Minimal Out-of-Equilibrium Metabolism for Synthetic Cells: A Membrane Perspective

Abstract

Life-like systems need to maintain a basal metabolism, which includes importing a variety of building blocks required for macromolecule synthesis, exporting dead-end products, and recycling cofactors and metabolic intermediates, while maintaining steady internal physical and chemical conditions (physicochemical homeostasis). A compartment, such as a unilamellar vesicle, functionalized with membrane-embedded transport proteins and metabolic enzymes encapsulated in the lumen meets these requirements. Here, we identify four modules designed for a minimal metabolism in a synthetic cell with a lipid bilayer boundary: energy provision and conversion, physicochemical homeostasis, metabolite transport, and membrane expansion. We review design strategies that can be used to fulfill these functions with a focus on the lipid and membrane protein composition of a cell. We compare our bottom-up design with the equivalent essential modules of JCVI-syn3a, a top-down genome-minimized living cell with a size comparable to that of large unilamellar vesicles. Finally, we discuss the bottlenecks related to the insertion of a complex mixture of membrane proteins into lipid bilayers and provide a semiquantitative estimate of the relative surface area and lipid-to-protein mass ratios (i.e., the minimal number of membrane proteins) that are required for the construction of a synthetic cell.

Keywords: JCVI-syn3a; bottom-up synthetic cells; energy conservation; membrane composition; metabolite transport; minimal metabolism; out-of-equilibrium; physicochemical homeostasis.

Figure 1

Engineering of synthetic cells. Top-down approaches create minimal cells by deleting nonessential sequences from the genomes of living organisms. These are then transplanted into host cells devoid of genetic material. Bottom-up strategies assemble nonliving building blocks into synthetic systems to obtain life-like properties.

Figure 2

Genome size as a function of cell volume. The cell volumes of prokaryotes were obtained from ref (20). For each bacterial species, the respective genome sizes were collected from the NCBI Assembly database (Table S1). The blue line indicates the volume of an LUV with a diameter of 0.4 μm, while the green and red lines report volumes of GUVs of 1.0 and 2.0 μm in diameter, respectively. (A) Data for cell volumes up to 5 μm³. (B) Zoom-in of the data for cell volumes up to 1 μm³.

Figure 3

Lipid species are building blocks for synthetic cells. The biomimetic phosphatidylcholine analogue is from ref (99).

Figure 4

Probabilities of encapsulating soluble components as a function of the vesicle radius. The cumulative probabilities of a vesicle to contain one molecule with a certain (or larger) abundance were calculated from the Poisson probability mass function. The cumulative probability of a vesicle to contain multiple molecules was obtained from the independent probabilities of each molecule (see Supporting Information). (A) Probability of encapsulating one type of molecule at 5 μM concentration. Abundances (=0), (≥1), and (≥10) reflect the probability of finding zero, one (or more), and ten (or more) copies per vesicle. (B) Probability of encapsulating multiple types of molecules (1 to 250), each at 5 μM concentration and each with an abundance of 10 (or more) copies per vesicle.

Figure 5

Energy and redox power provision. Membrane proteins used in the top-down approach and bottom-up designs of building minimal life-like systems. Left panel. Visualization of JCVI-syn3a membrane proteins annotated for energy conservation.^,, Right panel. Overview of membrane proteins proposed for the engineering of energy and redox cofactor provision in bottom-up constructed synthetic cells. Reaction stoichiometries are not specified.

Figure 6

Physicochemical homeostasis. Membrane proteins used in the top-down approach and bottom-up designs of building minimal life-like systems. Left panel. Visualization of JCVI-syn3a membrane proteins annotated for physicochemical homeostasis.^,, Right panel. Overview of membrane proteins proposed for the engineering of physicochemical homeostasis in bottom-up constructed synthetic cells. Reaction or transport stoichiometries are not shown.

Figure 7

Permeability coefficient of metabolites. Permeability coefficients for amino acids, glycerol, weak acids, and water have been determined in vesicles composed of DOPC and POPC lipids at 20 °C.^,, The permeability coefficient of ammonia was determined under identical conditions in vesicles composed of DOPE/DOPC/DOPG (50:12:38 molar). Permeability coefficients for sodium, potassium, and carbon dioxide were taken from different studies. For permeability coefficients lower than 1 × 10^–5 cm/s, membrane transporters are arguably needed in synthetic systems, while compounds with higher permeability coefficients can rely on passive diffusion.

Figure 8

Membrane transport. Membrane proteins used in the top-down approach and bottom-up designs of minimal life-like systems. Left panel. Visualization of JCVI-syn3a membrane proteins annotated for membrane transport.^,, Right panel. Overview of membrane proteins proposed for the engineering of membrane transport in bottom-up constructed synthetic cells. Although JCVI-syn3a uses, in many cases, ATP-driven transport systems, we envision that structurally simpler proton- or sodium coupled transporters could be used in the bottom-up constructed synthetic cell. Transport stoichiometries are not shown.

Figure 9

Membrane expansion. Membrane proteins used in the top-down approach and bottom-up designs of minimal life-like systems. Left panel. Visualization of JCVI-syn3a membrane proteins annotated for membrane expansion.^,, Right panel. Overview of membrane proteins proposed for the engineering of membrane expansion in bottom-up constructed synthetic cells. Stoichiometries are not represented.

Figure 10

Membrane surface area demand for a minimal metabolism in synthetic cells. (A) Relative protein surface occupancy as a function of the vesicle radius. The relative protein surface occupancy represents the cumulative section area of the JCVI-syn3a membrane proteins. In one scenario, the JCVI-syn3a membrane protein abundance is taken into account (abundance of JCVI-syn3a, which corresponds to a doubling time of ∼2 h); the limit case where each protein is accounted for just once is also reported (abundance = 1). The physiological range of relative protein surface occupancies found in biological membranes is indicated (red shade). LUVs with r_average ≈ 230 nm fall in this range even if the protein abundance is taken into account (dashed black lines). (B) Lipid-to-protein mass ratio as a function of the vesicle radius. The total membrane protein mass was taken from JCVI-syn3a (abundance of JCVI-syn3a and doubling time of ∼2 h). The limit case of one copy number for each protein (abundance = 1) is also shown. A realistic range of lipid-to-protein mass ratios based on current technologies is shown (blue shade); the lower limit is imposed by technical limitations that affect reconstitution. LUVs with r_average ≈ 230 nm do not fall in the feasible range, and a radius >0.8 μm is required for a lipid-to-protein mass ratio of 20:1 w/w (dashed black lines).