MaxSynBio: Design of Artificial Cells from Functional Modules

The exciting field of Synthetic Biology focuses on producing engineered, predictable cells or other biological systems to carry out desired functions, whereby these functions can be modified, enhanced or entirely new. Conceptually, Synthetic Biology relies on breaking down the complexity of biological systems into smaller subsystems and functional modules, followed by (re-)designing and (re-)assembling them into larger units. This can be considered as an analogue to the design of a biotechnological or chemical plant (Rollié et al., 2012). With respect to this, the PSE Group has introduced its expertise in the assembly and control of chemical, energy and production processes in MaxSynBio– a research consortium with eight other Max Planck Institutes and research groups from the Friedrich-Alexander University in Erlangen-Nuremberg and the University of Bordeaux, which is jointly funded by the Max Planck Society and the Federal Ministry of Education and Research in Germany.

The primary scientific goal of MaxSynBio is a true bottom-up engineering approach for mimicking the fundamental structural and functional principles of real cells towards better understanding of living systems (Fig. 1). The near future in Synthetic Biology will still lean heavily on the top-down approach, i.e. minimal systems derived from an existing organism. The bottom-up approach however – starting from first principles – might allow us to refine and further improve these minimal systems with alternative synthetic or biological components and pave the way for establishing a radically new generation of biotechnological production processes.

The MaxSynBio consortium focuses on selected life processes, which are of fundamental importance for the replication and functionality of living cells, such as growth, division, morphogenesis, signaling and motility, summarized under the umbrella term “proliferome” (Schwille et al., 2018). The specific responsibilities of the PSE group within the MaxSynBio consortium include:

Fig. 1: Top-down and bottom-up approaches to synthetic biology (from: Sundmacher, K. and Schwille, P. (2014) Research Perspectives of the Max Planck Society).
  • Compartmentalization and growth (tools for production, functionalization, characterization, and controlled increase of compartment size)
  • Establishment and characterization of energy and cofactor regeneration modules for artificial cells (light- or chemically-driven)
  • Establishment and characterization of metabolic (e.g. for CO2 fixation) and transport modules 
  • Coupling of the energy supply modules with different energy sink subsystems
  • General methodology for integration of functional parts and modules and modular design of synthetic cells, based on assembly standards and mathematical modeling

and the general workflow is depicted in Fig. 2.

Fig. 2: Workflow for the bottom-up assembly of an artificial mitochondrion. Left: characterization of functional parts through performance standards such as activity, stability, etc.; Middle: integration of functional parts into functional modules for metabolism, energy and transport through assembly standards; Right: integration of modules into hypothetical artificial mitochondrion system through assembly standards and computer-aided optimization.

Compartmentalization is a major landmark of living systems – to establish gradients for energy generation or to spatially segregate metabolic reactions from the environment – and therefore the reconstitution of living processes is inherently associated with the creation and manipulation of compartments. Phospholipid membranes closely imitate the architecture and function of natural membranes and they have been widely used in planar (e.g. as supported bilayers) and closed (vesicles) architectures as model systems. Recently, amphiphilic copolymer-based analogues of cellular membranes have expanded the chemical dimensions of supramolecular structures, which allows for further tailoring to technological, biological and medical applications. Within the compartmentalization technology platform, we are developing various methods for production and characterization of nano- and microcompartments of different chemical compositions, including conventional and microfluidic tools (Weiss et al., 2018). In addition, we establish different protocols for membrane functionalization and studying the respective interactions (e.g. with membrane proteins) (Fig. 3).

Fig. 3: Protein-functionalized microcompartments. (A) Bending rigidity for protein-free (w/o bo3; white area) and protein-functionalized (w/ bo3; gray area) liposomes, hybrids, and polymersomes. (B) bo3-polymer-GUVs with encapsulated pH-sensitive dye (green) trapped in microfluidic chip. Scale bar, 50 μm. (C) Successful insertion and homogenous distribution of bo3 oxidase-ATTO 514 (green) in bo3-polymer-GUVs. Polymer labeled with rhodamine (PDMS-g-PEO-Rho, red) was used to visualize the memrbane. Scale bar, 10 μm.

Active processes in living systems need continuous supply of energy and materials and the energy supply and storage are closely connected to the cellular metabolism. Following the bottom-up approach, we have obtained and characterized the basic biochemical machinery for energy transduction and conversion – light- and chemically-driven proton pumps and ATP synthase – and are constantly expanding the library of purified membrane proteins (other oxidases, porins, fusogenic peptides, etc.). We are integrating energy-converting parts in three types of nano- and microcompartments – liposomes, hybrid vesicles and polymersomes – and characterizing them with respect to activity, reconstitution efficiency and orientation, whereby experimental optimization resulted in retention of enzymatic activity in semisynthetic and synthetic nanocompartments (Fig. 4, Otrin et al., 2017). We have also demonstrated encapsulated cofactor regeneration by a hydrophobic redox mediator as a synthetic analog for NADH dehydrogenase and cytochrome c reductase (Fig. 5, Wang et al., 2018). We are also exploring various metabolic pathways and ways to integrate them with energy supply modules. In parallel, we use the respiratory chain from E. coli membranes as a natural counterpart of bottom-up-assembled modules and combine it with a simple metabolism (Beneyton et al., 2018). Additionally, we describe the energy and metabolic modules through mathematical models, which provides possibilities for optimization and a quantitative framework for their integration.

Fig. 4: Reconstituting minimal phosphorylation machinery in polymer and hybrid membranes. Above: homogenous lipid/polymer hybrid membrane in microcontainers; Below: respiratory-driven ATP synthesis in lipid, hybrid and polymer nanocontainers.
Fig. 5: NAD regeneration module. (A) Complexes I and III – natural functional parts for NADH oxidation and transmembrane electron transfer; (B) a synthetic analogue inserted into phospholipid bilayer.

Recent Publications

Xu D., Kleineberg C., Vidaković-Koch T., Wegner S. V. (2020). Multistimuli sensing adhesion unit for the self-positioning of minimal synthetic cells. Small 16 (35): 2002440

Marušič N., Otrin O., Zhao Z., Lira R.B., Kyrilis F.L., Hamdi F., Kastritis P.L., Vidaković-Koch T., Ivanov I., Sundmacher K., and Dimova R. (2020). Constructing artificial respiratory chain in polymer compartments: Insights into the interplay between bo3 oxidase and the membrane. Proceedings of National Academy of Sciences 117 (26): 15006-15017

Kleineberg C., Wölfer C., Abbasnia A., Pischel D., Bednarz C., Ivanov I., Heitkamp T., Börsch M., Sundmacher K., Vidaković-Koch T. (2020). Light-driven ATP regeneration in diblock/grafted hybrid vesicles. ChemBioChem 21 (15): 2149-2160

Otrin L., Kleineberg C., de Silva L.C., Landfester K., Ivanov I., Wang M, Bednarz C., Sundmacher K., Vidaković-Koch T. (2019). Artifical organelles for energy regeneration. Advanced Biosystems 3 (6): 1800323

Ivanov I., Lira R.B., Tang T.-Y.D., Franzmann T., Klosin A., de Silva L.C., Hyman A., Landfester K., Lipowsky R., Sundmacher K., and Dimova R. (2019). Directed growth of biomimetic microcompartments. Advanced Biosystems 3: 1800314

Krafft D., López Castellanos S., Lira R.B., Dimova R., Ivanov I., Sundmacher K. (2019). Compartments for synthetic cells: Osmotically assisted separtion of oil from double emulsions in microfluidic chip. ChemBioChem 20: 2604-2608

Beneyton T., Krafft D., Bednarz C., Kleineberg C.,Woelfer C., Ivanov I., Vidaković-Koch T., Sundmacher K., and Baret J. C. (2018). Out-of-equilibrium microcompartments for the bottom-up integration of metabolic functions. Nature Communications 9: 2391

Wang M., Wölfer C., Otrin L., Ivanov I., Vidaković-Koch T., and Sundmacher K. (2018). Transmembrane NADH Oxidation with Tetracyanoquinodimethane. Langmuir 34 (19): 5435-5443

Weiss M., Frohnmayer J. P., Benk L. T., Haller B., Janiesch J. W., Heitkamp T., Börsch M., Lira R. B., Dimova R., Lipowsky R., Bodenschatz E., Baret J. C.,  Vidakovic-Koch T., Sundmacher K., Platzman I., Spatz J. P. (2018). Sequential bottom-up assembly of mechanically stabilized synthetic cells by microfluidics. Nature Materials 17(1): 89-96

Schwille P., Spatz J., Landfester K., Bodenschatz E., Herminghaus S., Sourjik V., Erb T. J., Bastiaens P., Lipowsky R., Hyman A., Dabrock P., Baret J. C., Vidaković-Koch T., Bieling P., Dimova R., Mutschler H., Robinson T., Tang T. D., Wegner S., and Sundmacher K. (2018). MaxSynBio: Avenues Towards Creating Cells from the Bottom Up. Angewandte Chemie International Edition 57 (41):13382-13392

Otrin L., Marušič N., Bednarz C., Vidaković-Koch T., Lieberwirth I., Landfester K., and Sundmacher K. (2017). Toward Artificial Mitochondrion: Mimicking Oxidative Phosphorylation in Polymer and Hybrid Membranes. Nano Letters 17 (11): 6816-6821

Rollié S., Mangold M. and Sundmacher K. (2012). Designing biological systems: systems engineering meets synthetic biology. Chemical Engineering Science, 69 (1): 1-29

Collaborations

Dr. Rumiana Dimova, Prof. Reinhard Lipowsky, Max Planck Institute of Colloids and Interfaces, Potsdam, Germany

Prof. Jean-Christophe Baret, University of Bordeaux, Bordeaux, France

Prof. Tobias Erb, Max Planck Institute for Terrestrial Microbiology, Marburg, Germany

Prof. Katharina Landfester, Max Planck Institute for Polymer Research, Mainz, Germany

Prof. Reinhard Jahn, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany

Prof. Robert J. Flassig, Brandenburg University of Applied Sciences, Brandenburg, Germany

Prof. Ramin Golestanian, Max Planck Institute for Dynamics and Self-Organization, Göttingen, Germany

Prof. Michael Börsch, Jena University Hospital, Jena, Germany

Prof. Panagiotis L. Kastritis, Martin Luther University Halle-Wittenberg, Halle, Germany

Prof. Seraphine Wegner, Institute of Physiological Chemistry and Phatobiochemistry, Münster, Germany

Prof. Eberhard Bodenschatz, Max Planck Institute for Dynamics and Self-organization, Göttingen, Germany

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