Synthesising a minimal cell with artificial metabolic pathways

The study aims to build a synthetic minimal cell—an artificial vesicle system that reproduces through a network of chemical transformations regulated by an information polymer—capturing core features of living systems (metabolism and reproduction) in a simplified framework. Contemporary cells rely on integrated pathways: energy production, genetic information processing (DNA replication and protein synthesis), and membrane synthesis leading to growth and division. Traditional reconstructions emulate these biological pathways using DNA-encoded proteins within vesicles, but their complexity obscures the contribution of physico-chemical processes. An alternative is to construct minimal cell-like systems where vesicles reproduce based on instructions encoded in nucleic acids or analogous information molecules. The authors previously developed a system where vesicle growth and division are coupled to synthesis of a semi-synthetic information polymer (PAN-ES) templated by AOT vesicles; however, that system lacked an energy production unit and sustainable reproduction. The present work introduces an integrated artificial metabolic pathway with three units—energy production, information polymer synthesis, and membrane growth—to achieve sustained, recursive vesicle reproduction. The approach quantitatively links chemical reaction networks and membrane mechanics to vesicle reproduction, addressing how non-living matter can be organized to mimic life-like reproduction cycles.

The work builds on simplified models of living systems (e.g., Gánti’s chemoton-like schemes) and prior minimal cell research where vesicle growth/division was driven by enzymatic lipid synthesis, template polymerization, or encapsulated biochemical reactions. Previous studies demonstrated autopoietic reproduction of fatty acid vesicles, DNA-coupled vesicle self-reproduction, and enzymatic production of membrane components to induce budding and division. The authors’ earlier AOT-templated polyaniline (PANI-ES) system showed that information polymers can be synthesized on vesicle surfaces and promote membrane growth, but it lacked an upstream energy production step and was not sustainable. Prior work also identified membrane physical parameters (spontaneous curvature, Gaussian curvature rigidity) and inverse-cone lipids (e.g., cholesterol or DLPE) as key to achieving limiting shapes and division. The present study integrates these insights with a defined catalytic cascade (GOD→H2O2→HRPC) and controlled osmotic strategies to sustain multi-generational vesicle reproduction.

- System design (three units): 1) Energy production: D-glucose is oxidized by glucose oxidase (GOD) using dissolved O2 to generate H2O2 (energy currency) and gluconic acid. 2) Information polymer synthesis: H2O2 oxidizes horseradish peroxidase isoenzyme C (HRPC), which catalyzes aniline oxidation to aniline radical cations. On AOT vesicle membranes, specific interactions (hydrogen bonding with the sulfonate head group) template linear para-N–C coupling to form PANI-ES on the outer leaflet. 3) Membrane growth: PANI-ES on AOT vesicles binds external AOT molecules via hydrogen bonding, reducing their hydrophilicity and promoting their incorporation into the membrane, increasing surface area; flip-flop equilibrates leaflets. - Implementation in bulk (LUVs): • AOT LUV suspensions (20 mM NaH2PO4, pH 4.3) containing aniline and HRPC were mixed with an energy production solution containing GOD and D-glucose to initiate the cascade. Formation of PANI-ES was confirmed by UV/Vis/NIR absorption (characteristic polaron band near 1000 nm) and Raman spectroscopy (ν(C=N+) ~1345 cm−1). Controls with DOPC LUVs or without vesicles lacked these signatures. Kinetics of PANI-ES formation were monitored by absorbance at 1000 nm, completing in ~150 s under the tested conditions. - Implementation on GUVs and membrane growth: • AOT GUVs were prepared in conditions optimal for cascade polymerization (e.g., 40 mM aniline, 0.92 μM HRPC in 20 mM NaH2PO4, pH 4.3). A double micro-injection delivered 100 mM D-glucose containing 20 mM AOT micelles to a target GUV to trigger the cascade and supply membrane molecules. Vesicle morphology was monitored by phase-contrast microscopy; deformation from spherical to prolate accompanied growth. Growth curves under varied conditions were compared to kinetic model predictions. - Enabling division via membrane composition: • Cholesterol (inverse-cone shape lipid) was incorporated into AOT GUVs (AOT:Chol ~9:1 mol ratio) to enable deformation to limiting shapes and division. To prevent depletion of Chol during successive generations, mixed micelles of SDBS + Chol were supplied by micro-injection to maintain Chol content in membranes. AOT micelles were co-supplied for sustained membrane growth. - Osmotic strategy for volume recovery (long-term swelling): • To reconcile increasing membrane area with nearly constant internal volume, an asymmetric osmosis strategy was used: GUVs encapsulated 100 mM D-sucrose (low permeability) and were placed in external 100 mM D-fructose (higher permeability) solution containing AOT. Fructose permeation inward, coupled with water influx, maintained an osmotic pressure difference for long-term volume increase. Under these conditions vesicle volume could increase almost linearly up to ~17-fold over ~700 s. During reproduction experiments, micro-injected D-glucose and AOT/SDBS+Chol micelles enabled growth and division while osmotic swelling recovered daughter vesicle volumes. - Kinetic and physical modeling: • A reduced reaction scheme (Eqs. 1–5) captured: GOD-catalyzed H2O2 production; HRPC-mediated aniline activation; initiation and propagation of PANI-ES formation on AOT membranes; and conversion of free membrane area into bilayer area through PANI-ES-mediated AOT uptake. The resulting ODEs (Eqs. 6–16) describe time evolution of energy currency, monomer, polymer, enzyme intermediates, and membrane area; model outputs matched absorbance kinetics at 1000 nm and membrane growth data. • Membrane mechanics were analyzed using the spontaneous curvature model to relate reduced volume and curvature to limiting shapes and division, and the role of Gaussian curvature rigidity differences between components in neck destabilization. - Vesicle and micelle preparation; instrumentation: • GUVs: hydration of dry lipid films (AOT or AOT+Chol) followed by incubation to form GUVs (radii ~30–50 μm). LUVs: freeze–thaw cycles then extrusion through 100 nm membranes. Mixed micelles (SDBS+Chol) prepared from chloroform stocks, dried, rehydrated, and dispersed. • Spectroscopy: UV/Vis/NIR (JASCO V-730) at 25 °C; micro-Raman (Renishaw inVia, 532 nm) with confocal mapping to localize PANI-ES on GUV surfaces. • Microscopy: phase-contrast imaging (Zeiss Axio Vert) with image-based 3D reconstruction to estimate surface area and volume. Double micro-injection delivered D-glucose and micelles to target GUVs under optimized cascade conditions.

- Successful coupling of energy production to information polymer synthesis on AOT vesicles: • GOD-catalyzed oxidation of D-glucose produced H2O2 that drove HRPC-catalyzed polymerization of aniline on AOT vesicles to yield PANI-ES. Evidence: UV/Vis/NIR peak at ~1000 nm (delocalized polarons) and Raman ν(C=N+) at ~1345 cm−1. Polymerization completed on the order of ~150 s under the tested conditions. • Controls with DOPC LUVs or without vesicles showed no characteristic IR/NIR absorption or Raman bands, indicating the necessity of AOT templating for specific PANI-ES formation. - PANI-ES promotes membrane growth: • Micro-injection of 100 mM D-glucose containing 20 mM AOT micelles into AOT GUVs under cascade conditions led to growth and prolate deformation. Quantitative membrane area growth matched predictions of the kinetic model; in contrast, omission of D-glucose or of aniline/HRPC/GOD resulted in minimal or no growth. - Enabling shape transformation and division with cholesterol and sustained supply: • Binary AOT+Chol (9:1) GUVs coupled to PANI-ES synthesis achieved growth to limiting shapes and division. To avoid depletion, external supply of SDBS+Chol mixed micelles maintained Chol levels, enabling repeated divisions. • Observations without osmotic volume recovery: across 47 observations, second-generation vesicles were produced 38 times, third-generation 27 times, and fourth-generation 21 times. - Achieving recursive reproduction with volume recovery: • Using asymmetric osmosis (encapsulated 100 mM D-sucrose; external 100 mM D-fructose + AOT) enabled near-linear long-term volume increase (up to ~17× in ~700 s), supporting recovery of daughter vesicle volumes. • Integrated protocol (AOT+Chol GUVs; double micro-injection of D-glucose plus AOT micelles and SDBS+Chol micelles): complete reproduction cycles including growth, deformation to limiting shape, division, and volume recovery were achieved. In a representative experiment, a mother GUV divided at 67 s into two daughters; one daughter further divided into two granddaughters. By 200 s, the total normalized surface area and volume of the offspring (#2a, #2b, #3b) reached ~2.98 and ~3.02, respectively. • Across 50 experiments under recursive conditions, second-generation vesicles formed in 45 cases, third-generation in 22 cases, and fourth-generation in 14 cases. - Modeling support: • The kinetic ODE model (Eqs. 6–16) reproduced the time courses of PANI-ES formation (absorbance at 1000 nm) and membrane growth. Membrane physics analysis identified conditions (reduced spontaneous curvature around c ≈ 3 and appropriate reduced volume) necessary for symmetric limiting shapes and spontaneous division, with Gaussian curvature rigidity differences aiding neck destabilization.

The findings demonstrate that a minimal artificial metabolic network—comprising energy production (glucose → H2O2), information polymer synthesis (PANI-ES templated by AOT), and membrane growth—can drive recursive vesicle reproduction when coupled with appropriate membrane composition and osmotic control. Specific interactions between aniline radical cations and the AOT sulfonate headgroups encode a para-N–C sequence in PANI-ES (information), which in turn promotes AOT uptake and membrane growth (function/fitness), paralleling genotype–phenotype relationships in biology. Thermodynamically, the enthalpic gain from specific hydrogen bonding in PANI-ES–AOT complexes compensates sequence-ordering entropy loss, stabilizing the encoded polymer. Membrane mechanics clarify how growth leads to division: with an inverse-cone component (e.g., cholesterol) and sufficient reduced spontaneous curvature (c ≈ 3) at appropriate reduced volume, vesicles deform to the limiting shape of two spheres connected by a neck and then divide. Differences in Gaussian curvature rigidities between membrane components promote compositional segregation at the neck, lowering the energy barrier for scission. The balance between AOT uptake (driven by PANI-ES) and flip-flop rates regulates curvature; excessive uptake relative to flip-flop shifts divisions from symmetric to asymmetric. Long-term volume recovery is essential for recursion; asymmetric osmosis using a permeable external osmolyte (fructose) and a less permeable internal osmolyte (sucrose) sustains water influx and volume growth to match increased surface area. Overall, the system provides a quantitative, physically interpretable pathway from chemical transformations to compartment growth and division, offering a simplified analogue to biological reproduction and insights into protocell-like behavior.

This work establishes a synthetic minimal cell that integrates three core functional units—energy production, information polymer synthesis, and membrane growth—into an artificial metabolic pathway capable of driving recursive vesicle reproduction. The GOD–HRPC cascade converts glucose to H2O2 and then to templated PANI-ES on AOT vesicles, which catalyzes AOT incorporation and membrane growth. With cholesterol supplied continuously and osmotic conditions engineered for sustained volume increase, vesicles reproducibly undergo growth, deformation to limiting shapes, division, and multi-generational reproduction (up to four generations in multiple trials). The reactions are captured by a kinetic ODE model, and the membrane transformations are rationalized by elasticity theory and curvature energetics. Future work should refine control over reduced spontaneous curvature and volume to sustain symmetric or controlled asymmetric divisions, improve autonomous supply of membrane components (beyond micro-injection), integrate in situ lipid synthesis, and explore feedback mechanisms between information polymers and membrane composition to approach more life-like, self-regulating minimal cells.

- Dependence on external supply: The system requires micro-injection of glucose and membrane/mixed micelles (AOT and SDBS+Chol); it lacks autonomous in situ lipid synthesis. - Component depletion and composition drift: Without continuous supply, inverse-cone lipid (cholesterol) depletes over generations, halting division; maintaining precise membrane composition is challenging. - Volume–area mismatch: Growth increases surface area faster than volume; recursive reproduction necessitates engineered osmotic conditions (sucrose/fructose) to recover volumes. - Division symmetry control: Divisions tend to become asymmetric when uptake exceeds flip-flop rates; systematic regulation of reduced spontaneous curvature and reduced volume is required for sustainable, controlled reproduction. - Experimental variability: While multi-generational reproduction was observed, success rates decline with generation number (e.g., 45/50 for second, 22/50 for third, 14/50 for fourth under recursive conditions), indicating sensitivity to conditions and limited robustness. - Model simplifications: Kinetic and mechanical models use reduced schemes and assumptions (steady state, homogeneity) that may not capture all spatial/temporal heterogeneities.