Difference between revisions of "Team:TU Darmstadt/Project/Bio/InVitroDegradation/sec2"
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− | 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 (1). Also the Xylose Monomer is suitable for generation of photopolymers and therefore of use in our project. 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). | + | 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 (1). Also the Xylose Monomer is suitable for generation of photopolymers and therefore of use in our project. For xylan degradation three enzymes were chosen, which cleave either the xylan main chain (xynA (2)) or side chains (aes(3), 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 <em>et al.</em> (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. | 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 <em>et al.</em> (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. | ||
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− | <li> | + | <li> Sixta, Herbert, ed. (2006). Handbook of pulp 1. Weinheim, Germany: Wiley-VCH Verlag GmbH & Co. KGaA. pp. 28–30. ISBN 978-3-527-30999-3. |
+ | </li> | ||
+ | <li> | ||
+ | M.G. Paice, R. Bourbonnais, M. Desrochers, L. Jurasek, M. Yaguchi, A xylanase gene from Bacillus subtilis: nucleotide sequence and comparison with B. pumilus gene. Arch. Microbiol., 144, 201-206(1986) | ||
+ | </li> | ||
+ | <li> | ||
+ | R1 Peist, A Koch, P Bolek, S Sewitz, T Kolbus, W Boos, Characterization of the aes gene of Escherichia coli encoding an enzyme with esterase activity. J Bacteriol. 1997, 179:7679-7686. | ||
+ | |||
</li> | </li> | ||
<li> 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).</li> | <li> 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).</li> | ||
<li> R. J. Conrado <em>et al.</em>, DNA-guided assembly of biosynthetic pathways promotes improved catalytic efficiency. Nucleic Acids Res 40, 1879-1889 (2012). | <li> R. J. Conrado <em>et al.</em>, DNA-guided assembly of biosynthetic pathways promotes improved catalytic efficiency. Nucleic Acids Res 40, 1879-1889 (2012). | ||
</li> | </li> | ||
+ | <li> | ||
+ | J. E. Dueber <em>et al.</em>, Synthetic protein scaffolds provide modular control over metabolic flux. Nat Biotechnol 27, 753-759 (2009). | ||
</li> | </li> | ||
− | |||
</ol> | </ol> |
Revision as of 23:32, 18 September 2015
Contents
Xylan Degradation
Introduction
Experimental setup
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.
Results
In vitro bioreactor
- Sixta, Herbert, ed. (2006). Handbook of pulp 1. Weinheim, Germany: Wiley-VCH Verlag GmbH & Co. KGaA. pp. 28–30. ISBN 978-3-527-30999-3.
- M.G. Paice, R. Bourbonnais, M. Desrochers, L. Jurasek, M. Yaguchi, A xylanase gene from Bacillus subtilis: nucleotide sequence and comparison with B. pumilus gene. Arch. Microbiol., 144, 201-206(1986)
- R1 Peist, A Koch, P Bolek, S Sewitz, T Kolbus, W Boos, Characterization of the aes gene of Escherichia coli encoding an enzyme with esterase activity. J Bacteriol. 1997, 179:7679-7686.
- 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).
- J. E. Dueber et al., Synthetic protein scaffolds provide modular control over metabolic flux. Nat Biotechnol 27, 753-759 (2009).