Construction of Out-of-Equilibrium Metabolic Networks in Nano- and Micrometer-Sized Vesicles.

We present a method to incorporate into vesicles complex protein networks, involving integral membrane proteins, enzymes, and fluorescence-based sensors, using purified components. This method is relevant for the design and construction of bioreactors and the study of complex out-of-equilibrium metabolic reaction networks. We start by reconstituting (multiple) membrane proteins into large unilamellar vesicles (LUVs) according to a previously developed protocol. We then encapsulate a mixture of purified enzymes, metabolites, and fluorescence-based sensors (fluorescent proteins or dyes) via freeze-thaw-extrusion and remove non-incorporated components by centrifugation and/or size-exclusion chromatography. The performance of the metabolic networks is measured in real time by monitoring the ATP/ADP ratio, metabolite concentration, internal pH, or other parameters by fluorescence readout. Our membrane protein-containing vesicles of 100-400 nm diameter can be converted into giant-unilamellar vesicles (GUVs), using existing but optimized procedures. The approach enables the inclusion of soluble components (enzymes, metabolites, sensors) into micrometer-size vesicles, thus upscaling the volume of the bioreactors by orders of magnitude. The metabolic network containing GUVs are trapped in microfluidic devices for analysis by optical microscopy.

Biochemical and structural insight into the chemical resistance and cofactor specificity of the formate dehydrogenase from Starkeya novella.

Formate dehydrogenases (Fdhs) mediate the oxidation of formate to carbon dioxide and concomitant reduction of nicotinamide adenine dinucleotide (NAD+ ). The low cost of the substrate formate and importance of the product NADH as a cellular source of reducing power make this reaction attractive for biotechnological applications. However, the majority of Fdhs are sensitive to inactivation by thiol-modifying reagents. In this study, we report a chemically resistant Fdh (FdhSNO ) from the soil bacterium Starkeya novella strictly specific for NAD+ . We present its recombinant overproduction, purification and biochemical characterization. The mechanistic basis of chemical resistance was found to be a valine in position 255 (rather than a cysteine as in other Fdhs) preventing the inactivation by thiol-modifying compounds. To further improve the usefulness of FdhSNO as for generating reducing power, we rationally engineered the protein to reduce the coenzyme nicotinamide adenine dinucleotide phosphate (NADP+ ) with better catalytic efficiency than NAD+ . The single mutation D221Q enabled the reduction of NADP+ with a catalytic efficiency kCAT /KM of 0.4 s-1 ·mm-1 at 200 mm formate, while a quadruple mutant (A198G/D221Q/H379K/S380V) resulted in a fivefold increase in catalytic efficiency for NADP+ compared with the single mutant. We determined the cofactor-bound structure of the quadruple mutant to gain mechanistic evidence behind the improved specificity for NADP+ . Our efforts to unravel the key residues for the chemical resistance and cofactor specificity of FdhSNO may lead to wider use of this enzymatic group in a more sustainable (bio)manufacture of value-added chemicals, as for instance the biosynthesis of chiral compounds.

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

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.