Advances in engineering and applications of synthetic phase-separated membraneless organelles in biotechnology

Membraneless organelles (MLOs) formed through liquid-liquid phase separation (LLPS) constitute crucial dynamic microenvironments within cells, capable of selectively concentrating specific molecules and regulating biochemical reactions. Based on the working mechanisms of natural MLOs, researchers have designed and constructed various synthetic MLOs. These MLOs have been applied in regulating enzyme activity, optimizing metabolic pathways, regulating gene expression, producing recombinant proteins, and developing functional biomaterials. Here, we systematically summarized the design strategies, characterization techniques, and client protein recruitment methods for synthetic MLOs, and categorically reviewed their application progress in the biotechnology field. We also discussed current challenges faced in the practical applications of synthetic MLOs and future research directions. This review aims to provide theoretical guidance and practical reference for the design and application of LLPS-driven synthetic MLOs, thereby promoting their innovative development in synthetic biology and biotechnology.

• Publication year

  2026

• Venue

  Synthetic and Systems Biotechnology

• Publication date

  2026-01-22

• Fields of study

  Medicine, Engineering

• Identifiers
  DOI 10.1016/j.synbio.2026.01.007PMID 41624985PMCID 12860260
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar

• No claims are published for this paper.

• No concepts are published for this paper.

• Emerging regulatory mechanisms and functions of biomolecular condensates: implications for therapeutic targets

  2025cited by this paper
• Current practices in the study of biomolecular condensates: a community comment

  2025cited by this paper
• Engineered membraneless organelles in Corynebacterium glutamicum for enhanced indigoidine biosynthesis and antimicrobial peptide production

  2025influential reference
• MambaPhase: deep learning for liquid–liquid phase separation protein classification

  2025cited by this paper
• Raman spectroscopy and imaging of protein droplet formation and aggregation.

  2025cited by this paper
• Light-induced programmable solid-liquid phase transition of biomolecular condensates for improved biosynthesis.

  2025cited by this paper
• Corelet™ Platform: Precision High Throughput Screening for Targeted Drug Discovery of Biomolecular Condensates.

  2025cited by this paper
• Liquid–liquid phase separation-assembled coacervate vesicles for biopharmaceutical delivery

  2025cited by this paper
• Functional Biomaterials Derived from Protein Liquid–Liquid Phase Separation and Liquid‐to‐Solid Transition

  2025cited by this paper
• Applications of Liquid–Liquid Phase Separation in Biosensing

  2025cited by this paper
• Size-controlled assembly of phase separated protein condensates with interfacial protein cages

  2025cited by this paper
• Clickable, Thermally Responsive Hydrogels Enabled by Recombinant Spider Silk Protein and Spy Chemistry for Sustained Neurotrophin Delivery

  2024cited by this paper
• The role of liquid-liquid phase separation in the disease pathogenesis and drug development.

  2024cited by this paper
• De novo engineering of programmable and multi-functional biomolecular condensates for controlled biosynthesis

  2024influential reference
• Spatial organization of translation and translational repression in two phases of germ granules

  2024cited by this paper
• Compartmentalization of pathway sequential enzymes into synthetic protein compartments for metabolic flux optimization in Escherichia coli.

  2024cited by this paper
• A protein pre-trained model-based approach for the identification of the liquid-liquid phase separation (LLPS) proteins.

  2024cited by this paper
• Unveiling intracellular phase separation: advances in optical imaging of biomolecular condensates.

  2024cited by this paper
• Phase-separating peptide coacervates with programmable material properties for universal intracellular delivery of macromolecules

  2024cited by this paper
• Revolutionizing orthopedic healthcare: a systematic review unveiling recombinant antimicrobial peptides

  2024cited by this paper
• Phase separation methods for protein purification: A meta‐analysis of purification performance and cost‐effectiveness

  2024cited by this paper
• Regulation of enzymatic reactions by chemical composition of peptide biomolecular condensates

  2024cited by this paper
• Direct observation of a condensate effect on super-enhancer controlled gene bursting.

  2024cited by this paper
• Self-assembly of stabilized droplets from liquid–liquid phase separation for higher-order structures and functions

  2024cited by this paper
• Recent advances in design and application of synthetic membraneless organelles.

  2024influential reference
• Liquid-liquid phase separation (LLPS) in synthetic biosystems

  2024influential reference
• Seq2Phase: language model-based accurate prediction of client proteins in liquid–liquid phase separation

  2023cited by this paper
• Surface tension measurement and calculation of model biomolecular condensates

  2023cited by this paper
• The liquid-to-solid transition of FUS is promoted by the condensate surface

  2023cited by this paper
• Phase-Separated Synthetic Organelles Based on Intrinsically Disordered Protein Domain for Metabolic Pathway Assembly in Escherichia coli.

  2023influential reference
• Study liquid–liquid phase separation with optical microscopy: A methodology review

  2023cited by this paper
• Phase-separated DropCRISPRa platform for efficient gene activation in mammalian cells and mice

  2023cited by this paper
• Engineering synthetic biomolecular condensates

  2023cited by this paper
• Asymmetric oligomerization state and sequence patterning can tune multiphase condensate miscibility

  2023cited by this paper
• Engineered elastin-like polypeptides: An efficient platform for enhanced cancer treatment

  2023cited by this paper
• In vivo liquid–liquid phase separation protects amyloidogenic and aggregation‐prone peptides during overexpression in Escherichia coli

  2022cited by this paper
• Programming cell-surface signaling by phase-separation-controlled compartmentalization

  2022influential reference
• The Evolution of Intein-Based Affinity Methods as Reflected in 30 years of Patent History

  2022cited by this paper
• A high-throughput method for exploring the parameter space of protein liquid-liquid phase separation

  2022cited by this paper
• Biomolecular condensate phase diagrams with a combinatorial microdroplet platform

  2022cited by this paper
• Synthetic protein condensates for cellular and metabolic engineering

  2022cited by this paper
• A brief guideline for studies of phase-separated biomolecular condensates

  2022cited by this paper
• Birnaviridae Virus Factories Show Features of Liquid-Liquid Phase Separation and Are Distinct from Paracrystalline Arrays of Virions Observed by Electron Microscopy

  2022cited by this paper
• Spatial engineering of E. coli with addressable phase-separated RNAs.

  2022influential reference
• Engineering DNA-based synthetic condensates with programmable material properties, compositions, and functionalities

  2022cited by this paper
• Condensed-phase signaling can expand kinase specificity and respond to macromolecular crowding

  2022cited by this paper
• Quantifying phase separation at the nanoscale by dual-color fluorescence cross-correlation spectroscopy (dcFCCS)

  2022cited by this paper
• HARLEY mitigates user bias and facilitates efficient quantification and co-localization analyses of foci in yeast fluorescence images

  2022cited by this paper
• Controlling synthetic membraneless organelles by a red-light-dependent singlet oxygen-generating protein

  2022cited by this paper
• Phase-Separated Multienzyme Compartmentalization for Terpene Biosynthesis in a Prokaryote.

  2022influential reference
• Mechanically Strong Proteinaceous Fibers: Engineered Fabrication by Microfluidics

  2021cited by this paper
• Quantitative theory for the diffusive dynamics of liquid condensates

  2021cited by this paper
• Synthetic Protein Condensates That Inducibly Recruit and Release Protein Activity in Living Cells.

  2021cited by this paper
• Mechanistic dissection of increased enzymatic rate in a phase-separated compartment

  2021cited by this paper
• Designer Membraneless Organelles Sequester Native Factors for Control of Cell Behavior

  2021influential reference
• Molecular Organization of the Early Stages of Nucleosome Phase Separation Visualized by Cryo-Electron Tomography

  2021cited by this paper
• Local thermodynamics govern formation and dissolution of Caenorhabditis elegans P granule condensates

  2021cited by this paper
• Liquid-Liquid Phase Separation: Unraveling the Enigma of Biomolecular Condensates in Microbial Cells

  2021cited by this paper
• Birnaviridae virus factories show features of liquid-liquid phase separation, and are distinct from paracrystalline arrays of virions observed by electron microscopy

  2021cited by this paper
• Phase-separating peptides for direct cytosolic delivery and redox-activated release of macromolecular therapeutics

  2021cited by this paper
• pH-Controlled Coacervate–Membrane Interactions within Liposomes

  2020cited by this paper
• Phase separation at the nanoscale quantified by dcFCCS

  2020cited by this paper
• Spider silk self-assembly via modular liquid-liquid phase separation and nanofibrillation

  2020cited by this paper
• The role of liquid-liquid phase separation in regulating enzyme activity.

  2020cited by this paper
• Behavior control of membrane-less protein liquid condensates with metal ion-induced phase separation

  2020cited by this paper
• Client proximity enhancement inside cellular membrane-less compartments governed by client-compartment interactions

  2020cited by this paper
• De novo engineering of intracellular condensates using artificial disordered proteins

  2020cited by this paper
• Clusters of bacterial RNA polymerase are biomolecular condensates that assemble through liquid–liquid phase separation

  2020cited by this paper
• RNA contributions to the form and function of biomolecular condensates

  2020cited by this paper
• Liquid-liquid phase separation in biology: mechanisms, physiological functions and human diseases

  2020cited by this paper
• Designer protein assemblies with tunable phase diagrams in living cells

  2020cited by this paper
• Comparative roles of charge, π, and hydrophobic interactions in sequence-dependent phase separation of intrinsically disordered proteins

  2020cited by this paper
• Phase-Separated Multienzyme Biosynthesis.

  2020cited by this paper
• Modular Thermal Control of Protein Dimerization

  2019cited by this paper
• Light-based control of metabolic flux through assembly of synthetic organelles

  2019influential reference
• Considerations and Challenges in Studying Liquid-Liquid Phase Separation and Biomolecular Condensates.

  2019influential reference
• Biomimetic composites with enhanced toughening using silk-inspired triblock proteins and aligned nanocellulose reinforcements

  2019cited by this paper
• Modular enzyme assembly for enhanced cascade biocatalysis and metabolic flux

  2019influential reference
• Exploiting mammalian low-complexity domains for liquid-liquid phase separation–driven underwater adhesive coatings

  2019cited by this paper
• Controllable protein phase separation and modular recruitment to form responsive membraneless organelles

  2018cited by this paper
• Visualizing Dynamics of Cell Signaling In Vivo with a Phase Separation-Based Kinase Reporter.

  2018cited by this paper
• High‐efficiency affinity precipitation of multiple industrial mAbs and Fc‐fusion proteins from cell culture harvests using Z‐ELP‐E2 nanocages

  2018cited by this paper
• Fusion of fibroblast growth factor 21 to a thermally responsive biopolymer forms an injectable depot with sustained anti‐diabetic action

  2018cited by this paper
• Visualizing Dynamics of Cell Signaling In Vivo with a Phase Separation-Based Kinase Reporter.

  2018cited by this paper
• Mapping Local and Global Liquid Phase Behavior in Living Cells Using Photo-Oligomerizable Seeds.

  2018influential reference
• Methods and Strategies to Quantify Phase Separation of Disordered Proteins.

  2018cited by this paper
• A Molecular Grammar Governing the Driving Forces for Phase Separation of Prion-like RNA Binding Proteins.

  2018cited by this paper
• Phase Transitions in Biological Systems with Many Components.

  2017cited by this paper
• Spatiotemporal Control of Intracellular Phase Transitions Using Light-Activated optoDroplets.

  2017cited by this paper
• Organization and Function of Non-dynamic Biomolecular Condensates.

  2017cited by this paper
• Genetic visualization of protein interactions harnessing liquid phase transitions

  2017cited by this paper
• Biomolecular condensates: organizers of cellular biochemistry

  2017cited by this paper
• Mussel adhesion – essential footwork

  2017cited by this paper
• One-week glucose control via zero-order release kinetics from an injectable depot of glucagon-like peptide-1 fused to a thermosensitive biopolymer

  2017cited by this paper
• RNA-Based Coacervates as a Model for Membraneless Organelles: Formation, Properties, and Interfacial Liposome Assembly.

  2016cited by this paper
• Compositional Control of Phase-Separated Cellular Bodies.

  2016cited by this paper
• Osteopontin Stabilizes Metastable States Prior to Nucleation during Apatite Formation

  2016cited by this paper
• The road to the synthesis of "difficult peptides".

  2016cited by this paper
• A Liquid-to-Solid Phase Transition of the ALS Protein FUS Accelerated by Disease Mutation.

  2015cited by this paper
• The disordered P granule protein LAF-1 drives phase separation into droplets with tunable viscosity and dynamics

  2015cited by this paper

• No citing papers are available for this paper.