal image analysis

PurposeThe primary aim of this study was to develop an open-source Python-based software for the automated analysis of dynamic cell behaviors in three-dimensional tumor models using non-confocal microscopy. This research seeks to address the existing gap in accessible tools for high-throughput analysis of cancer and endothelial cell dynamics in vitro, facilitating the rapid assessment of drug sensitivity. MethodsOur approach involved annotating over 1000 2 mm Z-stacks of cancer and endothelial cell co-culture model and training machine learning models to automatically calculate cell coverage, cancer invasion depth, and microvessel dynamics. Specifically, cell coverage area was computed using focus stacking and Gaussian mixture models to generate thresholded Z-projections. Cancer invasion depth was determined using a deep neural network binary classification model, measuring the distance between Z-planes with invaded cells. Lastly, microvessel dynamics were assessed through a U-Net Xception-style deep learning model for segmentation, a disperse algorithm for network graph representation, then persistent homology to quantify microvessel length and connectivity. Finally, we reanalyzed an image set from a high-throughput drug screen involving a chemotherapy agent on a 3D cervical and endothelial co-culture model. ResultsThe software accurately measured cell coverage, cancer invasion, and microvessel length, yielding drug sensitivity IC50 values with a 95% confidence level compared to manual calculations. Additionally, it significantly reduced the image processing time from weeks down to hours. ConclusionsOur free and open source software offers an automated solution for quantifying 3D cell behavior in tumor models using non-confocal microscopy, providing the broader Cellular and Molecular Bioengineering community with an alternative to standard confocal microscopy paired with proprietary software.

bioengineering↗

bioRxiv2024

Engineering a wolf spider A-family toxin towards increased antimicrobial activity but low toxicity

Peptides with insecticidal, antimicrobial and/or cytolytic activities, also known as spider venom antimicrobial peptides (AMPs), can be found in the venoms of RTA-clade spiders. They show translational potential as therapeutic leads. A set of 52 AMPs has been described in the Chinese wolf spider (Lycosa shansia), and many have been shown to exhibit antibacterial effects. Here we explored the potential to enhance their antimicrobial activity using bioengineering. We generated a panel of artificial derivatives of an A-family peptide and screened their activity against selected microbial pathogens, vertebrate cells and insects. In several cases, we increased the antimicrobial activity of the derivatives while retaining the low cytotoxicity of the parental molecule. Furthermore, we injected the peptides into adult Drosophila suzukii and found no evidence of insecticidal effects, confirming the low levels of toxicity. Our data therefore suggest that spider venom linear peptides can be modified into more potent antimicrobial agents that could help to battle infectious diseases in the future.

bioengineering↗

bioRxiv2024

zIncubascope: long-term quantitative imaging of multi-cellular assemblies inside an incubator

Recent advances in bioengineering have made it possible to develop increasingly complex biological systems to recapitulate organ functions as closely as possible in vitro. Monitoring the assembly and growth of multi-cellular aggregates, micro-tissues or organoids and extracting quantitative information is a crucial but challenging task required to decipher the underlying morphogenetic mechanisms. We present here an imaging platform designed to be accommodated inside an incubator which provides high-throughput monitoring of cell assemblies over days and weeks. We exemplify the capabilities of our system by investigating human induced pluripotent stem cells (hiPSCs) enclosed in spherical capsules, hiPSCs in tubular capsules and yeast cells in spherical capsules. Combined with a customized pipeline of image analysis, our solution provides insight into the impact of confinement on the morphogenesis of these self-organized systems.

bioengineering↗

bioRxiv2024

Biomimetic fractal topography enhances podocyte maturation in vitro

Cells and tissues in their native environment are organized into intricate fractal structures, which are rarely recapitulated in their culture in vitro. The extent to which fractal patterns that resemble complex topography in vivo influence cell maturation, and the cellular responses to such shape stimulation remain inadequately elucidated. Yet, the application of fractal cues (topographical stimulation via self-similar patterns) as an external input may offer a much-needed solution to the challenge of improving the differentiated cell phenotype in vitro. Here, we established fractality in podocytes, branching highly differentiated kidney cells, and glomerulus structure. Biomimetic fractal patterns derived from glomerular histology were used to generate topographical (2.5-D) substrates for cell culture. Podocytes grown on fractal topography were found to express higher levels of functional markers and exhibit enhanced cell polarity. To track morphological complexities of differentiated podocytes, we employed a fluorescent labelling assay where labelled individual cells are tracked within otherwise optically silent confluent cell monolayer to reveal cell-cell interdigitation. RNAseq analysis suggests enhanced ECM deposition and remodeling in podocytes grown on fractal topography compared to flat surface or non-fractal microcurvature, mediated by YAP signaling. The incorporation of fractal topography into standard tissue culture well plates as demonstrated here may serve as a user-friendly bioengineered platform for high-fidelity cell culture.

bioengineering↗

bioRxiv2024

Bioprinting of aptamer-based programmable bioinks to modulate multiscale microvascular morphogenesis in 4D

Dynamic growth factor presentation influences how individual endothelial cells assemble into complex vascular networks. Here, we developed programmable bioinks that facilitate dynamic VEGF presentation to guide vascular morphogenesis within 3D-bioprinted constructs. We leveraged aptamers high affinity for rapid VEGF sequestration in spatially confined regions and utilized aptamer-complementary sequence (CS) hybridization to tune VEGF release kinetics temporally, days after bioprinting. We show that spatial resolution of programmable bioink, combined with CS-triggered VEGF release, significantly influences alignment, organization, and morphogenesis of microvascular networks in bioprinted constructs. The presence of aptamer-tethered VEGF and the generation of instantaneous VEGF gradients upon CS-triggering restricted hierarchical network formation to the printed aptamer regions at all spatial resolutions. Network properties improved as the spatial resolution decreased, with low-resolution designs yielding the highest network properties. Specifically, CS-treated low-resolution designs exhibited significant vascular network remodeling, with increase in vessel density(1.35-fold), branching density(1.54-fold), and average vessel length(2.19-fold) compared to non-treated samples. Our results suggests that CS acts as an external trigger capable of inducing time-controlled changes in network organization and alignment on-demand within spatially localized regions of a bioprinted construct. We envision that these programmable bioinks will open new opportunities for bioengineering functional, hierarchically self-organized vascular networks within engineered tissues.

bioengineering↗

bioRxiv2024

Tailoring hydrophobicity and strength in spider silk-inspired coatings via thermal treatments

The advent of advanced coatings has transformed material functionalities, extending their roles from basic coverage and visual appeal to include unique properties such as self-healing, superior hydrophobicity, and antimicrobial action. However, the traditional dependency on petrochemical-derived materials for these coatings raises environmental concerns. This study proposes the use of renewable and alternative materials for coating development. We present the use of bioengineered spider silk-inspired protein (SSIP), produced through recombinant technology, as a viable, eco-friendly alternative due to their ease of processing under ambient pressure and the utilization of water as a solvent, alongside their exceptional physicochemical properties. Our research investigates the effects of different thermal treatments and protein concentrations on the mechanical strength and surface water repellency of coatings on silica bases. Our findings reveal a direct correlation between the temperature of heat treatment and the enhancements in surface hydrophobicity and mechanical strength, where elevated temperatures facilitate increased resistance to water and improved mechanical integrity. Consequently, we advocate SSIPs present a promising, sustainable choice for advanced coatings, providing a pathway to fine-tune coating recipes for better mechanical and hydrophobic properties with a reduced ecological footprint, finding potential uses in various fields such as electronics.

bioengineering↗

bioRxiv2024

Reversal of contractile defects by mediating calcium homeostasis in human mini-heart models of heart failure with preserved ejection fraction (HFpEF) leads to first-in-human gene therapy clinical trial

AimsHeart failure with preserved ejection fraction (HFpEF), is a global health problem lacking disease-modifying therapeutic options, reflecting a lack of predictive models for preclinical drug testing. Aligned with FDA Modernization Act 2.0, we aimed to create the first in vitro human-specific mini-heart models of HFpEF, and to test the efficacy of a candidate gene therapy to improve cardiac kinetics and correct the disease phenotype. Methods and ResultsHealthy human pluripotent stem cell-derived ventricular cardiomyocytes were used to bioengineer beating cardiac tissue strips and pumping cardiac chambers. When conditioned with transforming growth factor-{beta}1 and endothelin-1, these mini-heart models exhibited signature disease phenotypes of significantly elevated diastolic force and tissue stiffness, and slowed contraction and relaxation kinetics, with no significant deficit in systolic force or ejection fraction versus unconditioned controls. Bioinformatic analysis of bulk RNA sequencing data from HFpEF mini-heart models and patient ventricular samples identified downregulation of SERCA2a of the calcium signalling pathway as a key differentially expressed gene. After dosage optimization, AAV-mediated expression of SERCA2a abrogated the disease phenotype and improved the cardiac kinetics in HFpEF mini-Hearts. ConclusionsThese findings contributed to FDA approval of an ongoing first-in-human gene therapy clinical trial for HFpEF, with Fast Track designation. We conclude that such human-based disease-specific mini-heart platforms are relevant for target discovery and validation that can facilitate clinical translation of novel cardiac therapies. Translational PerspectiveHeart failure with preserved ejection fraction (HFpEF) is a significant and growing global health concern lacking disease-modifying therapeutic options, reflecting inadequate preclinical models of the disease. Aligned with FDA Modernization Act 2.0, we created the first in vitro human-specific mini-heart models of HFpEF, demonstrated phenotypic disease characteristics of elevated stiffness and slowed kinetics, showed transcriptomic consistency with HFpEF patient data, identified SERCA2a as a key downregulated gene, performed dosing titration of SERCA2a gene therapy, and showed improvement of cardiac kinetics post-treatment. The findings contributed to FDA approval of an ongoing first-in-human gene therapy clinical trial for HFpEF.

bioengineering↗

bioRxiv2024

Development of a Novel Method to Fabricate Highly Functional Human Purkinje Networks

BACKGROUNDIn this study, we present a method to bioengineer functional Purkinje networks using recent advances in laser-based bioprinting. METHODSA custom bioink as formulated using optimized concentrations of polyethylene glycol diacrylate (PEGDA), gelatin methacryloyl (GELMA), lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), and tartrazine. A standard triangle language (STL) model of Purkinje networks was developed based on the mammalian Purkinje network mapped out using India ink staining. A commercial bioprinter, the Lumen X, from CellInk, was used to bioprint Purkinje networks. The biocompatibility of the bioprinted Purkinje networks was tested using iPSCs from healthy donors. Negative molds of the Purkinje networks were designed to simulate interaction between Purkinje cells and adjacent cardiomyocytes using different degrees of overlap between the two cell types. The negative molds were also shown to be biocompatible, based on the culture of iPSCs derived from healthy donors. RESULTSWe were able to successfully bioprint over 100 Purkinje networks and demonstrate biocompatibility with iPSCs for up to 7 days. Three different configurations of the negative molds were designed and fabricated and all three shown to be biocompatible with iPSCs for up to 7 days. A co-culture system was developed by placing the Purkinje networks in proximity to the negative molds for all three configurations designed. CONCLUSIONOur results demonstrate the ability to bioprint Purkinje networks and molds and provide an in vitro system to study the functional interaction between Purkinje cells and adjacent cardiomyocytes.

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bioRxiv2025

Crosslinker-free in situ hydrogel induces self-aggregation of human dental pulp stem cells with enhanced antibacterial activity

Recently, injectable hydrogels have garnered significant attention in tissue engineering due to their controlled flowability, strong plasticity, adaptability, and good biocompatibility. However, research on readily injectable in situ-forming hydrogels capable of forming functional three-dimensional (3D) tissue condensations remains limited. This study explores the development and evaluation of a carboxymethyl chitosan (CMCTS) / oxidized hyaluronic acid (oHA) hydrogel incorporated with silver sulfadiazine (AgSD) for tissue engineering applications with inherent antibacterial activity. Through physicochemical analysis, the optimal formulation of CMCTS/oHA hydrogels was established. The hydrogel demonstrated excellent injectability, enabling minimally invasive in situ delivery. In vitro cytotoxicity assays identified 0.1% AgSD as the optimal concentration, supporting cell proliferation while exhibiting antimicrobial efficacy against S. mutans and E. faecalis. In vivo studies revealed complete hydrogel degradation and good biocompatibility, with no adverse tissue reactions. The hydrogels ability to form 3D cell aggregates and support tissue regeneration underscores its potential for future 3D tissue engineering applications. Consequently, the injectable CMCTS/oHA/AgSD hydrogel developed in this study holds significant potential for application in a wide range of bioengineering fields, including antibacterial substance delivery systems and 3D tissue engineering, indicating potential for future clinical application.

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bioRxiv2025

Stable, Easy-to-Handle, Fully Autologous Electrospun Polymer-Peptide Skin Equivalent for Severe Burn Injuries

Severe burn injuries represent a significant clinical challenge due to their complex healing process and the high risk of complications, including infection, scarring, and contracture formation. Current therapeutic approaches for burn wound treatment include autologous donor-site grafting and advanced cell therapy techniques like cultured epidermal autografts (CEA), which successfully facilitate wound closure through re-epithelialization. However, CEAs are limited by fragility, shrinkage, lack of a dermal layer, and risks of contamination. Here, aiming to overcome these limitations, we developed a personalized skin equivalent featuring an engineered scaffold composed of electrospun polycaprolactone (PCL) functionalized with the bioactive peptide fluorenylmethyloxycarbonyl-phenylalanine-arginine-glycine-aspartic acid (Fmoc-FRGD). This scaffold is designed to mimic the natural extracellular matrix (ECM), promoting cellular adhesion, integration, and proliferation while maintaining structural integrity. In-vitro analysis demonstrated the scaffolds ability to support multi-layered human skin cell growth, while in-vivo experiments confirmed its efficacy in facilitating wound closure and full-thickness skin regeneration in a murine model. This bioengineered skin equivalent is mechanically robust, easy to handle, fully autologous and exhibits no contraction, offering a transformative therapeutic alternative for the treatment of severe burn injuries.

bioengineering↗

bioRxiv2025

High-yield bioproduction of virus-free P4-EKORhE multi-lysin transducing particles as an antimicrobial gene therapeutic

A description of the construction of the bioengineered P4-EKORhE and a comprehensive method for producing very high yields (up to 1012 particles per millilitre) enable the use of virus-like particles to transduce genetically encoded antimicrobials through a combination of synthetic biology and optimised upstream and downstream processing. The final product, a gene-delivered antimicrobial in the form of the multi-lysins cassette, is fully functional before and after packaging within P4-EKORhE particles. The antimicrobial activity of the multilysins cassette, characterized by its lysis proteins, was tested in vivo in both pure bacterial Escherichia coli (E. coli) cultures and in a model of infection using A549 immortalised human epithelial tissue cell cultures. This work exemplifies several bioproduction methods and demonstrates how the virology of the P4 and P2 phages can be harnessed to establish a bioprocess for producing transducing particles at very high yields, avoiding contamination by the natural virus while maintaining the antimicrobial effectiveness of the final product.

bioengineering↗

bioRxiv2025

AI.zymes - A modular platform for evolutionary enzyme design

The ability to create new-to-nature enzymes would substantially advance bioengineering, medicine, and the chemical industry. Despite recent breakthroughs in protein design and structure prediction, designing biocatalysts with activities rivaling those of natural enzymes remains challenging. Here, we present AI.zymes, a modular platform integrating cutting-edge protein engineering algorithms within an evolutionary framework. By combining programs such as Rosetta, ESMFold, ProteinMPNN, and FieldTools in iterative rounds of design and selection, AI.zymes optimizes a broad range of catalytically relevant properties, such as transition state affinity and protein stability. Notably, AI.zymes can also improve properties that are not targeted by the employed design algorithms. For instance, AI.zymes enhanced electrostatic catalysis by iteratively selecting variants with stronger catalytic electric fields. AI.zymes was benchmarked by improving the promiscuous Kemp eliminase activity of ketosteroid isomerase, yielding a 7.7-fold increase in activity after zero-shot experimental validation of only 7 variants. Due to its modularity, AI.zymes can readily incorporate emerging design algorithms, paving the way for a unifying framework for enzyme design. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=114 SRC="FIGDIR/small/633707v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@f2ac2eorg.highwire.dtl.DTLVardef@61b2aorg.highwire.dtl.DTLVardef@e176edorg.highwire.dtl.DTLVardef@46d692_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2025

Lithographic structuring of thermoresponsive hydrogel on a micron scale

Hydrogel microstructures and microchannels are important tools in biophysical bottom-up approaches for studying the interactions between extracellular obstacles or boundaries and cells. In prior studies, several microstructuring techniques, cell sources, and gel types have been employed to mimic static cellular environments. However, the dynamic nature of the extracellular environment, which exerts forces on cells, plays an important role in tissue-level contexts. To date, only few approaches have been implemenmted to mimic these forces of the extracellular environment. Here, we present an approach for generating arrays of hydrogel microstructures, that expand and contract dynamically in response to temperature modulations. To do this, we use a thermoresponsive hydrogel and a custom-built lithographic setup. Through direct lithographic photopatterning of a liquid phase, we fabricate smart hydrogel microstructures at the micrometer scale (10-100 m) with well-defined geometries. These hydrogel microstructures display reversible size changes of over 70% triggered by temperature modulations. Our study represents a step towards mimicking the dynamic properties of biological boundaries and obstacles. The dynamic nature of smart hydrogel microstructures holds the potential to mimic the micron-scale dynamics of extracellular environments. Our results may thus be relevant for the fields of bioengineering and systems level biophysics offering opportunities for manipulating and exploring cellular interactions with dynamic obstacles.

bioengineering↗

bioRxiv2025

Database Chemistry for Genomics-Based Safety and Quality Evaluation of Biologics

Genomics-based safety and quality evaluation studies are advancing the bioindustry by enhancing various aspects, including viral safety, host cell protein (HCP) control, product heterogeneity control, cellular heterogeneity control, and process reproducibility. High-throughput instruments and genome-scale databases are essential in genomics, with the reference genome sequence being the most critical database. The completeness and accuracy of these genome sequences depend on DNA quality, sequencing instruments, read coverage, and assembly strategies. Significant efforts are being made to perfect genome assembly and continuously improve it. However, the quantitative impact of reference genome sequence accuracy on the safety of biologics is not yet fully understood. In this study, we compared and benchmarked six Chinese hamster genomes, including four newly sequenced genomes derived from Chinese hamster cell lines, from an industrial perspective. We also developed database assembly techniques to enhance the safety of biologics. We recommend using two or more independent reference genomes for viral safety studies. For HCP control, we suggest using protein sequences in which trypsin degradation peptides that overlap with high-risk proteins should be masked and unified. Additionally, we can predict microenvironments using single-cell transcriptome data. In bioengineering processes, any nucleotide samples have potential commercial benefits.

bioengineering↗

bioRxiv2025

A high throughput investigation of the binding specificity of carbohydrate-binding modules for synthetic and natural polymers

Carbohydrate-binding modules (CBMs) are non-catalytic domains that enhance enzyme binding to substrates, crucial for polysaccharide degradation. Type A CBMs also show potential for engineering plastic-degrading enzymes due to their high affinity for synthetic polymers. This study presents a high-throughput screening pipeline for characterizing the affinity and specificity of Type A CBMs to the synthetic polymers polyethylene terephthalate (PET), polystyrene (PS), and polyethylene (PE), and the natural polysaccharides cellulose, chitin, and starch. Approximately 800 CBMs from the families CBM2, CBM3, CBM10, and CBM64 were expressed as enhanced green fluorescent protein (EGFP)-fusion proteins and tested for binding using a modified holdup assay, which could produce up to 10,000 data points per day. The screening identified approximately 150 binders for PET and PE, around 250 for PS, and demonstrated family-specific binding patterns for avicel, chitin, and starch. Distinct patterns of substrate affinity and specificity were observed, which allowed for rationalizations of binding at the structural and phylogenetic levels. To demonstrate practical utility, CBMs with high PET affinity were fused to the PET hydrolase LCCICCG, enhancing enzymatic activity on crystalline PET powder by around 5-fold. Importantly, these CBM-enzyme fusions mitigated competitive binding to inert plastic impurities, improving performance in mixed plastic assays. This work significantly expands the known repertoire of CBMs capable of binding synthetic polymers, advances our understanding of CBM-substrate interactions, and provides knowledge for engineering enzymes to tackle plastic pollution, particularly in municipal solid waste where mixed plastics pose significant challenges. Significance StatementImproper recycling of plastic poses a severe environmental threat, with synthetic polymers persisting in ecosystems for decades. Enzymatic recycling of plastic offers a promising solution, with optimization of the enzymes and processes ongoing. To expand the toolkit for engineering enzymes via substrate-binding module fusion, we developed a high-throughput screening platform to identify carbohydrate-binding modules (CBMs) that selectively bind synthetic plastics such as PET, polystyrene, and polyethylene, as well as natural polysaccharides. By screening 797 CBMs, we discovered binding domains with distinct specificities, enabling the design of enzyme fusions that enhance plastic degradation, even in mixed plastic environments. This approach provides insights into protein-substrate interactions that can be leveraged for both waste management and bioengineering innovations.

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