Synthetic Cell Organelles for Bio-catalyzed Metabolic Reactions
- Figure 1. Concept of the bio-catalytic nanocompartment: The polymer vesicle (black) contains the enzyme phosphoglucomutase (yellow) that converts glucose-1-phosphate (blue) to glucose-6-phosphate (brown). Both compounds are able to enter and leave the nanocompartment through engineered α-hemolysin pores embedded in the polymer membrane. Reproduced by permission of The Royal Society of Chemistry .
- Figure 2. A. Transmission electron microscopy image of the polymer vesicles containing phosphoglucomutase (polymer stained with uranyl acetate for visualization). B. Fluorescence measurements showing that a fluorescent dye that is entrapped inside can only leave the polymer vesicles upon addition of the α-hemolysin pore (blue). Control experiments with detergent (red) and the polymer vesicles in buffer (black) show that they efficiently contain the entrapped dye molecules. Reproduced by permission of The Royal Society of Chemistry .
- Figure 3. Fluorescence measurements allow detection of the overall reaction performed by the bio-catalytic nanocompartment (nanoreactor). Inside the nanocompartment, glucose-6-phosphate is generated, which is then consumed by an established enzymatic assay performed in the surrounding solution that yields a fluorescent product nicotinamide adenine dinucleotide phosphate (NADPH). The reaction is initiated by addition of the substrate glucose-1-phosphate (ca. 500 s) and only when α-hemolysin pores are present, (purple curve) the reaction can proceed. Control reactions without α-hemolysin pore (black) show no product formation. Reproduced by permission of The Royal Society of Chemistry .
Combining natural biomolecules with synthetic polymer vesicles enables creation of bio-inspired, nanometer-sized catalytic compartments. Inside the nanocompartments, glucose meta-bolites can be converted by an enzyme and released through engineered pore proteins embedded in the polymer vesicle membrane.
Cell organelles are inner compartments inside cells that perform a complex set of biochemical reactions essential for life. Inspired by the concept of compartmentalization, that is separating reactions in confined spaces, synthetic bio-catalytic nanocompartments were created. The advantage of our synthetic system is the possibility to use all the options offered by modern polymer chemistry to fine-tune and control the different parameters. Notably, the nanocompartments are highly stable and permeability of the membrane can be controlled to allow passage of only selected small molecules by using pore proteins. The biocatalyst is retained in the interior and catalyzes the conversion of the metabolites into products that are released through pore proteins (see fig. 1).
Assembly of Synthetic Nanocompartments
In the presented study, polymer vesicles (polymersomes) are used as nanocompartment hull, and engineered α-hemolysin as pore protein [1,2]. Polymer vesicles applied as nanocompartments can be formed by self-assembly of an amphiphilic block copolymer in aqueous buffer. The polymer used in this study is a poly (2-methyloxazoline)6-b-poly(dimethylsiloxane)34-b-poly(2-methyloxazoline)6 triblock copolymer that can form a polymer compartment with a membrane that structurally resembles the lipid bilayer architecture found in cell membranes. Through light scattering, the diameter of the polymer vesicles formed with this polymer was measured and was found to be 100-200 nm, which is in agreement with transmission electron microscopy (fig. 2A). Importantly, biomimetic compartments formed from polymers are more robust and have thicker membranes than lipid compartments (liposomes). These properties already implicate that the compartments are more stable. At the same time, permeation of molecules across the polymer membrane is hindered.
Since this property prevents uncontrolled entry or release of molecules, it is used to the advantage in the design of functional nanocompartments.
Small molecules like starting materials and products of the reaction need to be transported across the polymer membrane. This was achieved by insertion of an engineered α-hemolysin pore protein . The functional insertion of this pore protein was confirmed by experiments with an entrapped fluorescent dye (carboxyfluorescein). Here, the dye is contained inside the nanocompartments at a concentration where the fluorescence is quenched. Upon insertion of the α-hemolysin in the polymer membrane, pores of ca. 1.9 nm in diameter are formed that allow the dye to diffuse out of the polymer vesicle along its concentration gradient. As can be seen from the data in figure 2B, this can be detected by an increase in fluorescence intensity. Importantly, the control experiments confirmed that dye molecules could only leave the polymeric nanocompartment when α-hemolysin is added.
To yield a functional nanocompartment, a biocatalyst was entrapped. Successful entrapment of the enzyme inside the polymer vesicles was detected by fluorescence correlation spectroscopy using dye-labeled enzymes. Clearly different diffusion times were observed for free dye (atto488) in solution (29 μs) and dye-labeled phosphoglucomutase (153 μs). The significantly larger polymer vesicles containing the labeled enzyme in their interior possess an increased diffusion time of 3 ms, confirming successful entrapment of the dye-labeled enzyme inside the nanocompartments. In addition, these measurements served to prove complete purification and removal of any enzyme remaining outside of the polymer vesicles.
As proof of concept, a simple metabolic biochemical reaction inside the nanocompartment was employed. The biocatalyst entrapped inside the polymer vesicles is phosphoglucomutase, an enzyme that plays a crucial part in the carbohydrate metabolism. Thus, it serves as an excellent paradigm to build a biomimetic nanocompartment. Phosphoglucomutase catalyzes the conversion of glucose-1-phosphate to glucose-6-phosphate and therefore mimics an important step in glucose catabolism. An established secondary enzymatic assay was used to monitor the formation of the reaction product. The reaction is initiated by addition of the substrate glucose-1-phosphate, which is then converted to glucose-6-phosphate. Subsequently, the glucose-6-phosphate is consumed in a second enzymatic reaction in solution, which yields NADPH, a product that allows spectrophotometric detection due to its autofluorescence. Importantly, the reaction can only occur when the α-hemolysin pore protein was added. Control experiments without addition of the α-hemolysin clearly show that no increase in fluorescence is detected (see fig. 3) and thus the enzymatic reaction cannot occur as the substrate cannot enter the nanocompartment.
This study highlights the potential of bio-catalytic nanocompartments composed of amphiphilic triblock copolymers and natural components like enzymes and pore proteins to recreate compartmentalized metabolic reactions. It was shown that the engineered α-hemolysin pore could insert and mediate transport even in a polymer membrane that is thicker and more entangled than lipid membranes, while the entrapped enzyme maintained its catalytic activity. In the future, these synthetic nanocompartments will not only allow to recreate compartmentalized metabolic reactions in vitro for in-depth study – one day, it may also offer an option for optimized preparation of valuable biomolecules when multiple steps are combined into one biosynthetic reaction pathway.
The authors would like to thank the support from SNF, the University of Basel and a postdoctoral research fellowship from the DAAD (G. G.-G.). Further, Samuel Lörcher and Wolfgang Meier are thanked for providing the polymer, Gabriele Persy and Christina Zelmer for TEM measurements and Shiksha Mantri and Hagan Bayley for a fruitful collaboration.
Gesine Gunkel-Grabole1, Mihai Lomora1, and Cornelia Palivan1
1Departement Chemie, Universität Basel, Basel, Switzerland.
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