Biomimetic reactors

Primary investigator: Pierre-Alain Monnard.

Amphiphile structures can be used to design bioreactor compartments that can either contain reaction systems in their aqueous core (Enclosed Bioreactors) or co-localize the reacting species within their boundaries (Interfacial Bioreactors).

Enclosed Bioreactors based on liposomes and vesicles

The use of liposomes as biomimetic enclosed bioreactors requires the combination of features, such as catalytic networks and energy harvesting capabilities, into an integrated system with a steady supply of substrates and energy. Although this integration has yet to be performed, this design can be achieved because functional complex enzymatic assemblies can be encapsulated in liposomes. Selective exchanges between their internal compartment and the external medium can occur via cellular-like mechanisms, such as carrier-promoted uptake/release of solutes, liposome fusion, and passive diffusion of solutes. Finally, the relative stability of liposomal bilayers also allows for the efficient embedding of functioning energy-transduction assemblies, thus the formation of energy gradients that can be then used to promote reactions.

Enclosed Bioreactors based on Emulsion compartments

Water-in-oil emulsions are prepared by mixing an aqueous phase and an apolar, oil phase containing the amphiphile (also named the emulsifier) yielding a complete compartmentalization of the aqueous phase. The number, size, and stability of emulsion compartments are influenced by the kind of amphiphile used, the ratio of water to amphiphiles, the ratio of water to oil, and the mechanical power (stirring rates) used in preparing the system. Size and stability of the compartments usually are the crucial parameters in bioreactor design. The size will define an essential aspect of the bioreactor functionality, namely the state of the water molecules. In microemulsions which are obtained at low ratios of water to charged amphiphiles such as di(ethylhexyl)sulfo-succinate (AOT), most of the water molecules are bound to the amphiphile interface and have the same properties as immobilized water (i.e., a low dielectric constant and a suppressed freezing point). As the ratio of water to amphiphiles increases and one forms larger structures, namely the so-called macroemulsions (emulsions with typical structures sizes of greater than 500 nm), the large fraction of water starts to behave as in a homogeneous aqueous medium, allowing "normal" function of the enzymes.

Figure 1. A) liposomal and b) emulsion bioreactors. On the right, the enclosed and interfacial concepts.

Before efficient liposome- or emulsion based micro- or nanoreactors are produced, several issues are to be clarified and procedures designed for

  1. an efficient encapsulation method to minimize the non-encapsulation and optimize the simultaneous encapsulation of all components in complex catalytic systems.
    An efficient encapsulation of complex catalytic assemblies or a library of molecules (1015 different DNA templates for a RNA selection) is crucial because a catalytic assembly will only function if it is complete. With libraries for molecular selection, each molecule can potential be the intended target, thus we must minimize the non-encapsulation. Furthermore, if distinct reactions have to occur in each reactor we have to ensure the encapsulation of a single system per compartment.
  2. the supply of the encapsulated enzymatic system with substrates;
    The boundaries of any compartment represent a barrier to the free diffusion of molecules, thus the supply of encapsulated enzymatic assemblies is essential. The influence of bilayer properties will be investigated with emphasis on achieving a steady substrate delivery by passive diffusion and with embedded macromolecule carriers, such as amphiphilic peptides, or RNA fragments.
  3. the effect of the compartmentalization on the activity of enzymatic systems and its products
    The compartmentalization impact on the catalytic activity of compartmentalized enzymes is to date not clearly understood. Compartment size, excluded volumes and interactions with the compartment boundaries will influence compartmentalized reactions.
  4. recovering intact reactors containing molecular products of interest.
    In selection reactions, the recovery of active molecules is always determining whether a selection scheme worked. Using the compartment as the recovery target, one will not only take advantage of the compartment size (100- to 1000 nm diameter vs a few nm for the molecules of interest), but also be able to label the compartment to simplify recovery.

Project example: RNA-selection bioreactors

One application of enclosed bioreactor we will pursue is related to the selection of RNAs with novel functions. Common RNA-selection methods in homogeneous media, such as SELEX (Systematic Evolution of Ligand by EXponential enrichment), are based on the screening of large libraries of RNA (1013 to 1016 different biopolymers synthesized from large libraries of random nucleic acid templates). Such a number of individual compartments is achievable in liposome suspension. By encapsulating and transcribing a single template per liposome (Scheme 1), we will however overcome a systemic limitation of homogeneous-phase selection: Molecular selections are only feasible if the phenotype molecules (the active molecules) remain linked to their genotype (DNA templates) to permit their identification and recovery. Although RNAs represent both genotype and phenotype, RNAs must either self-modify or be tethered to a single substrate molecule to permit their direct selection for novel catalytic activity. Thus, only an "intramolecular single-turnover" selection can be achieved in a homogeneous medium. In contrast, an entrapped transcription with a single template per liposome will maintain each catalytic-RNA phenotype and its products spatially isolated, allowing selections based on essential catalytic parameters, such as substrate-association and product-release rates, or multiple catalytic turnovers. An individual recovery of the encapsulated active molecules can then be performed by collecting the intact compartments using Microfluidics techniques. Indeed, liposomes could be in in-situ labeled as a fluorogenic tag that is non-fluorescent when linked to substrates would be released upon reaction and insert in the compartment bilayers, making the liposomes fluorescent and detectable.

Scheme 1. Liposome nano-scale bioreactor for RNA transcription and selection. A) Several T7 RNA polymerase molecules along with one template are entrapped in one liposome. Substrate molecules (NTPs) added in the external medium passively diffuse across the bilayers, e.g., using transient packing defects. B) Multiple RNA transcripts accumulate in the aqueous compartments. C) The RNA activity is monitored in situ.


Interfacial Bioreactors

In this case, the catalytic systems are directly interacting with a surface and their various components are "absorbed" on the reactor plane (a bilayer, a mineral surface). The main advantage of this set-up is its openness, however it is limited because of the type of interactions applied to localize the system on the bioreactor plane. We have however found that the co-localization in an interfacial reactor can impart new properties to a catalytic system (see the protocell project or our work on the positive interactions between bilayers and enzymes such as Terminal Deoxynucleotidyl transferase).

Monnard, P.-A., Oberholzer, T. and Luisi, P.L.: (1997) Encapsulation of polynucleotides in liposomes. Biochim. Biophys. Acta, 1329, 39-50.
Oberholzer, T., Meyer, E., Amato, I., Lustig, A. and Monnard P.-A.: (1999) Enzymatic reactions in liposomes using the detergent-induced liposome loading method. Biochim. Biophys. Acta, 1416, 57-68.
Monnard, P.-A., Berclaz, N., Conde-Frieboes, K. and Oberholzer, T.: (1999) Decreased solute entrapment in POPC liposomes prepared by freeze/thaw in the presence of physiological amounts of monovalent salts. Langmuir, 15, 7504-7509.
Monnard, P.-A. and Deamer, D.W.: (2001) Nutrient uptake by protocells: A liposome model system. Origins Life Evol. Biosphere, 31, 147-155.
Monnard, P.-A.: (2003) Liposome-entrapped polymerases, a model for microscale/nanoscale bioreactors. J. Membr. Biol., 191, 87-97.
Monnard, P.-A.: (2005) "Catalysis in abiotic structured media: An approach to selective synthesis of biopolymers." Mol. Cell. Life Sci., 62, 520-534.
Monnard, P.-A., Luptak, A. and Deamer, D.W.: (2007) "Models of primitive cellular life: Polymerases and templates in liposomes." Phys. Trans. R. Soc. B. 362, 1741-1750.
Monnard P.-A., Declue M. and Ziock, H.-J. (2008) "Amphiphile nanostructures as model for artificial cell-like entities" Current Nanoscience, 4, 71-87.
DeClue, M., Monnard P.-A., Bailey, J., Ziock, H., Boncella, J.W. and Rasmussen, S.: "Nucleobase controlled light driven metabolic formation of amphiphile containers for a minimal protocell." submitted.
Oberholzer, T. and Monnard, P.-A.: Liposome-assisted nucleic acid synthesis by terminal deoxynucleotidyl transferase. in preparation