Upscale metabolic engineering has highest potential achieving the production of biofuels, therapeutically products and specialty chemicals with genetically modified organisms. Still, time-consuming purification runs after fermentation and the limitations of in vivo reactions like toxicity of accumulated intermediates due to slow turnover rates of pathway enzymes or the influence of key enzymes on host metabolism can disfavor bioreactors (1,2). In our approach we tend to solve these problems in vitro by immobilizing all needed pathway enzymes in close proximity on the bioreactor surface using a protein scaffold (Dueber et al., 2009). In consequence, pathway enzymes remain in the bioreactor during purification and can be reused in the next fermentation. As a proof of principle we use the degradation of xylan, a group of hemicellulose, to β-D-xylose, which can be used as substrate for biosynthesis of all three targeted monomers for the synthesis of a photopolymer. The model degradation pathway is immobilized on a silica surface by using a fusion protein consisting of the protein scaffold by iGEM TU Darmstadt 2014 (1) and the Si4-tag by iGEM Leeds 2013 (2).

Xylan defines a group of hemicelluloses, polysaccharides based on xylose, mainly occurring in plant cell walls. In hardwoods up to 35 % of the whole hemicellulose content are Xylanes (4), also the Xylose Monomer is suitable for generation of photopolymers. For xylan degradation three enzymes were chosen, which cleave either the xylan main chain (xynA) or side chains (aes, Ruxyn1). The side chains of xylan vary by the type of utilized xylan. Xylan of hardwood mainly has acetyl groups, while softwood xylan lacks acetyl groups but has arabinose residues instead. Both xylan types are targeted by aes (degradation of acetyl residues) and Ruxyn1 (degradation of arabinose residues). 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 (3). 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.

Anzahl an Biobricks an Ergebnissen orientieren! Editieren! In this project the protein scaffold by iGEM TU Darmstadt 2014 (based on Dueber et al., 2009 (1)) was fused with a 15 amino acid long peptide (silica-tag) (2), binding to silica surfaces as shown by iGEM Leeds 2013. Green Fluorescent Protein (GFP) was fused with the silica-tag for proving silica-binding abilities of the peptide. For easy purification of both fusion proteins N-terminal polyhistidine tags were added For increasing the degradation efficiency of xylan, protein scaffolding was prepared by fusing the cognate binding peptides of the three protein domains GBD, SH3 and PDZ to the degradation enzymes Ruxyn1, aes and xynA, respectively. All coding sequences were assembled in an operon under the control of a T7 promoter system, which was co-transformed with the scaffolding plasmid into E. coli Top10. In the next step three different characterization elements for later on optimization are important:

1) binding efficiency of the silica tag to the silica surface and stability of boundary

2) binding efficiency of the enzyme-scaffold interaction sequence

3) Xylan degradation yield enrichment using the protein scaffold

1) To check the binding to a silica-surface and stability of boundary we fused the Si4-tag by iGEM Leeds 2013 (2) with GFP (BBa_E0040), YFP (BBa_E0030) and CFP (BBa_E0020) using Overlap PCR / Biobrick Assembly. After Expression and Purification we want to visualize the boundary to a silica-coated surface in different buffer conditions and time dependency off the boundary.

2) By linking the zinc finger ligand sequence to GFP (BBa_E0040), YFP (BBa_E0030) and CFP (BBa_E0020) and combining purified protein scaffold domains with the fluorescent proteins we want to control the boundary of ligand scaffold domain in different buffer conditions.

3) To verify the advantage of our protein scaffolding we want to compare the yield of degrading xylan only based on the three degradation enzymes in one composite to the combination of the composite and the protein-scaffold.

Not available yet.

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 0,22 μL). The solved polymer is transported into the reactor via the pipe on the left hand and is then degraded by the three enzymes Ruxyn1, aes and xynA, 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 membrane, 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.