bims-enlima 2025-01-26 papers

bims-enlima

Biomed News

on Engineered living materials

Issue of 2025–01–26
34 papers selected by
Rahul Kumar, Tallinna Tehnikaülikool

A screening setup to streamline in vitro engineered living material cultures with the host.
Light-Triggered Protease-Mediated Release of Actin-Bound Cargo from Synthetic Cells.
Constructing mechanosensitive signalling pathways de novo in synthetic cells.
Long-term homeostasis in microbial consortia via auxotrophic cross-feeding.
Bio-inspired interlocking metasurfaces.
Combinatorial Nanoparticle-Bound ssDNA Oligonucleotide Library Synthesized by Split-and-Pool Synthesis.
A practical workflow for cytocompatibility assessment of living therapeutic materials.
Environment signal dependent biocontainment systems for engineered organisms: Leveraging triggered responses and combinatorial systems.
A temperature-inducible protein module for control of mammalian cell fate.
Synthetic cells in tissue engineering.
Bistable Functions and Signaling Motifs in Systems Chemistry: Taking the Next Step Toward Synthetic Cells.
Biophysical assays to test cellular mechanosensing: moving towards high throughput.
Reprocessable and Recyclable Materials for 3D Printing via Reversible Thia-Michael Reactions.
Amphoteric Cross-Linked Cellulose Nanoparticles as a Platform for Immobilization of Proteins and Cells, Enabling Bioanalyte Sensing.
Accelerated enzyme engineering by machine-learning guided cell-free expression.
Giant KASH proteins and ribosomes synergistically establish cytoplasmic biophysical properties in vivo.
Fluorescein-based SynNotch adaptors for regulating gene expression responses to diverse extracellular and matrix-based cues.
Bioinspired hydrophobic pseudo-hydrogel for programmable shape-morphing.
A parallel bioreactor strategy to rapidly determine growth-coupling relationships for bioproduction: a mevalonate case study.
Beyond In Vivo, Pharmaceutical Molecule Production in Cell-Free Systems and the Use of Noncanonical Amino Acids Therein.
'Ship-in-a-Bottle' Integration of pH-Sensitive 3D Proteinaceous Meshes into Microfluidic Channels.
Directed Evolution of Silicatein Reveals Biomineralization Synergism between Protein Sequences.
Mechanobiology of 3D cell confinement and extracellular crowding.
Mature chromatin packing domains persist after RAD21 depletion in 3D.
Machine learning reveals novel compound for the improved production of chitooligosaccharides in Escherichia coli.
Shear-Stress Initiates Signal Two of NLRP3 Inflammasome Activation in LPS-Primed Macrophages through Piezo1.
Tools for Intersectional Optical and Chemical Tagging on Cell Surfaces.
Daily briefing: AI-designed proteins offer lifeline for treating snake bites.
Metabolic Engineering of Yeast.
Cancer cells 'poison' the immune system with tainted mitochondria.
Opportunities in Bottlebrush Block Copolymers for Advanced Materials.
Bottlebrush Pastes as a Platform for Solvent-Free, Injectable, and Shape-Persistent Materials with Tissue-Mimetic Viscoelasticity.
Precision control of cellular functions with a temperature-sensitive protein.
The FIRE biosensor illuminates iron regulatory protein activity and cellular iron homeostasis.

Mater Today Bio. 2025 Feb;30 101437

A screening setup to streamline in vitro engineered living material cultures with the host.

Krupansh Desai, Shrikrishnan Sankaran, Aránzazu Del Campo, Sara Trujillo.

  Engineered living materials (ELMs), which usually comprise bacteria, fungi, or animal cells entrapped in polymeric matrices, offer limitless possibilities in fields like drug delivery or biosensing. Determining the conditions that sustain ELM performance while ensuring compatibility with ELM hosts is essential before testing them in vivo. This is critical to reduce animal experimentation and can be achieved through in vitro investigations. Currently, there are no standards that ensure ELM compatibility with host tissues. Towards this goal, we designed a 96-well plate-based screening method to streamline ELM growth across culture conditions and determine their compatibility potential in vitro. We showed proliferation of three bacterial species encapsulated in hydrogels over time and screened six different cell culture media. We fabricated ELMs in bilayer and monolayer formats and tracked bacterial leakage as a measure of ELM biocontainment. After screening, an appropriate medium was selected that sustained growth of an ELM, and it was used to study cytocompatibility in vitro. ELM cytotoxicity on murine fibroblasts and human monocytes was studied by adding ELM supernatants and measuring cell membrane integrity and live/dead staining, respectively, proving ELM cytocompatibility. Our work illustrates a simple setup to streamline the screening of compatible environmental conditions of ELMs with the host.

Keywords:  Biocompatibility; Engineered living materials; In vitro culture; Living therapeutics

DOI:  https://doi.org/10.1016/j.mtbio.2024.101437
Adv Biol (Weinh). 2025 Jan 18. e2400539

Light-Triggered Protease-Mediated Release of Actin-Bound Cargo from Synthetic Cells.

Mousumi Akter, Hossein Moghimianavval, Gary D Luker, Allen P Liu.

  Synthetic cells offer a versatile platform for addressing biomedical and environmental challenges, due to their modular design and capability to mimic cellular processes such as biosensing, intercellular communication, and metabolism. Constructing synthetic cells capable of stimuli-responsive secretion is vital for applications in targeted drug delivery and biosensor development. Previous attempts at engineering secretion for synthetic cells have been confined to non-specific cargo release via membrane pores, limiting the spatiotemporal precision and specificity necessary for selective secretion. Here, a protein-based platform termed TEV Protease-mediated Releasable Actin-binding Protein (TRAP) is designed and constructed for selective, rapid, and triggerable secretion in synthetic cells. TRAP is designed to bind tightly to reconstituted actin networks and is proteolytically released from bound actin, followed by secretion via cell-penetrating peptide membrane translocation. TRAP's efficacy in facilitating light-activated secretion of both fluorescent and luminescent proteins is demonstrated. By equipping synthetic cells with a controlled secretion mechanism, TRAP paves the way for the development of stimuli-responsive biomaterials, versatile synthetic cell-based biosensing systems, and therapeutic applications through the integration of synthetic cells with living cells for targeted delivery of protein therapeutics.

Keywords:  actin; cell‐penetrating peptide; light‐triggered GUV export; protease‐mediated secretion; synthetic cell

DOI:  https://doi.org/10.1002/adbi.202400539
Biochem Soc Trans. 2025 Jan 21. pii: BST20231285. [Epub ahead of print]

Constructing mechanosensitive signalling pathways de novo in synthetic cells.

James W Hindley.

  Biological mechanotransduction enables cells to sense and respond to mechanical forces in their local environment through changes in cell structure and gene expression, resulting in downstream changes in cell function. However, the complexity of living systems obfuscates the mechanisms of mechanotransduction, and hence the study of these processes in vitro has been critical in characterising the function of existing mechanosensitive membrane proteins. Synthetic cells are biomolecular compartments that aim to mimic the organisation, functionality and behaviours of biological systems, and represent the next step in the development of in vitro cell models. In recent years, mechanosensitive channels have been incorporated into synthetic cells to create de novo mechanosensitive signalling pathways. Here, I will discuss these developments, from the molecular parts used to construct existing pathways, the functionality of such systems, and potential future directions in engineering synthetic mechanotransduction. The recapitulation of mechanotransduction in synthetic biology will facilitate an improved understanding of biological signalling through the study of molecular interactions across length scales, whilst simultaneously generating new biotechnologies that can be applied as diagnostics, microreactors and therapeutics.

Keywords:  mechanotransduction; membranes; synthetic biology; synthetic cells; transmembrane proteins

DOI:  https://doi.org/10.1042/BST20231285
bioRxiv. 2025 Jan 08. pii: 2025.01.08.631749. [Epub ahead of print]

Long-term homeostasis in microbial consortia via auxotrophic cross-feeding.

Nicolas E Grandel, Amanda M Alexander, Xiao Peng, Caroline Palamountain, Razan N Alnahhas, Andrew J Hirning, Krešimir Josić, Matthew R Bennett.

Synthetic microbial consortia are collections of multiple strains or species of engineered organisms living in a shared ecosystem. Because they can separate metabolic tasks among different strains, synthetic microbial consortia have myriad applications in developing biomaterials, biomanufacturing, and biotherapeutics. However, synthetic consortia often require burdensome control mechanisms to ensure that the members of the community remain at the correct proportions. This is especially true in continuous culture systems in which slight differences in growth rates can lead to extinctions. Here, we present a simple method for controlling consortia proportions using cross-feeding in continuous auxotrophic co-culture. We use mutually auxotrophic E. coli with different essential gene deletions and regulate the growth rates of members of the consortium via cross-feeding of the missing nutrients in each strain. We demonstrate precise regulation of the co-culture steady-state ratio by exogenous addition of the missing nutrients. We also model the co-culture's behavior using a system of ordinary differential equations that enable us to predict its response to changes in nutrient concentrations. Our work provides a powerful tool for consortia proportion control with minimal metabolic costs to the constituent strains.

DOI: https://doi.org/10.1101/2025.01.08.631749
Bioinspir Biomim. 2025 Jan 23.

Bio-inspired interlocking metasurfaces.

Ophelia Bolmin, Philip Noell, Brad Boyce.

  Interlocking metasurfaces (ILMs) are patterned arrays of mating features that enable the joining of bodies by constraining motion and transmitting force. They offer an alternative to traditional joining solutions such as mechanical fasteners, welds, and adhesives. This study explores the development of bio-inspired ILMs using a problem-driven bioinspired design (BID) framework. We develop a taxonomy of attachment solutions that considers both biological and engineered systems and derive conventional design principles for ILM design. We develop two engineering implementations to demonstrate concept development using the taxonomy and ILM conventional design principle through the BID framework: one for rapidly assembled bridge truss members and another for modular microrobots. These implementations highlight the potential of BID to enhance performance, functionality, and tunability in ILMs.

Keywords:  Bio-inspired; Interlocking; Metasurfaces; attachment; bio-inspired process; joining; metamaterial

DOI:  https://doi.org/10.1088/1748-3190/adadbb
ACS Appl Bio Mater. 2025 Jan 20. 8(1): 844-853

Combinatorial Nanoparticle-Bound ssDNA Oligonucleotide Library Synthesized by Split-and-Pool Synthesis.

John V L Nguyen, Ahlem Meziadi, Christina Nassif, Dillon Da Fonte, Lidija Malic, Maryam Tabrizian.

  Synthetic ssDNA oligonucleotides hold great potential for various applications, including DNA aptamers, DNA digital data storage, DNA origami, and synthetic genomes. In these contexts, precise control over the synthesis of the ssDNA strands is essential for generating combinatorial sequences with user-defined parameters. Desired features for creating synthetic DNA oligonucleotides include easy manipulation of DNA strands, effective detection of unique DNA sequences, and a straightforward mechanism for strand elongation and termination. In this study, we present a split-and-pool method for generating synthetic DNA oligonucleotides on nanoparticles, enabling the creation of scalable combinatorial libraries. Our approach involves coupling DNA to nanoparticles, ligating double-digested fragments for orientation-specific synthesis, and attaching a final single-digested fragment to ensure strand termination. We assess the quality of our method by characterizing both the DNA and the nanoparticles used as solid supports, confirming that our method produces scalable, combinatorial nanoparticle-bound ssDNA libraries with controllable strand lengths.

Keywords:  DNA library; DNA-functionalized nanoparticles; aptamer; molecular engineering; nucleic acid engineering; oligonucleotide synthesis

DOI:  https://doi.org/10.1021/acsabm.4c01681
Biomater Adv. 2025 Jan 10. pii: S2772-9508(25)00009-3. [Epub ahead of print]169 214182

A practical workflow for cytocompatibility assessment of living therapeutic materials.

Joëlle Aurelie Mekontso, Usama Farrukh, Sara Trujillo, Aránzazu Del Campo.

  Living Therapeutic Materials (LTMs) are a promising alternative to polymeric drug carriers for long term release of biotherapeutics. LTMs contain living drug biofactories that produce the drug using energy sources from the body fluids. To clarify their application potential, it is fundamental to adapt biocompatibility and cytotoxicity assays applied from non-living biomaterials and therapeutics to evaluate how LTMs interact with host cells. Here, we have established a first step in this direction, by developing a practical workflow to parallelize in vitro assessment of minimal safety and cytocompatibility properties of bacterial LTMs. It allows systematic monitoring and quantification of the dynamic evolution of the bacterial population (growth, metabolic activity) in parallel to quantify the response of different mammalian cells to LTM supernatants with regards to cytotoxicity and release of pro-inflammatory cytokines over a period of 7 days using a maximum of 10 samples. The protocol was tested with a Pluronic-based thin film containing ClearColi. The results show no cytotoxic effects of ClearColi containing hydrogels in three mammalian cell lines, and no induction of pro-inflammatory cytokines under the tested conditions. This workflow represents a first step in establishing a roadmap for the safety assessment of LTMs, and investigation of biocompatibility potential of future living therapeutic devices.

Keywords:  Bacterial hydrogels; Cytocompatibility; Living therapeutic materials; Pluronic; Workflow

DOI:  https://doi.org/10.1016/j.bioadv.2025.214182
Synth Syst Biotechnol. 2025 Jun;10(2): 356-364

Environment signal dependent biocontainment systems for engineered organisms: Leveraging triggered responses and combinatorial systems.

Shreya Varma, Khushi Ash Gulati, Janani Sriramakrishnan, Riyaa Kedar Ganla, Ritu Raval.

  As synthetic biology advances, the necessity for robust biocontainment strategies for genetically engineered organisms (GEOs) grows increasingly critical to mitigate biosafety risks related to their potential environmental release. This paper aims to evaluate environment signal-dependent biocontainment systems for engineered organisms, focusing specifically on leveraging triggered responses and combinatorial systems. There are different types of triggers-chemical, light, temperature, and pH-this review illustrates how these systems can be designed to respond to environmental signals, ensuring a higher safety profile. It also focuses on combinatorial biocontainment to avoid consequences of unintended GEO release into an external environment. Case studies are discussed to demonstrate the practical applications of these systems in real-world scenarios.

Keywords:  Biocontainment; Combinatorial systems; Engineered organisms; Genetic circuits; Kill switches; Synthetic biology; Triggered responses

DOI:  https://doi.org/10.1016/j.synbio.2024.12.005
Nat Methods. 2025 Jan 23.

A temperature-inducible protein module for control of mammalian cell fate.

William Benman, Zikang Huang, Pavan Iyengar, Delaney Wilde, Thomas R Mumford, Lukasz J Bugaj.

Inducible protein switches are currently limited for use in tissues and organisms because common inducers cannot be controlled with precision in space and time in optically dense settings. Here, we introduce a protein that can be reversibly toggled with a small change in temperature, a stimulus that is both penetrant and dynamic. This protein, called Melt (Membrane localization using temperature) oligomerizes and translocates to the plasma membrane when temperature is lowered. We generated a library of Melt variants with switching temperatures ranging from 30 °C to 40 °C, including two that operate at and above 37 °C. Melt was a highly modular actuator of cell function, permitting thermal control over diverse processes including signaling, proteolysis, nuclear shuttling, cytoskeletal rearrangements and cell death. Finally, Melt permitted thermal control of cell death in a mouse model of human cancer. Melt represents a versatile thermogenetic module for straightforward, non-invasive and spatiotemporally defined control of mammalian cells with broad potential for biotechnology and biomedicine.

DOI: https://doi.org/10.1038/s41592-024-02572-4
Curr Opin Biotechnol. 2025 Jan 21. pii: S0958-1669(24)00188-5. [Epub ahead of print]92 103252

Synthetic cells in tissue engineering.

Anna Burgstaller, Sara Madureira, Oskar Staufer.

Tissue functions rely on complex structural, biochemical, and biomechanical cues that guide cellular behavior and organization. Synthetic cells, a promising new class of biomaterials, hold significant potential for mimicking these tissue properties using simplified, nonliving building blocks. Advanced synthetic cell models have already shown utility in biotechnology and immunology, including applications in cancer targeting and antigen presentation. Recent bottom-up approaches have also enabled synthetic cells to assemble into 3D structures with controlled intercellular interactions, creating tissue-like architectures. Despite these advancements, challenges remain in replicating multicellular behaviors and dynamic mechanical environments. Here, we review recent advancements in synthetic cell-based tissue formation and introduce a three-pillar framework to streamline the development of synthetic tissues. This approach, focusing on synthetic extracellular matrix integration, synthetic cell self-organization, and adaptive biomechanics, could enable scalable synthetic tissues engineering for regenerative medicine and drug development.

DOI: https://doi.org/10.1016/j.copbio.2024.103252
Acc Chem Res. 2025 Jan 22.

Bistable Functions and Signaling Motifs in Systems Chemistry: Taking the Next Step Toward Synthetic Cells.

Indrajit Maity, Nathaniel Wagner, Dharm Dev, Gonen Ashkenasy.

ConspectusA key challenge in modern chemistry research is to mimic life-like functions using simple molecular networks and the integration of such networks into the first functional artificial cell. Central to this endeavor is the development of signaling elements that can regulate the cell function in time and space by producing entities of code with specific information to induce downstream activity. Such artificial signaling motifs can emerge in nonequilibrium systems, exhibiting complex dynamic behavior like bistability, multistability, oscillations, and chaos. However, the de novo, bottom-up design of such systems remains challenging, primarily because the kinetic characteristics and energy aspects yielding bifurcation have not yet been globally defined. We herein review our recent work that focuses on the design and functional analysis of peptide-based networks, propelled by replication reactions and exhibiting bistable behavior. Furthermore, we rationalize and discuss their exploitation and implementation as variable signaling motifs in homogeneous and heterogeneous environments.The bistable reactions constitute reversible second-order autocatalysis as positive feedback to generate two distinct product distributions at steady state (SS), the low-SS and high-SS. Quantitative analyses reveal that a phase transition from simple reversible equilibration dynamics into bistability takes place when the system is continuously fueled, using a reducing agent, to keep it far from equilibrium. In addition, an extensive set of experimental, theoretical, and simulation studies highlight a defined parameter space where bistability operates.Analogous to the arrangement of protein-based bistable motifs in intracellular signaling pathways, sequential concatenation of the synthetic bistable networks is used for signal processing in homogeneous media. The cascaded network output signals are switched and erased or transduced by manipulating the order of addition of the components, allowing it to reach "on demand" either the low-SS or high-SS. The pre-encoded bistable networks are also useful as a programming tool for the downstream regulation of nanoscale materials properties, bridging together the Systems Chemistry and Nanotechnology fields. In such heterogeneous cascade pathways, the outputs of the bistable network serve as input signals for consecutive nanoparticle formation reaction and growth processes, which-depending on the applied conditions-regulate various features of (Au) nanoparticle shape and assembly.Our work enables the design and production of various signaling apparatus that feature higher complexity than previously observed in chemical networks. Future studies, briefly discussed at the end of the Account, will be directed at the design and analysis of more elaborate functionality, such as bistability under flow conditions, multistability, and oscillations. We propose that a profound understanding of the design principles facilitating the replication-based bistability and related functions bear implications for exploring the origin of protein functionality prior to the highly evolved replication-translation-transcription machinery. The integration of our peptide-based signaling motifs within future synthetic cells seems to be a straightforward development of the two alternating states as memory and switch elements for controlling cell growth and division and even communication among different cells. We furthermore suggest that such systems can be introduced into living cells for various biotechnology applications, such as switches for cell temporal and spatial manipulations.

DOI: https://doi.org/10.1021/acs.accounts.4c00703
Biophys Rev. 2024 Dec;16(6): 875-882

Biophysical assays to test cellular mechanosensing: moving towards high throughput.

Marta Cubero-Sarabia, Anna Maria Kapetanaki, Massimo Vassalli.

  Mechanosensitivity is the ability of cells to sense and respond to mechanical stimuli. In order to do this, cells are endowed with different components that allow them to react to a broad range of stimuli, such as compression or shear forces, pressure, and vibrations. This sensing process, mechanosensing, is involved in fundamental physiological mechanisms, such as stem cell differentiation and migration, but it is also central to the development of pathogenic states. Here, we review the approaches that have been proposed to quantify mechanosensation in living cells, with a specific focus on methodologies that enable higher experimental throughput. This aspect is crucial to fully understand the nuances of mechanosensation and how it impacts the physiology and pathology of living systems. We will discuss traditional methods for studying mechanosensing at the level of single cells, with particular attention to the activation of the mechanosensitive ion channel piezo1. Moreover, we will present recent attempts to push the analysis towards higher throughput.

Keywords:  Atomic force microscopy; Cell mechanics; Mechanobiology; Mechanosensitivity; Piezo1

DOI:  https://doi.org/10.1007/s12551-024-01263-w
Angew Chem Int Ed Engl. 2025 Jan 20. e202423522

Reprocessable and Recyclable Materials for 3D Printing via Reversible Thia-Michael Reactions.

Yong-Liang Su, Liang Yue, McKinley K Paul, Joseph Kern, Kaitlyn S Otte, Rampi Ramprasad, H Jerry Qi, Will R Gutekunst.

  The development of chemically recyclable polymers for sustainable 3D printing is crucial to reducing plastic waste and advancing towards a circular polymer economy. Here, we introduce a new class of polythioenones (PCTE) synthesized via Michael addition-elimination ring-opening polymerization (MAEROP) of cyclic thioenone (CTE) monomers. The designed monomers are straightforward to synthesize, scalable and highly modular, and the resulting polymers display mechanical performance superior to commodity polyolefins such as polyethylene and polypropylene. The material was successfully employed in 3D printing using fused-filament fabrication (FFF), showcasing excellent printability and mechanical recyclability. Notably, PCTE-Ph retains its tensile strength and thermal stability after multiple mechanical recycling cycles. Furthermore, PCTE-Ph can be depolymerized back to its original monomer with a 90% yield, allowing for repolymerization and establishing a successful closed-loop life cycle, making it a sustainable alternative for additive manufacturing applications.

Keywords:  3D printing; Michael addition-elimination; Ring-opening polymerization; chemically recyclable polymer; fused-filament fabrication

DOI:  https://doi.org/10.1002/anie.202423522
ACS Appl Mater Interfaces. 2025 Jan 23.

Amphoteric Cross-Linked Cellulose Nanoparticles as a Platform for Immobilization of Proteins and Cells, Enabling Bioanalyte Sensing.

Surya Dev Mishra, Jyotsna Kawadkar, Pradyumna A Joshi, Kamal Vats, Aasheesh Srivastava, Ram Kumar Mishra, Vishal Rai.

  Cellulosic nanomaterials have significantly promoted the development of sensing devices, drug delivery, and bioreactor processes. Their synthetic flexibility makes them a prominent choice for immobilizing biomolecules or cells. In this work, we developed a practical and user-friendly approach to accessing cellulose nanoparticles (CNPs). The synthetic route is convenient and does not require a separate purification protocol. These particles are extensively characterized with FTIR, PXRD, TGA, DLS, and SEM. Later, we functionalized them with two chemically orthogonal handles: hydroxylamine and aldehyde. While the prior engaged glycan on the bacterial surface, the latter could capture an antibiotic to promote an in vitro controlled drug release. Besides, their dense functionalization enables efficient inter-CNP reactions, resulting in an amphoteric covalent cross-linked CNP capable of immobilizing proteins and cells. Also, it enables orthogonal dual immobilization to offer proximity control. Its capabilities were validated by installing an aldehyde-equipped bacterium and an activable fluorophore to offer a platform for detecting H2S, a secretory reductant. It conveniently extends to H2S detection in chicken eggs. Overall, the probe-, enzyme-, and bacterial cell-equipped amphoteric cross-linked CNP offers the potential to support bioprocesses for producing enzymes, secondary metabolites, vitamins, and hormones.

Keywords:  H2S sensing; bacterial immobilization; cellulose nanoparticles; dual-immobilization; enzyme immobilization

DOI:  https://doi.org/10.1021/acsami.4c17239
Nat Commun. 2025 Jan 20. 16(1): 865

Accelerated enzyme engineering by machine-learning guided cell-free expression.

Grant M Landwehr, Jonathan W Bogart, Carol Magalhaes, Eric G Hammarlund, Ashty S Karim, Michael C Jewett.

Enzyme engineering is limited by the challenge of rapidly generating and using large datasets of sequence-function relationships for predictive design. To address this challenge, we develop a machine learning (ML)-guided platform that integrates cell-free DNA assembly, cell-free gene expression, and functional assays to rapidly map fitness landscapes across protein sequence space and optimize enzymes for multiple, distinct chemical reactions. We apply this platform to engineer amide synthetases by evaluating substrate preference for 1217 enzyme variants in 10,953 unique reactions. We use these data to build augmented ridge regression ML models for predicting amide synthetase variants capable of making 9 small molecule pharmaceuticals. Over these nine compounds, ML-predicted enzyme variants demonstrate 1.6- to 42-fold improved activity relative to the parent. Our ML-guided, cell-free framework promises to accelerate enzyme engineering by enabling iterative exploration of protein sequence space to build specialized biocatalysts in parallel.

DOI: https://doi.org/10.1038/s41467-024-55399-0
bioRxiv. 2025 Jan 12. pii: 2025.01.10.632479. [Epub ahead of print]

Giant KASH proteins and ribosomes synergistically establish cytoplasmic biophysical properties in vivo.

Xiangyi Ding, Hongyan Hao, Daniel Elnatan, Patrick Neo Alinaya, Shilpi Kalra, Abby Kaur, Sweta Kumari, Liam J Holt, G W Gant Luxton, Daniel A Starr.

Understanding how cells control their biophysical properties during development remains a fundamental challenge. While cytoplasmic macromolecular crowding affects multiple cellular processes in single cells, its regulation in living animals remains poorly understood. Using genetically encoded multimeric nanoparticles for in vivo rheology, we discovered that C. elegans tissues maintain distinct cytoplasmic biophysical properties that differ from those observed across diverse systems, including bacteria, yeast species, and cultured mammalian cells. We identified two conserved mechanisms controlling cytoplasmic macromolecular diffusion: ribosome concentration, a known regulator of cytoplasmic crowding, works in concert with a previously unknown function for the giant KASH protein ANC-1 scaffolding the endoplasmic reticulum. These findings reveal mechanisms by which tissues establish and maintain distinct cytoplasmic biophysical properties, with implications for understanding cellular organization across species.
One-Sentence Summary: Living tissues maintain unique intracellular biophysical properties under the control of cytoplasmic constraints and crowding.

DOI: https://doi.org/10.1101/2025.01.10.632479
Nat Commun. 2025 Jan 20. 16(1): 852

Fluorescein-based SynNotch adaptors for regulating gene expression responses to diverse extracellular and matrix-based cues.

Jeremy C Tran, Christopher J Kuffner, Alexander M Marzilli, Ryan Emily Miller, Zachary E Silfen, Jeffrey B McMahan, D Christopher Sloas, Christopher S Chen, John T Ngo.

Synthetic Notch (SynNotch) receptors function like natural Notch proteins and can be used to install customized sense-and-respond capabilities into mammalian cells. Here, we introduce an adaptor-based strategy for regulating SynNotch activity via fluorescein isomers and analogs. Using an optimized fluorescein-binding SynNotch receptor, we describe ways to chemically control SynNotch signaling, including an approach based on a bio-orthogonal chemical ligation and a spatially controllable strategy via the photo-patterned uncaging of an o-nitrobenzyl-caged fluorescein conjugate. We further show that fluorescein-conjugated extracellular matrix (ECM)-binding peptides can be used to regulate SynNotch activity depending on the folding state of collagen-based ECM networks. To demonstrate the utility of these tools, we apply them to activate dose-dependent gene expression responses and to induce myogenic-like phenotypes in multipotent fibroblasts with spatiotemporal and microenvironmental control. Overall, we introduce an optimized fluorescein-binding SynNotch as a versatile tool for regulating transcriptional responses to ligands based on the clinically-approved fluorescein dye.

DOI: https://doi.org/10.1038/s41467-025-56148-7
Nat Commun. 2025 Jan 21. 16(1): 875

Bioinspired hydrophobic pseudo-hydrogel for programmable shape-morphing.

Zhigang Wang, Haotian Hu, Zefan Chai, Yuhang Hu, Siyuan Wang, Cheng Zhang, Chunjie Yan, Jun Wang, Wesley Coll, Tony Jun Huang, Xianchen Xu, Heng Deng.

Inspired by counterintuitive water "swelling" ability of the hydrophobic moss of the genus Sphagnum (Peat moss), we prepared a hydrophobic pseudo-hydrogel (HPH), composed of a pure hydrophobic silicone elastomer with a tailored porous structure. In contrast to conventional hydrogels, HPH achieves absorption-induced volume expansion through surface tension induced elastocapillarity, presenting an unexpected absorption-induced volume expansion capability in hydrophobic matrices. We adopt a theoretical framework elucidating the interplay of surface tension induced elastocapillarity, providing insights into the absorption-induced volume expansion behavior. By systematically programming the pore structure, we demonstrate tunable, anisotropic, and programmable absorption-induced expansion. This leads to dedicated self-shaping transformations. Incorporating magnetic particles, we engineer HPH-based soft robots capable of swimming, rolling, and walking. This study demonstrates a unusual approach to achieve water-responsive behavior in hydrophobic materials, expanding the possibilities for programmable shape-morphing in soft materials and soft robotic applications.

DOI: https://doi.org/10.1038/s41467-025-56291-1
Biotechnol Biofuels Bioprod. 2025 Jan 17. 18(1): 6

A parallel bioreactor strategy to rapidly determine growth-coupling relationships for bioproduction: a mevalonate case study.

Alec Banner, Joseph Webb, Nigel Scrutton.

   BACKGROUND: The climate crisis and depleting fossil fuel reserves have led to a drive for 'green' alternatives to the way we manufacture chemicals, and the formation of a bioeconomy that reduces our reliance on petrochemical-based feedstocks. Advances in Synthetic biology have provided the opportunity to engineer micro-organisms to produce compounds from renewable feedstocks, which could play a role in replacing traditional, petrochemical based, manufacturing routes. However, there are few examples of bio-manufactured products achieving commercialisation. This may be partially due to a disparity between academic and industrial focus, and a greater emphasis needs to be placed on economic feasibility at an earlier stage. Terpenoids are a class of compounds with diverse use across fuel, materials and pharmaceutical industries and can be manufactured biologically from the key intermediate mevalonate.
RESULTS: Here, we report on a method of utilising parallel bioreactors to rapidly map the growth-coupling relationship between the specific product formation rate, specific substrate utilisation rate and specific growth rate. Using mevalonate as an example product, a maximum product yield coefficient of 0.18 gp/gs was achieved at a growth rate ( μ ) of 0.34 h-1. However, this process also led to the formation of the toxic byproduct acetate, which can slow growth and cause problems during downstream processing. By using gene editing to knock out the ackA-pta operon and poxB from E. coli BW25113, we were able to achieve the same optimum production rate, without the formation of acetate.
CONCLUSIONS: We demonstrated the power of using parallel bioreactors to assess productivity and the growth-coupling relationship between growth rate and product yield coefficient of mevalonate production. Using genetic engineering, our resultant strain demonstrated rapid mevalonate formation without the unwanted byproduct acetate. Mevalonate production is quantified and reported in industrially relevant units, including key parameters like conversion efficiency that are often omitted in early-stage publications reporting only titre in g/L.

Keywords:  Fermentation; Growth-coupling; Mevalonate; Parallel bioreactor; Scale-up

DOI:  https://doi.org/10.1186/s13068-024-02599-x
Chem Rev. 2025 Jan 22.

Beyond In Vivo, Pharmaceutical Molecule Production in Cell-Free Systems and the Use of Noncanonical Amino Acids Therein.

Marco G Casteleijn, Ulrike Abendroth, Anne Zemella, Ruben Walter, Rashmi Rashmi, Rainer Haag, Stefan Kubick.

Throughout history, we have looked to nature to discover and copy pharmaceutical solutions to prevent and heal diseases. Due to the advances in metabolic engineering and the production of pharmaceutical proteins in different host cells, we have moved from mimicking nature to the delicate engineering of cells and proteins. We can now produce novel drug molecules, which are fusions of small chemical drugs and proteins. Currently we are at the brink of yet another step to venture beyond nature's border with the use of unnatural amino acids and manufacturing without the use of living cells using cell-free systems. In this review, we summarize the progress and limitations of the last decades in the development of pharmaceutical protein development, production in cells, and cell-free systems. We also discuss possible future directions of the field.

DOI: https://doi.org/10.1021/acs.chemrev.4c00126
Nanomaterials (Basel). 2025 Jan 10. pii: 104. [Epub ahead of print]15(2):

'Ship-in-a-Bottle' Integration of pH-Sensitive 3D Proteinaceous Meshes into Microfluidic Channels.

Daniela Serien, Koji Sugioka, Aiko Narazaki.

  Microfluidic sensors incorporated onto chips allow sensor miniaturization and high-throughput analyses for point-of-care or non-clinical analytical tools. Three-dimensional (3D) printing based on femtosecond laser direct writing (fs-LDW) is useful for creating 3D microstructures with high spatial resolution because the structures are printed in 3D space along a designated laser light path. High-performance biochips can be fabricated using the 'ship-in-a-bottle' integration technique, in which functional microcomponents or biomimetic structures are embedded inside closed microchannels using fs-LDW. Solutions containing protein biomacromolecules as a precursor can be used to fabricate microstructures that retain their native protein functions. Here, we demonstrate the ship-in-a-bottle integration of pure 3D proteinaceous microstructures that exhibit pH sensitivity. We fabricated proteinaceous mesh structures with gap sizes of 10 and 5 μm. The sizes of these gaps changed when exposed to physiological buffers ranging from pH of 4 to 10. The size of the gaps in the mesh can be shrunk and expanded repeatedly by changing the pH of the surrounding buffer. Fs-LDW enables the construction of microscopic proteinaceous meshes that exhibit dynamic functions such as pH sensing and might find applications for filtering particles in microfluidic channels.

Keywords:  3D printing; femtosecond laser direct writing; microfluidic integration; pH-actuation

DOI:  https://doi.org/10.3390/nano15020104
ACS Omega. 2025 Jan 14. 10(1): 334-343

Directed Evolution of Silicatein Reveals Biomineralization Synergism between Protein Sequences.

Toriana N Vigil, Mary-Jean C Rowson, Abigail J Frost, Abigail R Janiga, Bryan W Berger.

Biomineralization is a green synthesis route for a variety of metal nanoparticles. Silicatein is a biomineralization protein originally found in marine sponge Tethya aurantia that converts inorganic precursors to metal oxide nanoparticles. In this work, we investigate the popular catalytic triad hypothesis and implement directed evolution with the aim to improve the solubility and kinetics of silicatein to enable increased nanoparticle synthesis. Site-directed mutagenesis with catalytic triad residues did not abolish biomineralization activity, aligning with the results seen in one previous study. Recombinant production of silicatein and mutants in Escherichia coli following library generation and a survival screen yielded several mutant proteins with augmented biomineralization activity. Sequence analysis of these mutant proteins reveals multiple sequences within a single cell that contribute to enhanced biomineralization. Combined with the sequence analysis of silicateins from different marine sponges, these results suggest the protein is permissive to wide sequence variations and that multiple protein sequences act synergistically for enhanced biomineralization.

DOI: https://doi.org/10.1021/acsomega.4c06359
Biophys Rev. 2024 Dec;16(6): 833-849

Mechanobiology of 3D cell confinement and extracellular crowding.

Gabriela Da Silva André, Céline Labouesse.

  Cells and tissues are often under some level of confinement, imposed by the microenvironment and neighboring cells, meaning that there are limitations to cell size, volume changes, and fluid exchanges. 3D cell culture, increasingly used for both single cells and organoids, inherently impose levels of confinement absent in 2D systems. It is thus key to understand how different levels of confinement influences cell survival, cell function, and cell fate. It is well known that the mechanical properties of the microenvironment, such as stiffness and stress relaxation, are important in activating mechanosensitive pathways, and these are responsive to confinement conditions. In this review, we look at how low, intermediate, and high levels of confinement modulate the activation of known mechanobiology pathways, in single cells, organoids, and tumor spheroids, with a specific focus on 3D confinement in microwells, elastic, or viscoelastic scaffolds. In addition, a confining microenvironment can drastically limit cellular communication in both healthy and diseased tissues, due to extracellular crowding. We discuss potential implications of extracellular crowding on molecular transport, extracellular matrix deposition, and fluid transport. Understanding how cells sense and respond to various levels of confinement should inform the design of 3D engineered matrices that recapitulate the physical properties of tissues.

Keywords:  Compressive stresses; Confinement; Macromolecular crowding; Mechanotransduction; Organoid; Tumor spheroid

DOI:  https://doi.org/10.1007/s12551-024-01244-z
Sci Adv. 2025 Jan 24. 11(4): eadp0855

Mature chromatin packing domains persist after RAD21 depletion in 3D.

Wing Shun Li, Lucas M Carter, Luay Matthew Almassalha, Ruyi Gong, Emily M Pujadas-Liwag, Tiffany Kuo, Kyle L MacQuarrie, Marcelo Carignano, Cody Dunton, Vinayak Dravid, Masato T Kanemaki, Igal Szleifer, Vadim Backman.

Understanding chromatin organization requires integrating measurements of genome connectivity and physical structure. It is well established that cohesin is essential for TAD and loop connectivity features in Hi-C, but the corresponding change in physical structure has not been studied using electron microscopy. Pairing chromatin scanning transmission electron tomography with multiomic analysis and single-molecule localization microscopy, we study the role of cohesin in regulating the conformationally defined chromatin nanoscopic packing domains. Our results indicate that packing domains are not physical manifestation of TADs. Using electron microscopy, we found that only 20% of packing domains are lost upon RAD21 depletion. The effect of RAD21 depletion is restricted to small, poorly packed (nascent) packing domains. In addition, we present evidence that cohesin-mediated loop extrusion generates nascent domains that undergo maturation through nucleosome posttranslational modifications. Our results demonstrate that a 3D genomic structure, composed of packing domains, is generated through cohesin activity and nucleosome modifications.

DOI: https://doi.org/10.1126/sciadv.adp0855
N Biotechnol. 2025 Jan 17. pii: S1871-6784(25)00006-8. [Epub ahead of print]

Machine learning reveals novel compound for the improved production of chitooligosaccharides in Escherichia coli.

Friederike Mey, Gaetan De Waele, Wouter Demeester, Chiara Guidi, Dries Duchi, Tomek Diederen, Hanne Kochuyt, Kirsten Van Huffel, Nicola Zamboni, Willem Waegeman, Marjan De Mey.

  In order to improve predictability of outcome and reduce costly rounds of trial-and-error, machine learning models have been of increasing importance in the field of synthetic biology. Besides applications in predicting genome annotation, process parameters and transcription initiation frequency, such models have also been of help for pathway optimization. The latter is a common strategy in metabolic engineering and improves the production of a desirable compound by optimizing enzyme expression levels of the production pathway. However, engineering steps might not lead to sufficient improvement, and bottlenecks may remain hidden among the hundreds of metabolic reactions occurring in a living cell, especially if the production pathway is highly interconnected with other parts of the cell's metabolism. Here, we use the synthesis of chitooligosaccharides (COS) to show that the production from such complex pathways can be improved by using machine learning models and feature importance analysis to find new compounds with an impact on COS production. We screened Escherichia coli libraries of engineered transcription regulators with an expected broad range of metabolic diversity and trained several machine learning models to predict COS production titers. Subsequent feature analysis led to the finding of iron, whose addition we could show improved COS production in vivo up to 2-fold. Additionally, the analysis revealed important clues for future engineering steps.

Keywords:  Synthetic biology; global regulator engineering; machine learning; metabolic engineering; metabolomics

DOI:  https://doi.org/10.1016/j.nbt.2025.01.005
ACS Appl Mater Interfaces. 2025 Jan 21.

Shear-Stress Initiates Signal Two of NLRP3 Inflammasome Activation in LPS-Primed Macrophages through Piezo1.

Adam Fish, James Forster, Vaishali Malik, Ashish Kulkarni.

  The innate immune system is tightly regulated by a complex network of chemical signals triggered by pathogens, cellular damage, and environmental stimuli. While it is well-established that changes in the extracellular environment can significantly influence the immune response to pathogens and damage-associated molecules, there remains a limited understanding of how changes in environmental stimuli specifically impact the activation of the NLRP3 inflammasome, a key component of innate immunity. Here, we demonstrated how shear stress can act as Signal 2 in the NLRP3 inflammasome activation pathway by treating LPS-primed immortalized bone marrow-derived macrophages (iBMDMs) with several physiologically relevant magnitudes of shear stress to induce inflammasome activation. We demonstrated that magnitudes of shear stress within 1.0 to 50 dyn/cm2 were able to induce ASC speck formation, while 50 dyn/cm2 was sufficient to induce significant calcium signaling, gasdermin-D cleavage, caspase-1 activity, and IL-1β secretion, all hallmarks of inflammasome activation. Utilizing NLRP3 and caspase-1 knockout iBMDMs, we demonstrated that the NLRP3 inflammasome was primarily activated as a result of shear stress exposure. Quantitative polymerase chain reaction (qPCR), ELISA, and a small molecule inhibitor study aided us in demonstrating that expression of Piezo1, NLRP3, gasdermin-D, IL-1β, and CCL2 secretion were all upregulated in iBMDMs treated with shear stress. This study provides a foundation for further understanding the interconnected pathogenesis of chronic inflammatory diseases and the ability of shear stress to play a role in their progression.

Keywords:  NLRP3; inflammasome; macrophages; piezo 1; shear-stress

DOI:  https://doi.org/10.1021/acsami.4c18845
ACS Chem Biol. 2025 Jan 21.

Tools for Intersectional Optical and Chemical Tagging on Cell Surfaces.

Sarah Innes-Gold, Hanzeng Cheng, Luping Liu, Adam E Cohen.

We present versatile tools for intersectional optical and chemical tagging of live cells. Photocaged tetrazines serve as "photo-click" adapters between recognition groups on the cell surface and diverse chemical payloads. We describe two new functionalized photocaged tetrazine structures which add a light-gating step to three common cell-targeting chemical methods: HaloTag/chloroalkane labeling, nonspecific primary amine labeling, and antibody labeling. We demonstrate light-gated versions of these three techniques in live cultured cells. We then explore two applications: monitoring tissue flows on the surface of developing zebrafish embryos, and combinatorial multicolor labeling and sorting of optically defined groups of cells. Photoclick adapters add optical control to cell tagging schemes, with modularity in both tag and cell attachment chemistry.

DOI: https://doi.org/10.1021/acschembio.4c00756
Nature. 2025 Jan 16.

Daily briefing: AI-designed proteins offer lifeline for treating snake bites.

Jacob Smith.

DOI: https://doi.org/10.1038/d41586-025-00163-7
Annu Rev Biophys. 2025 Jan 21.

Metabolic Engineering of Yeast.

Shuobo Shi, Yu Chen, Jens Nielsen.

Microbial cell factories have been developed to produce various compounds in a sustainable and economically viable manner. The yeast Saccharomyces cerevisiae has been used as a platform cell factory in industrial biotechnology with numerous advantages, including ease of operation, rapid growth, and tolerance for various industrial stressors. Advances in synthetic biology and metabolic models have accelerated the design-build-test-learn cycle in metabolic engineering, significantly facilitating the development of yeast strains with complex phenotypes, including the redirection of metabolic fluxes to desired products, the expansion of the spectrum of usable substrates, and the improvement of the physiological properties of strain. Strains with enhanced titer, rate, and yield are now competing with traditional petroleum-based industrial approaches. This review highlights recent advances and perspectives in the metabolic engineering of yeasts for the production of a variety of compounds, including fuels, chemicals, proteins, and peptides, as well as advancements in synthetic biology tools and mathematical modeling.

DOI: https://doi.org/10.1146/annurev-biophys-070924-103134
Nature. 2025 Jan 22.

Cancer cells 'poison' the immune system with tainted mitochondria.

Asher Mullard.



Keywords:  Cancer; Cell biology; Immunology

DOI:  https://doi.org/10.1038/d41586-025-00176-2
ACS Nano. 2025 Jan 21. 19(2): 1884-1910

Opportunities in Bottlebrush Block Copolymers for Advanced Materials.

Dipankar Saha, Connor L Witt, Rida Fatima, Takumi Uchiyama, Varun Pande, Dong-Po Song, Hua-Feng Fei, Benjamin M Yavitt, James J Watkins.

  Bottlebrush block copolymers (BBCPs) are a unique class of materials that contain a backbone with densely grafted and chemically distinct polymeric side chains. The nonlinear architecture of BBCPs provides numerous degrees of freedom in their preparation, including control over key parameters such as grafting density, side chain length, block arrangement, and overall molecular weight. This uniquely branched structure provides BBCPs with several important distinctions from their linear counterparts, including sterically induced side chain and backbone conformations, rapid and large self-assembled nanostructures, and reduced or eliminated entanglement effects (assuming sufficient grafting density and that the molecular weight of the side chains is below their respective entanglement molecular weight). These distinctions allow access to large domain sizes, very rapid assembly, and the ability to preferentially add additives and/or precursors to one domain, thereby enabling the efficient fabrication of a wide range of advanced materials and devices. BBCPs have been utilized to create finely controlled and well-ordered nanostructures for use in applications, such as photonic crystals, drug delivery systems, energy conversion, energy storage devices, and key components in surface coatings. To further deploy BBCPs as templates for the formation of precise nanostructures, having a thorough understanding of their synthesis, self-assembly, and templating is necessary. To explore and understand the self-assembly and subsequent applications of BBCPs, this review emphasizes the physics of self-assembly for BBCPs (including architectural, rheological, and thermodynamic considerations) and structure-property relationships between BBCPs and their resulting nanostructures. Lastly, we provide an overview of current research trends using BBCPs in energy storage, energy conversion, photonic, 3D printing, and drug delivery applications. We aim to provide researchers with the fundamentals of BBCP self-assembly in their use as nanostructured materials to continue their development of advanced materials.

Keywords:  Bottlebrush block copolymers; drug delivery; energy conversion; energy storage; morphology; photonics; self-assembly; structure−property; templating

DOI:  https://doi.org/10.1021/acsnano.4c12021
ACS Appl Mater Interfaces. 2025 Jan 22.

Bottlebrush Pastes as a Platform for Solvent-Free, Injectable, and Shape-Persistent Materials with Tissue-Mimetic Viscoelasticity.

Jessica Garcia, Foad Vashahi, Akmal Z Umarov, Georgiy G Ageev, Ioannis Moutsios, Dimitri A Ivanov, Andrey V Dobrynin, Sergei S Sheiko.

  Architecturally hindered crystallization of bottlebrush graft copolymers offers a reaction- and solvent-free pathway for creating injectable elastomers with tissue-mimetic softness. Currently, injectable materials involve solvents and chemical reactions, leading to uncontrolled swelling, leaching of unreacted moieties, and side reactions with tissue. To address this issue, bottlebrush copolymers with a poly(ethylene glycol) (PEG) amorphous block and crystallizable poly(lactic acid) (PLA) grafted chains (A-g-B) were synthesized, with grafted chains of controlled length arranged along the backbone at controlled spacing. The densely grafted PEG brush is leveraged to architecturally control both the rate and degree of crystallization of PLA grafts, offering tunability of mechanical properties as a function of architecture and time in a single-component solvent-free system covering a broad range of aggregation states comprising fluid-, paste-, and elastomer-like behaviors with modulus ranging from 1 to 50 kPa. The PLA-g-PEG pastes are particularly interesting, as they combine solvent-free injectability and time-controlled formation of shape-persistent elastomers at constant temperature. This molecular paste platform may advance reconstructive surgery, drug depots, and tissue engineering.

Keywords:  bottlebrush polymers; crystallization; elastomers; pastes; polymer networks; viscoelasticity

DOI:  https://doi.org/10.1021/acsami.4c19850
Nat Methods. 2025 Jan 23.

Precision control of cellular functions with a temperature-sensitive protein.

DOI: https://doi.org/10.1038/s41592-024-02573-3
Cell Rep Methods. 2025 Jan 11. pii: S2667-2375(24)00350-3. [Epub ahead of print] 100960

The FIRE biosensor illuminates iron regulatory protein activity and cellular iron homeostasis.

Carolyn Sangokoya.

  On Earth, iron is abundant, bioavailable, and crucial for initiating the first catalytic reactions of life from prokaryotes to plants to mammals. Iron-complexed proteins are critical to biological pathways and essential cellular functions. While it is well known that the regulation of iron is necessary for mammalian development, little is known about the timeline of how specific transcripts network and interact in response to cellular iron regulation to shape cell fate, function, and plasticity in the developing embryo and beyond. Here, we present a ratiometric genetically encoded dual biosensor called FIRE (Fe-IRE [iron-responsive element]) to evaluate iron regulatory protein (IRP)-binding activity and cellular iron status in live cells, allowing for the study and dissection of dynamic changes in cellular iron and IRP activity over developmental time. FIRE reveals a previously unrecognized foundational timeline of IRP activity and cellular iron homeostasis during stem cell pluripotency transition and early differentiation.

Keywords:  CP: Metabolism; CP: Molecular biology; IRPs; biosensor; embryoid; embryonic stem cells; iron; iron homeostasis; iron regulatory protein; pluripotent stem cells; post-transcriptional regulation; stem cell biology

DOI:  https://doi.org/10.1016/j.crmeth.2024.100960