Light-driven ATP production promotes mRNA biosynthesis inside hybrid multi-compartment artificial protocells

AbstractThe construction of energetically autonomous artificial protocells is one of the most urgent and challenging requirements in bottom-up synthetic biology. Here we show a hybrid multi-compartment approach to build Artificial Simplified-Autotroph Protocells (ASAPs) in an effective manner. Chromatophores obtained from Rhodobacter sphaeroides accomplish the photophosphorylation of ADP to ATP functioning as nanosized photosynthetic organellae when encapsulated inside artificial giant phospholipid vesicles. Under continuous illumination chromatophores produce ATP that in turn sustains the transcription of a DNA gene by T7 RNA polymerase inside ASAPs. Cryo-EM and time-resolved spectroscopy were used for characterizing the chromatophore morphology and the orientation of the photophosphorylation proteins, which allow high ATP production rates (up to ~100 ATP/s per ATP synthase). mRNA biosynthesis inside individual vesicles has been determined by confocal microscopy. The hybrid multi-compartment approach here proposed appears at the same time convenient and effective, and thus very promising for the construction of full-fledged artificial protocells.

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Chromatophores efficiently promote light-driven ATP synthesis and DNA transcription inside hybrid multicompartment artificial cells

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2012170118 ◽

2021 ◽

Vol 118 (7) ◽

pp. e2012170118

Author(s):

Emiliano Altamura ◽

Paola Albanese ◽

Roberto Marotta ◽

Francesco Milano ◽

Michele Fiore ◽

...

Keyword(s):

Electron Microscopy ◽

Rhodobacter Sphaeroides ◽

Atp Synthesis ◽

Phospholipid Vesicles ◽

Artificial Cells ◽

Efficient Manner ◽

Time Resolved ◽

Atp Production ◽

Cryo Electron Microscopy ◽

Rna Biosynthesis

The construction of energetically autonomous artificial protocells is one of the most ambitious goals in bottom-up synthetic biology. Here, we show an efficient manner to build adenosine 5′-triphosphate (ATP) synthesizing hybrid multicompartment protocells. Bacterial chromatophores from Rhodobacter sphaeroides accomplish the photophosphorylation of adenosine 5′-diphosphate (ADP) to ATP, functioning as nanosized photosynthetic organellae when encapsulated inside artificial giant phospholipid vesicles (ATP production rate up to ∼100 ATP∙s−1 per ATP synthase). The chromatophore morphology and the orientation of the photophosphorylation proteins were characterized by cryo-electron microscopy (cryo-EM) and time-resolved spectroscopy. The freshly synthesized ATP has been employed for sustaining the transcription of a DNA gene, following the RNA biosynthesis inside individual vesicles by confocal microscopy. The hybrid multicompartment approach here proposed is very promising for the construction of full-fledged artificial protocells because it relies on easy-to-obtain and ready-to-use chromatophores, paving the way for artificial simplified-autotroph protocells (ASAPs).

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Synthetic biology: the many facets of T7 RNA polymerase

Molecular Systems Biology ◽

10.15252/msb.20145492 ◽

2014 ◽

Vol 10 (7) ◽

pp. 745 ◽

Cited By ~ 13

Author(s):

David L Shis ◽

Matthew R Bennett

Keyword(s):

Synthetic Biology ◽

Rna Polymerase ◽

T7 Rna Polymerase ◽

The Many

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Implementation of a novel optogenetic tool in mammalian cells based on a split T7 RNA polymerase

10.1101/2021.10.27.466068 ◽

2021 ◽

Author(s):

Sara Dionisi ◽

Armin Baumschlager ◽

Karol Piera ◽

Mustafa Khammash

Keyword(s):

Gene Expression ◽

Synthetic Biology ◽

Rna Polymerase ◽

Mammalian Cells ◽

Stem Cell Differentiation ◽

T7 Rna Polymerase ◽

Control Gene ◽

Protein Inhibition ◽

Control Gene Expression ◽

Gene Expression Dynamics

Optogenetic tools are widely used to control gene expression dynamics both in prokaryotic and eukaryotic cells. These tools are used in a variety of biological applications from stem cell differentiation to metabolic engineering. Despite some tools already available in bacteria, no light-inducible system currently exists to orthogonally control gene expression in mammalian cells. Such a tool would be particularly important in synthetic biology, where orthogonality is advantageous to achieve robust activation of synthetic networks. Here we implement, characterize and optimize a new orthogonal optogenetic tool in mammalian cells based on a previously published system in bacteria called Opto-T7RNAPs. The tool consists of a split T7 RNA polymerase coupled with the blue light-inducible magnets system (mammalian OptoT7, mOptoT7). In our study we exploited the T7 polymerase's viral origins to tune our system's expression level, reaching up to 20-fold change activation over the dark control. mOptoT7 is used here to generate mRNA for protein expression, shRNA for protein inhibition and Pepper aptamer for RNA visualization. Moreover, we show that mOptoT7 can mitigate gene expression burden when compared to other optogenetic constructs. These properties make mOptoT7 a new powerful tool to use when orthogonality and viral-like RNA species are desired in both synthetic biology and basic science applications.

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