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  • Out-of-equilibrium microcompartments for the bottom-up integration of metabolic functions.

Out-of-equilibrium microcompartments for the bottom-up integration of metabolic functions.

Nature communications (2018-06-21)
Thomas Beneyton, Dorothee Krafft, Claudia Bednarz, Christin Kleineberg, Christian Woelfer, Ivan Ivanov, Tanja Vidaković-Koch, Kai Sundmacher, Jean-Christophe Baret
ABSTRACT

Self-sustained metabolic pathways in microcompartments are the corner-stone for living systems. From a technological viewpoint, such pathways are a mandatory prerequisite for the reliable design of artificial cells functioning out-of-equilibrium. Here we develop a microfluidic platform for the miniaturization and analysis of metabolic pathways in man-made microcompartments formed of water-in-oil droplets. In a modular approach, we integrate in the microcompartments a nicotinamide adenine dinucleotide (NAD)-dependent enzymatic reaction and a NAD-regeneration module as a minimal metabolism. We show that the microcompartments sustain a metabolically active state until the substrate is fully consumed. Reversibly, the external addition of the substrate reboots the metabolic activity of the microcompartments back to an active state. We therefore control the metabolic state of thousands of independent monodisperse microcompartments, a step of relevance for the construction of large populations of metabolically active artificial cells.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Fluorescein di(β-D-galactopyranoside)
Sigma-Aldrich
Adenosine 5′-triphosphate disodium salt hydrate, 99%
Sigma-Aldrich
Glucose-6-phosphate Dehydrogenase from Leuconostoc mesenteroides, recombinant, expressed in E. coli, lyophilized powder, ≥550 units/mg protein (biuret)
Sigma-Aldrich
Citrate Synthase from porcine heart, ammonium sulfate suspension, ≥100 units/mg protein
Sigma-Aldrich
Glycerokinase from Cellulomonas sp., lyophilized powder, 25-75 units/mg protein
Sigma-Aldrich
β-Nicotinamide adenine dinucleotide, reduced disodium salt hydrate, ≥97% (HPLC)
Sigma-Aldrich
β-Galactosidase from Escherichia coli, Grade VI, lyophilized powder, ≥250 units/mg protein
Sigma-Aldrich
α-Glycerophosphate Dehydrogenase from rabbit muscle, Type I, ammonium sulfate suspension, 100-300 units/mg protein