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

Abstract

The 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.

Charge Recombination Kinetics of Bacterial Photosynthetic Reaction Centres Reconstituted in Liposomes: Deterministic Versus Stochastic Approach

“…In conclusion, stochastic simulations can be suitable to get insights in the time behaviour of small compartmentalized chemically reacting systems with a randomly disperse composition and highly useful in the characterization of artificial lipid systems that mimic the behaviour of real cells [42,43].…”

Interfacing Living and Synthetic Cells as an Emerging Frontier in Synthetic Biology

“…extracted chromatophores from Rhodobacter sphaeroides and used them as photosynthesising organelles within artificial cells (Figure 4 C). Under illumination, these converted ADP to ATP, which in turn sustained the transcription of DNA to mRNA, paving the way for continual regeneration of energy‐carrying molecules using an external energy source [51] . Similarly, there have been recent efforts to appropriate thylakoid membranes and to couple these to a synthetic enzymatic cycle that fixes carbon dioxide within water‐in‐oil droplets, thus achieving a photosynthetic anabolic reaction in a cell‐like construct [52] …”

Interface Engineering in Multiphase Systems toward Synthetic Cells and Organelles: From Soft Matter Fundamentals to Biomedical Applications

“…[348] The ATP regeneration provides energy for various internal biosynthesis, for instance, RNA synthesis, protein synthesis and metabolism (Figure 19a-c). [11,[360][361][362] A lipid vesicle orchestrating a light-driven ATP production and ATP consumption by polymerization of actin monomers is achieved. The polymerization of actin monomers can regulate the morphology and motility of living cells.…”

“…[11] ATP-producing organelles have also been incorporated in vesicle to provide energy for RNA transcription and GFP protein synthesis (Figure 19a). [360][361][362] Including the ATP synthesis in a subcompartmental of synthetic cells is increasingly important for synthetic cells to perform complex tasks, and it substantially prolongs the activities of synthetic cells. [9,268,325,338,341] Very recently, a chloroplastmimicking synthetic compartment of combined light-driven energy modules and enzyme cascade modules for light harvest and carbon dioxide fixation.…”