Physicochemical considerations for bottom-up synthetic biology

Śmigiel, Wojciech M.; Lefrançois, Pauline; Poolman, Bert

doi:10.1042/etls20190017

Cited by 21 publications

(26 citation statements)

References 113 publications

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“…56,57 The permeation of formic acid through the phospholipid membrane is a crucial aspect of our pathway design, which can also be used in biomimicking systems with low permeability such as polymerosomes wherein transporters are not functional. 58 A potential next application of our redox system is its integration with synthetic metabolism in vesicles or droplet systems, 51,59,60 thereby generating a higher level of complexity combining ATP as fuel 57,61 and nicotinamide cofactors for redox homeostasis. Recently, water-in-oil droplets containing a pathway for CO 2 fixation 62 (cycle CETCH version 7.0) have yielded the production of glycolate in a light-driven manner by coupling the compartmentalization of thylakoid membranes and formation of ATP and NADPH in the aqueous lumen.…”

Section: ■ Discussionmentioning

confidence: 99%

Minimal Pathway for the Regeneration of Redox Cofactors

Partipilo

Ewins

Frallicciardi

et al. 2021

JACS Au

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Effective metabolic pathways are essential for the construction of in vitro systems mimicking the biochemical complexity of living cells. Such pathways require the inclusion of a metabolic branch that ensures the availability of reducing equivalents. Here, we built a minimal enzymatic pathway confinable in the lumen of liposomes, in which the redox status of the nicotinamide cofactors NADH and NADPH is controlled by an externally provided formate. Formic acid permeates the membrane where a luminal formate dehydrogenase uses NAD + to form NADH and carbon dioxide. Carbon dioxide diffuses out of the liposomes, leaving only the reducing equivalents in the lumen. A soluble transhydrogenase subsequently utilizes NADH for reduction of NADP + thereby making NAD + available again for the first reaction. The pathway is functional in liposomes ranging from a few hundred nanometers in diameter (large unilamellar vesicles) up to several tens of micrometers (giant unilamellar vesicles) and remains active over a period of 7 days. We demonstrate that the downstream biochemical process of reduction of glutathione disulfide can be driven by the transfer of reducing equivalents from formate via NAD(P)H, thereby providing a versatile set of electron donors for reductive metabolism.

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Section: ■ Discussionmentioning

confidence: 99%

Minimal Pathway for the Regeneration of Redox Cofactors

Partipilo

Ewins

Frallicciardi

et al. 2021

JACS Au

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“…The osmotic pressure and pH are critical homeostatic parameters meticulously maintained on the cellular level [ 1 ]. Whereas osmotic pressure is believed to be constant within the cell volume, the pH may vary between membranous organelles as required by specific local metabolic activities [ 2 ].…”

Section: Introductionmentioning

confidence: 99%

The Effect of the Osmotically Active Compound Concentration Difference on the Passive Water and Proton Fluxes across a Lipid Bilayer

Przybyło

Drabik

Doskocz

et al. 2021

IJMS

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The molecular details of the passive water flux across the hydrophobic membrane interior are still a matter of debate. One of the postulated mechanisms is the spontaneous, water-filled pore opening, which facilitates the hydrophilic connection between aqueous phases separated by the membrane. In the paper, we provide experimental evidence showing that the spontaneous lipid pore formation correlates with the membrane mechanics; hence, it depends on the composition of the lipid bilayer and the concentration of the osmotically active compound. Using liposomes as an experimental membrane model, osmotically induced water efflux was measured with the stopped-flow technique. Shapes of kinetic curves obtained at low osmotic pressure differences are interpreted in terms of two events: the lipid pore opening and water flow across the aqueous channel. The biological significance of the dependence of the lipid pore formation on the concentration difference of an osmotically active compound was illustrated by the demonstration that osmotically driven water flow can be accompanied by the dissipation of the pH gradient. The application of the Helfrich model to describe the probability of lipid pore opening was validated by demonstrating that the probability of pore opening correlates with the membrane bending rigidity. The correlation was determined by experimentally derived bending rigidity coefficients and probabilities of lipid pores opening.

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“…However, when compared to biological cells, these synthetic systems often lack a transport of reactants and products between the inside of vesicles and their external environment. Consequences are that reaction dynamics cannot be studied due to a fast runout of encapsulated substrates (contained in nanolitre volumes), or when reactions stop when reaching their thermodynamic equilibrium 18 . To overcome this major limitation and reach steady-state activities inside these reactors, a continuous transport of substrate(s) from the outside to the inside of the compartment is required.…”

Section: Introductionmentioning

confidence: 99%