In vitro Bioreactor
In previous studies it was shown that the spatial organization of pathway enzymes can increase pathway activities significantly. Once again inspiring examples are found in nature. For example the pathway enzymes of the phenylpropanoid pathway assemble to multi enzyme complexes by binding to the endoplasmatic reticulum membrane. In consequence, intermediates are transferred more efficiently between active sites in a metabolic channel and product titers are increased. The heterologous expression of a substrate channeling pathway often leads to loss of channeling abilities, because the scaffolding structure, like the ER membrane, may not exist in the heterologous host. For preventing the loss of the channeling activity, two synthetic channeling approaches were developed – protein scaffolding (1) and DNA scaffolding (3). Both approaches tend to bring fusion proteins between a pathway enzyme and a scaffold tag into close proximity on either DNA plasmid or multi domain protein. While zinc finger domains, specifically binding DNA sequences, are used as scaffold tag in DNA scaffolding, Dueber et al. (1) constructed an artificial multi domain protein consisting of interaction domains of metazoan signaling proteins (GBD, SH3, PDZ domains). These interaction domains highly affine bind cognate peptide ligands, which are fused with pathway enzymes, and thus create an artificial multi enzyme complex. Last years iGEM Team TU Darmstadt has worked on the protein scaffold and optimized it for the production in E. coli. Based on this success we worked on a new in vitro method immobilizing the protein scaffold on a silica surface, using a Si4-Tag (2), that was brought to the registry by the iGEM Leeds 2013.
We did not want to stop our reasoning on laboratory scale, because we believe the combination of protein scaffolding with immobilization on silica-surfaces has potential to generate a novel in vitro reactor type for degrading polymeric waste. The concept is based on mimicking known organelles with high membrane-protein activity and surface area like the mitochondrium or endoplasmatic reticulum. Like the surface area of these organelles, we also tried to maximize the surface area of the reactor. Also we wanted to simplify the purification of degradation products as well as generating separated reaction areas inside of the reactor. In this example we use two different reaction rooms separated by an ultrafiltration membrane (pore size 0.22μm). In our scenario xylan is transported into the reactor via the pipe on the left hand and is then degraded by the three enzymes Ruxyn1 (BBa_K805012), aes(BBa_K1216002) and xynA(BBa_K1175005), attached to the silica-tagged protein scaffold. As the titer of monomers rises the more flux is oriented into the second reaction chamber past the barrier, while the polymeric structure remains in the first due to the small pore size. Also enzymes could not pass the membrane. Different Scaffold / Enzyme mixtures with the same basic protein-scaffold domains are usable with separated pipes for fresh enzyme supplies. Finally, this concept could offer the possibility of high yield surface reactions and simple handling of reaction conditions while avoiding known disadvantages of in vivo production.
- J. E. Dueber et al., Synthetic protein scaffolds provide modular control over metabolic flux. Nat Biotechnol 27, 753-759 (2009).
- R. R. Naik, L. L. Brott, S. J. Clarson, M. O. Stone, Silica-precipitating peptides isolated from a combinatorial phage display peptide library. J Nanosci Nanotechnol 2, 95-100 (2002).
- R. J. Conrado et al., DNA-guided assembly of biosynthetic pathways promotes improved catalytic efficiency. Nucleic Acids Res 40, 1879-1889 (2012).