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Synthetic metabolism for in vitro acetone biosynthesis driven by ATP regeneration.

ABSTRACT: In vitro ketone production continues to be a challenge due to the biochemical features of the enzymes involved-even when some of them have been extensively characterized (e.g. thiolase from Clostridium acetobutylicum), the assembly of synthetic enzyme cascades still face significant limitations (including issues with protein aggregation and multimerization). Here, we designed and assembled a self-sustaining enzyme cascade with acetone yields close to the theoretical maximum using acetate as the only carbon input. The efficiency of this system was further boosted by coupling the enzymatic sequence to a two-step ATP-regeneration system that enables continuous, cost-effective acetone biosynthesis. Furthermore, simple methods were implemented for purifying the enzymes necessary for this synthetic metabolism, including a first-case example on the isolation of a heterotetrameric acetate:coenzyme A transferase by affinity chromatography.

SUBMITTER: Kozaeva E

PROVIDER: S-EPMC9627730 | biostudies-literature | 2022 Nov

REPOSITORIES: biostudies-literature

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Synthetic metabolism for in vitro acetone biosynthesis driven by ATP regeneration.

Kozaeva Ekaterina E Nieto-Domínguez Manuel M Hernández Abril D AD Nikel Pablo I PI

RSC chemical biology 20220916 11

In vitro ketone production continues to be a challenge due to the biochemical features of the enzymes involved-even when some of them have been extensively characterized (e.g. thiolase from Clostridium acetobutylicum), the assembly of synthetic enzyme cascades still face significant limitations (including issues with protein aggregation and multimerization). Here, we designed and assembled a self-sustaining enzyme cascade with acetone yields close to the theoretical maximum ...[more]

PMID: 36349222

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Project description:In vitro reconstructed minimal respiratory chains are powerful tools to investigate molecular interactions between the different enzyme components and how they are influenced by their environment. One such system is the coreconstitution of the terminal cytochrome bo3 oxidase and the ATP synthase from Escherichia coli into liposomes, where the ATP synthase activity is driven through a proton motive force (pmf) created by the bo3 oxidase. The proton pumping activity of the bo3 oxidase is initiated using the artificial electron mediator short-chain ubiquinone and electron source DTT. Here, we extend this system and use either complex II or NDH-2 and succinate or NADH, respectively, as electron entry points employing the natural long-chain ubiquinone Q8 or Q10. By testing different lipid compositions, we identify that negatively charged lipids are a prerequisite to allow effective NDH-2 activity. Simultaneously, negatively charged lipids decrease the overall pmf formation and ATP synthesis rates. We find that orientation of the bo3 oxidase in liposomal membranes is governed by electrostatic interactions between enzyme and membrane surface, where positively charged lipids yield the desired bo3 oxidase orientation but hinder reduction of the quinone pool by NDH-2. To overcome this conundrum, we exploit ionizable lipids, which are either neutral or positively charged depending on the pH value. We first coreconstituted bo3 oxidase and ATP synthase into temporarily positively charged liposomes, followed by fusion with negatively charged empty liposomes at low pH. An increase of the pH to physiological values renders these proteoliposomes overall negatively charged, making them compatible with quinone reduction via NDH-2. Using this strategy, we not only succeeded in orienting the bo3 oxidase essentially unidirectionally into liposomes but also found up to 3-fold increased ATP synthesis rates through the usage of natural, long-chain quinones in combination with the substrate NADH compared to the synthetic electron donor/mediator pair.

Dataset Information

Synthetic metabolism for in vitro acetone biosynthesis driven by ATP regeneration.

Publications

Synthetic metabolism for <i>in vitro</i> acetone biosynthesis driven by ATP regeneration.

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