Synthetic Biology Research Articles

Engineered bacteria that self-assemble bioglass polysilicate coatings display enhanced light focusing

Cutting-edge photonic devices frequently rely on microparticle components to focus and manipulate light. Conventional methods used to produce these microparticle components frequently offer limited control of their structural properties or require low-throughput nanofabrication of more complex structures. Here, we employ a synthetic biology approach to produce environmentally friendly, living microlenses with tunable structural properties. We engineered Escherichia coli bacteria to display the silica biomineralization enzyme silicatein from aquatic sea sponges. Our silicatein-expressing bacteria can self-assemble a shell of polysilicate "bioglass" around themselves. Remarkably, the polysilicate-encapsulated bacteria can focus light into intense nanojets that are nearly an order of magnitude brighter than unmodified bacteria. Polysilicate-encapsulated bacteria are metabolically active for up to 4 mo, potentially allowing them to sense and respond to stimuli over time. Our data demonstrate that synthetic biology offers a pathway for producing inexpensive and durable photonic components that exhibit unique optical properties.

Spontaneous Emergence of Lipid Vesicles in a Coacervate-Based Compartmentalized System.

The spontaneous emergence of lipid vesicles in the absence of evolved biological machinery represents a major challenge for bottom-up synthetic biology. We show that coacervate microdroplets could create a compartmentalized environment that enriches lipid molecules and facilitates their spontaneous assembly into lipid vesicles. These vesicles can escape from the coacervate microdroplets in a continuous process under non-equilibrium conditions, resembling a constant production process akin to a "primitive enzyme" factory assembly line. These findings significantly extend our understanding of the intricate interaction between lipid molecules and coacervate microdroplets, shedding light on the emergence of cellular systems and offering a new perspective on the conditions necessary for the development of life on Earth.

Modular Access to C2’‐Aryl/Alkenyl Nucleosides with Electrochemical Stereoselective Cross‐Coupling

AbstractChemically modified oligonucleotides have garnered significant attention in medicinal chemistry, chemical biology, and synthetic biology due to their enhanced stability in vivo compared to naturally occurring oligonucleotides. However, current methods for synthesizing modified nucleosides, particularly at the C2’‐position, are limited in terms of efficiency, modularity, and selectivity. Herein, we report a new approach for the synthesis of highly functionalized C2’‐α‐aryl/alkenyl nucleosides via an electrochemical nickel‐catalyzed cross‐coupling of 2’‐bromo nucleosides with a variety of (hetero)aryl and alkenyl iodides. This method affords a diverse array of C2’‐ α‐aryl/alkenyl nucleosides with excellent stereoselectivity, broad substrate scope, and good functional group compatibility. We further synthesized oligonucleotides incorporating C2’‐aryl‐modified thymidine moieties and demonstrated that their annealed double‐stranded DNAs exhibit decreased melting temperatures (Tm). Additionally, oligonucleotides with C2’‐aryl modifications at the 3’ end showed enhanced resistance to 3’‐exonuclease degradation and C2’‐aryl modifications did not impede the cellular uptake process, highlighting the potential of these modified oligonucleotides for therapeutic applications.

Discovery and Functional Identification of 2,3-Oxidosqualene Cyclases and Cytochrome P450s in Triterpenoid Metabolic Pathways of Actinidia eriantha.

Actinidia eriantha Benth, known as the "king of fruits", is rich in triterpenoid compounds, particularly ursane-type and oleanane-type triterpenic acids. These secondary metabolites have been widely applied in medicine, cosmetics, agriculture, and other fields. To date, key enzyme genes involved in triterpenoid metabolic pathways in A. eriantha remain unexplored. This study employed transcriptome sequencing analysis combined with synthetic biology approaches involving heterologous expression in yeast to identify crucial genes responsible for the biosynthesis of triterpenoid components in A. eriantha: Two 2,3-oxidosqualene cyclases (AeOSC2 and AeOSC3) were characterized to catalyze the formation of major triterpene scaffolds, α-amyrin [precursor of ursolic acid (UA)], β-amyrin [precursor of oleanolic acid (OA)], and ψ-taraxasterol, and two cytochrome P450s (AeCYP716A8 and AeCYP716A9) mediating three-step oxidation at the C-28 position of ursane-type and oleanane-type triterpene scaffolds to form UA, OA, and intermediate oxidation products. We successfully reconstructed the biosynthetic pathway of ursane- and oleanane-type triterpenoids from A. eriantha in a heterologous yeast host and elucidated the two-step enzymatic reactions involved in triterpenoid biosynthesis. These findings lay the foundation for further understanding the biosynthesis of key active components in A. eriantha.

Discovery, Biomanufacture, and Derivatization of Licorice Triterpenoids.

Triterpenoids are the major active constituents of licorice, a well-known traditional medicinal herb. Licorice triterpenoids, represented by glycyrrhizin and glycyrrhetic acid, have a high structural diversity and are excellent lead compounds for the development of potent pharmaceuticals. However, their further application can be limited by insufficient activities, low bioavailability, and the presence of side effects, as well as the inefficiency of traditional plant extraction processes for compound production. To address these issues, researchers are focusing on rare triterpenoid components in the genus Glycyrrhiza and developing derivatives to preserve or enhance the original physiological activities with improved bioavailability and reduced side effects. At the same time, synthetic biology offers opportunities to shorten the production cycle, create eco-friendly manufacturing processes, and reduce the cost of producing licorice triterpenoids. Although much progress has been achieved in this field in recent years, there is still a lack of a comprehensive review to summarize the overall characteristics of licorice triterpenoids rather than glycyrrhizin and glycyrrhetinic acid. Based on this, our review comprehensively outlines the structures, origins, and pharmacological activities of licorice triterpenoids and predicts their pharmacological activities using the drugCIPHER algorithm. Furthermore, this paper reviews the advances and strategies for the biomanufacturing of licorice triterpenoids using synthetic biology methods and outlines the perspectives and structure-activity relationships for the derivatization of licorice triterpenoids. This review provides new insights into the discovery and synthesis of pharmaceuticals derived from natural triterpenes.

Evolution of Vitamin E Production: From Chemical Synthesis and Plant Extraction to Microbial Cell Factories.

Vitamin E, comprising tocopherols and tocotrienols, is an essential antioxidant known for its numerous health benefits. This review traces the evolution of vitamin E production, from traditional chemical synthesis and plant extraction methods to cutting-edge microbial cell factories. Chemical synthesis, while well-established, fails to produce specific stereoisomers, and its application is limited to animal feed due to concerns about chemical residues and limited bioactivity. Plant extraction, although yielding natural vitamin E, is constrained by resource availability and high cultivation costs. Recent advancements in metabolic engineering and synthetic biology have revolutionized vitamin E bioproduction, particularly through the use of engineered microbial cell factories. This review highlights the progress of vitamin E biosynthesis in plants and microorganisms and the key metabolic engineering strategies adopted. We also discuss the existing challenges and future perspectives. When these challenges are overcome, microbial cell factories present a sustainable and effective method to fulfill the increasing demand for high-quality vitamin E.

An independent biosynthetic route to frame a xanthanolide-type sesquiterpene lactone in Asteraceae.

Xanthanolides, also described as seco-guaianolides, are unique sesquiterpene lactones (STLs) with diverse bioactivities. Most of xanthanolides are 12,8-olides based on the position of their lactone ring. The biosynthetic pathway leading to xanthanolides has hitherto been elusive, especially how nature creates the xanthane skeleton is a long-standing question. This study reports the elucidation of a complete biosynthetic pathway to the important 12,8-xanthanolide 8-epi-xanthatin. The xanthane-type backbone is directly derived from the central precursor germacrene-type sesquiterpene, germacrene A acid, via oxidative rearrangement, catalyzed by an unusual cytochrome P450. Subsequently, a 12,8-lactone ring is formed within this xanthane-type backbone resulting in xanthanolides. The biosynthetic pathway for xanthanolides contrasts with the previously unified biosynthetic route for diverse 12,6-guaianolides, in which a 12,6-lactone ring formation precedes the transformation of a germacrene-type skeleton into a guaiane-type structure. The discovery of the full biosynthetic pathway of 8-epi-xanthantin opens new opportunities for producing xanthanolides in microbial organisms using synthetic biology strategies.

Efficient astaxanthin production: Advanced strategies to improve microbial fermentation

AbstractAstaxanthin is a very valuable chemical with strong antioxidant effects, including anti‐cancer, anti‐inflammatory, eye protection, and other properties. The rapid development of synthetic biology has facilitated microbial astaxanthin production, offering environmental benefits, mild reaction conditions, and alignment with consumer demand for natural compounds. Accordingly, this review introduces the latest progress in the production of astaxanthin using different microorganisms including native microbes like Haematococcus pluvialis and Xanthophyllomyces dendrorhous, as well as engineered microbes like Yarrowia lipolytica and Escherichia coli. Methods for improving astaxanthin production through fermentation process regulation and metabolic engineering are reviewed and directions for future work are proposed.

Facile and versatile PDMS-glass capillary double emulsion formation device coupled with rapid purification toward microfluidic giant liposome generation

The exceptional ability of liposomes to mimic a cellular lipid membrane makes them invaluable tools in biomembrane studies and bottom-up synthetic biology. Microfluidics provides a promising toolkit for creating giant liposomes in a controlled manner. Nevertheless, challenges associated with the microfluidic formation of double emulsions, as precursors to giant liposomes, limit the full exploration of this potential. In this study, we propose a PDMS-glass capillary hybrid device as a facile and versatile tool for the formation of double emulsions which not only eliminates the need for selective surface treatment, a well-known problem with PDMS formation chips, but also provides fabrication simplicity and reusability compared to the glass-capillary formation chips. These advantages make the presented device a versatile tool for forming double emulsions with varying sizes (spanning two orders of magnitude in diameter), shell thickness, number of compartments, and choice of solvents. We achieved robust thin shell double emulsion formation by operating the hybrid chip in double dripping mode without performing hydrophilic/phobic treatment a priori. In addition, as an alternative to the conventional, time-consuming density-based separation method, a tandem separation chip is developed to deliver double emulsions free of any oil droplet contamination in a continuous and rapid manner without any need for operator handling. The applicability of the device was demonstrated by forming giant liposomes using the solvent extraction method. This easy-to-replicate, flexible, and reliable microfluidic platform for the formation and separation of double emulsion templates paves the way for the high-throughput microfluidic generation of giant liposomes and synthetic cells, opening exciting avenues for biomimetic research.The presented giant liposome assembly line features a novel treatment-free hybrid chip for double emulsion formation coupled with a high throughput separation chip for sample purification.

Advancing microbiota therapeutics: the role of synthetic biology in engineering microbial communities for precision medicine

Over recent years, studies on microbiota research and synthetic biology have explored novel approaches microbial manipulation for therapeutic purposes. However, fragmented information is available on this aspect with key insights scattered across various disciplines such as molecular biology, genetics, bioengineering, and medicine. This review aims to the transformative potential of synthetic biology in advancing microbiome research and therapies, with significant implications for healthcare, agriculture, and environmental sustainability. By merging computer science, engineering, and biology, synthetic biology allows for precise design and modification of biological systems via cutting edge technologies like CRISPR/Cas9 gene editing, metabolic engineering, and synthetic oligonucleotide synthesis, thus paving the way for targeted treatments such as personalized probiotics and engineered microorganisms. The review will also highlight the vital role of gut microbiota in disorders caused by its dysbiosis and suggesting microbiota-based therapies and innovations such as biosensors for real-time gut health monitoring, non-invasive diagnostic tools, and automated bio foundries for better outcomes. Moreover, challenges including genetic stability, environmental safety, and robust regulatory frameworks will be discussed to understand the importance of ongoing research to ensure safe and effective microbiome interventions.

Light-activated channelrhodopsins: a revolutionary toolkit for the remote control of plant signalling.

Channelrhodopsins (CHRs), originating within algae and protists, are membrane-spanning ion channel proteins that are directly activated and/or deactivated by specific wavelengths of light. Since 2005, CHRs have been deployed as genetically encoded optogenetic tools to rapidly advance understanding of neuronal networks. CHRs provide the opportunity to finely tune ion transport across membranes and regulate membrane potential. These are fundamental biochemical signals, which in plants can be translated into physiological and developmental responses such as changes in photosynthesis, growth, turgor, vascular hydraulics, phosphorylation or reactive oxygen species (ROS) status, gene expression, or even cell death. Exploration of CHR family diversity and structure-function engineering has led to the expansion of the CHR optogenetic toolbox, offering unparalleled opportunities to precisely control and understand electrical and secondary messenger signalling in higher plants. In this Tansley Insight, we provide an overview of the recent progress in the application of CHR optogenetics in higher plants and discuss their possible uses in the remote control of plant biology, illuminating a new future domain for plant research enabled through synthetic biology.

Delivery of Encapsulated Intelligent Engineered Probiotic for Inflammatory Bowel Disease Therapy.

Engineered bacterial therapy holds enormous potential for treating intestinal diseases, employing synthetic biology techniques to achieve localized drug delivery within intestines. However, effective delivery of engineered bacteria to lesion sites and ensuring sustained colonization remain challenging. Here, a mucus encapsulated microsphere gel (MM) delivery system is developed to encapsulate genetically engineered bacteria capable of detecting and treating enteritis. The MM delivery system features an external mucosal coating composed of hyaluronic acid and epigallocatechin gallate, along with internal microspheres of highly biocompatible polyserine modified alginates encapsulating with the engineered probiotics. The MM delivery system effectively protects engineered bacteria harsh environment in stomach and significantly improves intestinal adhesion of the probiotics, extending colonization up to 24h, and does not affect the entry of biomarker or release of Avcystatin. It exhibits notable diagnostic and therapeutic efficacy in inflammatory bowel disease models, thus facilitating the advancement of live biotherapeutic products toward clinical application.

Molecular Decoration and Unconventional Double Bond Migration in Irumamycin Biosynthesis

Irumamycin (Iru) is a complex polyketide with pronounced antifungal activity produced by a type I polyketide (PKS) synthase. Iru features a unique hemiketal ring and an epoxide group, making its biosynthesis and the structural diversity of related compounds particularly intriguing. In this study, we performed a detailed analysis of the iru biosynthetic gene cluster (BGC) to uncover the mechanisms underlying Iru formation. We examined the iru PKS, including the domain architecture of individual modules and the overall spatial structure of the PKS, and uncovered discrepancies in substrate specificity and iterative chain elongation. Two potential pathways for the formation of the hemiketal ring, involving either an olefin shift or electrocyclization, were proposed and assessed using 18O-labeling experiments and reaction activation energy calculations. Based on our findings, the hemiketal ring is likely formed by PKS-assisted double bond migration and TE domain-mediated cyclization. Furthermore, putative tailoring enzymes mediating epoxide formation specific to Iru were identified. The revealed Iru biosynthetic machinery provides insight into the complex enzymatic processes involved in Iru production, including macrocycle sculpting and decoration. These mechanistic details open new avenues for a targeted architecture of novel macrolide analogs through synthetic biology and biosynthetic engineering.

Modular Access to C2'-Aryl/Alkenyl Nucleosides with Electrochemical Stereoselective Cross-Coupling.

Chemically modified oligonucleotides have garnered significant attention in medicinal chemistry, chemical biology, and synthetic biology due to their enhanced stability in vivo compared to naturally occurring oligonucleotides. However, current methods for synthesizing modified nucleosides, particularly at the C2'-position, are limited in terms of efficiency, modularity, and selectivity. Herein, we report a new approach for the synthesis of highly functionalized C2'-a-aryl/alkenyl nucleosides via an electrochemical nickel-catalyzed cross-coupling of 2'-bromo nucleosides with a variety of (hetero)aryl and alkenyl iodides. This method affords a diverse array of C2'- a-aryl/ alkenyl nucleosides with excellent stereoselectivities, broad substrate scope, and good functional group compatibility. We further synthesized oligonucleotides incorporating C2'-aryl-modified thymidine moieties and demonstrated that their annealed double-stranded DNAs exhibit decreased melting temperatures (Tm). Additionally, oligonucleotides with C2'-aryl modifications at the 3' end showed enhanced resistance to 3'-exonuclease degradation and C2'-aryl modifications did not impede the cellular uptake process, highlighting the potential of these modified oligonucleotides for therapeutic applications.

Optimisation of coumaric acid production from aromatic amino acids in Kluyveromyces marxianus

Yeasts are attractive hosts for the production of heterologous products due to their genetic tractability and relative ease of growth. While the baker’s yeast Saccharomyces cerevisiae is a powerful workhorse of the biotechnology industry, the species has metabolic limitations and it is critical that we develop alternative platforms that will facilitate the development of bioprocesses that rely on sustainable feedstocks. In this study, we used synthetic biology tools to construct coumaric acid–producing strains of Kluyveromyces marxianus, a yeast whose physiological traits render it attractive for biotechnology applications. Coumaric acid is a building block in the synthesis of many different families of aromatics and is a key precursor for the synthesis of complect phenylpropanoid molecules, including many flavours and aromas. The starting point for this work was a K. marxianus chassis strain that has increased flux towards the synthesis of tyrosine and phenylalanine, the aromatic amino acids that can serve as starting points for coumaric acid synthesis. Following principles of synthetic biology, a modular approach was taken to identify the best solution to different metabolic possibilities and these were then combined in different ways. For the first step, it was established that the route from phenylalanine was superior to that from tyrosine and the combined overexpression of PlPAL, AtC4H and AtCPR1 delivered the highest yield of coumaric acid. Next, it was established that while Pdc5 and Aro10 both had phenylpyruvate decarboxylase activity, inactivation of ARO10 was sufficient to prevent flux loss in the pathway. Since phenylalanine is the starting point, efforts were made to improve efficiency of its production. It was found that glutamate was a preferred nitrogen source for coumaric acid production, and this knowledge was used to engineer a strain that overexpressed S. cerevisiae GDH1 and delivered higher yields of coumaric acid. Ultimately, this strategy led to the development of strains that has yields of up to 48mg coumaric acid /g glucose. Strains were evaluated in bioreactors to investigate the effects of different process parameters. These analyses indicated that engineered strains face some redox balance challenges and further work will be required overcome these to develop strains that can perform well under industrial conditions.

CTGCT, Centre of Excellence for the Technologies of Gene and Cell Therapy: Collaborative Translation of Scientific Discoveries into Advanced Treatments for Neurological Rare Genetic Diseases and Cancer

The emerging field of precision medicine relies on scientific breakthroughs to understand disease mechanisms and develop cutting-edge technologies to overcome underlying genetic and functional aberrations. The establishment of the Centre of Excellence for the Technologies of Gene and Cell Therapy (CTGCT) at the National Institute of Chemistry (NIC) in Ljubljana represents a significant step forward, as it is the first centre of its kind in Slovenia. The CTGCT is poised to spearhead advances in cancer immunotherapy and personalised therapies for neurological and other rare genetic diseases. The centre’s overarching mission is to extend beyond the NIC’s scientific excellence in basic research and bring new therapeutic solutions toward clinical application. The CTGCT aims to develop a broad pipeline of biomedical tools, including innovative synthetic biology tools, gene editing and splicing technologies, RNA-based technologies, immune regulation engineering and novel viral and non-viral delivery systems. The CTGCT is supported by partner institutions from the UK, the Netherlands and Germany, which already have academic good manufacturing practice (GMP) facilities for the manufacture of advanced therapy medicinal products (ATMPs) and is committed to active collaboration with clinicians and patient organizations at all stages of development to improve access to gene and cell therapies (GCTs) for patients. The Centre also seeks to collaborate with national and international academic and industrial partners, and the newly established GMP facilities will address a critical bottleneck in the translation of GCTs from research to practice. Finally, CTGCT's translational research and technology transfer units will ensure the impactful dissemination of research and innovation activities in Slovenia, throughout the Western Balkans and Eastern Europe region, and beyond. With its comprehensive approach and forward-looking vision, the CTGCT will drive transformative advances in gene and cell therapies for the benefit of patients on a global scale.

An efficient and easily obtainable butelase variant for chemoenzymatic ligation and modification of peptides and proteins

The expanding field of site-specific ligation of proteins and peptides has catalyzed the development of novel methods that enhance molecular modification. Among these methods, enzymatic strategies have emerged as dominant due to their specificity and efficiency in modifying proteins under mild conditions. Asparaginyl endopeptidase is a group of cyclotide-producing cysteine proteases from plants. These plant cysteine proteases, known for their specificity, effectively recognize the tripeptide motif (Asx-Xaa-Yaa) and cleave at the C-terminal side of Asx residues, forming acyl-enzyme intermediates that facilitate transpeptidation. Butelase 1 stands out as the most efficient AEP for protein engineering, yet challenges in its expression and purification limit its accessibility for widespread research and industrial use. To address these challenges, we engineered a new, catalytically efficient variant of Butelase 1, Butelase AY, by mutating the gatekeeping residues Val237Ala and Thr238Tyr within the LAD-1 region. These modifications significantly enhanced the stability and yield of Butelase AY, allowing for successful application in various peptide and protein engineering tasks. Butelase AY was tested on the peptide GLGKY, the globular protein GFP, and the intrinsically disordered protein α-synuclein, effectively labeling them with a fluorescent probe. Notably, Butelase AY maintained its efficiency with substrates containing unnatural amino acids, making it a promising candidate for biorthogonal applications. Importantly, the mutations did not compromise the enzyme’s specificity, as it continued to process model peptides and native protein substrates with N-term NHV recognition motifs effectively. In conclusion, Butelase AY presents a novel recombinant tool for diverse protein labeling and modifications, particularly in biorthogonal strategies. This innovation has the potential to expand applications in biotechnology and therapeutic development, ultimately revolutionizing protein engineering and its utility in synthetic biology.

Electric stimulation: a versatile manipulation technique mediated microbial applications.

Electric stimulation (ES) is a versatile technique that uses an electric field to manipulate microorganisms individually. Over the past several decades, the capabilities of ES have expanded from bioremediation to the precise motion control of cells and microorganisms. However, there is limited information on the underlying mechanisms, latest advancement and broader microbial applications of ES in various fields, such as the production of extracellular polymers with upgraded properties. This review article summarizes recent advancements in ES and discusses it as a unique external manipulation technique for microorganisms with wide applications in bioremediation, industry, biofilm deactivation, disinfection, and controlled biosynthesis. One specific application of ES discussed in this review is the extracellular biosynthesis, regulation, and organization of extracellular polymers, such as bacterial cellulose nanofibrils, curdlan, and microbial nanowires. Overall, this review aims to provide a platform for microbial biotechnologists and synthetic biologists to leverage the manipulation of microorganisms using ES for bio-based applications, including the production of extracellular polymers with enhanced properties. Researchers can engineer, manipulate, and control microorganisms for various applications by harnessing the potential of electric fields.

Genetic engineering of Nannochloropsis oceanica to produce canthaxanthin and ketocarotenoids

BackgroundCanthaxanthin is a ketocarotenoid with high antioxidant activity, and it is primarily produced by microalgae, among which Nannochloropsis oceanica, a marine alga widely used for aquaculture. In the last decade, N. oceanica has become a model organism for oleaginous microalgae to develop sustainable processes to produce biomolecules of interest by exploiting its photosynthetic activity and carbon assimilation properties. N. oceanica can accumulate lipids up to 70% of total dry weight and contains the omega-3 fatty acid eicosapentaenoic acid (EPA) required for both food and feed applications. The genome sequence, other omics data, and synthetic biology tools are available for this species, including an engineered strain called LP-tdTomato, which allows homologous recombination to insert the heterologous genes in a highly transcribed locus in the nucleolus region. Here, N. oceanica was engineered to induce high ketocarotenoid and canthaxanthin production.ResultsWe used N. oceanica LP-tdTomato strain as a background to express the key enzyme for ketocarotenoid production, a β-carotene ketolase (CrBKT) from Chlamydomonas reinhardtii. Through the LP-tdTomato strain, the transgene insertion by homologous recombination in a highly transcribed genomic locus can be screened by negative fluorescence. The overexpression of CrBKT in bkt transformants increased the content of carotenoids and ketocarotenoids per cell, respectively, 1.5 and 10-fold, inducing an orange/red color in the bkt cell cultures. Background (LP) and bkt lines productivity were compared at different light intensities from 150 to 1200 µmol m-2 s-1: at lower irradiances, the growth kinetics of bkt lines were slower compared to LP, while higher productivity was measured for bkt lines at 1200 µmol m-2 s-1. Despite these results, the highest canthaxanthin and ketocarotenoids productivity were obtained upon cultivation at 150 µmol m-2 s-1.ConclusionsThrough targeted gene redesign and heterologous transformation, ketocarotenoids and canthaxanthin content were significantly increased, achieving 0.3% and 0.2% dry weight. Canthaxanthin could be produced using CO2 as the only carbon source at 1.5 mg/L titer. These bkt-engineered lines hold potential for industrial applications in fish or poultry feed sectors, where canthaxanthin and ketocarotenoids are required as pigmentation agents.

Self-organized spatial targeting of contractile actomyosin rings for synthetic cell division

A key challenge for bottom-up synthetic biology is engineering a minimal module for self-division of synthetic cells. Actin-based cytokinetic rings are considered a promising structure to produce the forces required for the controlled excision of cell-like compartments such as giant unilamellar vesicles (GUVs). Despite prior demonstrations of actin ring targeting to GUV membranes and myosin-induced constriction, large-scale vesicle deformation has been precluded due to the lacking spatial control of these contractile structures. Here we show the combined reconstitution of actomyosin rings and the bacterial MinDE protein system within GUVs. Incorporating this spatial positioning tool, able to induce active transport of membrane-attached diffusible molecules, yields self-organized equatorial assembly of actomyosin rings in vesicles. Remarkably, the synergistic effect of Min oscillations and the contractility of actomyosin bundles induces mid-vesicle deformations and vesicle blebbing. Our system showcases how functional machineries from various organisms may be combined in vitro, leading to the emergence of functionalities towards a synthetic division system.