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Membraneless organelles formed by liquid-liquid phase separation of proteins or nucleic acids are involved in diverse biological processes in eukaryotes. However, such cellular compartments have yet to be discovered or created synthetically in prokaryotes. Here, we report the formation of liquid protein condensates inside the cells of prokaryotic Escherichia coli upon heterologous overexpression of intrinsically disordered proteins such as spider silk and resilin. In vitro reconstitution under conditions that mimic intracellular physiologically crowding environments of E. coli revealed that the condensates are formed via liquid-liquid phase separation. We also show functionalization of these condensates via targeted colocalization of cargo proteins to create functional membraneless compartments able to fluoresce and to catalyze biochemical reactions. The ability to form and functionalize membraneless compartments may serve as a versatile tool to develop artificial organelles with on-demand functions in prokaryotes for applications in synthetic biology.
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http://dx.doi.org/10.1038/s41589-020-0579-9 | DOI Listing |
Adv Biol Regul
September 2025
Laboratory of Cancer Cell Architecture, Institute of Biochemistry and Experimental Oncology, First Faculty of Medicine, Charles University, Prague, Czech Republic. Electronic address:
Biomolecular condensates (BMCs) are membrane-less organelles formed through liquid-liquid phase separation, primarily driven by multivalent interactions between scaffold and client molecules. These dynamic compartments enable cells to spatially and temporally organize biochemical reactions by locally concentrating specific biomolecules, thereby enhancing the frequency of productive molecular interactions and increasing reaction rates. BMCs are integral to normal cellular physiology, with well-characterized examples including the nucleolus and Cajal bodies.
View Article and Find Full Text PDFCellular biochemistry arises from various interactions between macromolecules, including proteins, nucleic acids, and lipids. These make up membrane-bound organelles, membrane-less compartments, and molecular assemblies and scaffolds. Changes due to stimuli or disease can significantly impact cell fate and metabolism.
View Article and Find Full Text PDFSynth Syst Biotechnol
December 2025
Key Laboratory of High Magnetic Field and Ion Beam Physical Biology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, 230031, China.
Liquid-liquid phase separation (LLPS)-driven membraneless organelles (MLOs) have been employed to enhance metabolic efficiency in various microbial cell factories. However, their application in the industrial bacterium has not been explored. Here, we report the formation of liquid protein condensates in using the RGG domain of LAF-1.
View Article and Find Full Text PDFAngew Chem Int Ed Engl
August 2025
School of Chemistry and Chemical Engineering, Shanxi University, Taiyuan, 030006, China.
Advancing the design and construction of artificial protocells with organized complexity, diverse functionality and practical applicability is urgently demanded in vitro synthetic biology and bioengineering but remains a grand challenge. Here, we present a versatile Pickering emulsion-based encapsulation approach to transform membraneless coacervate compartments into robust multicompartmental hybrid microreactors, which concurrently assimilate the expected attributes of hierarchically compartmentalized structure, molecularly crowded environment, selectively permeable ability and mechanically reinforced stability. Single or multiple biological and non-biological catalytic species can be spatially sequestered in specific domains of the hybrid microreactor, enabling spatiotemporal regulation of individual biocatalysis or divergent cascades with high reaction efficiency.
View Article and Find Full Text PDFBio Protoc
August 2025
Deptartment of Medicine, University of Colorado School of Medicine, Aurora, CO, USA.
Protein synthesis and degradation (i.e., turnover) forms an important part of protein homeostasis and has been implicated in many age-associated diseases.
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