Projects / Programmes
New applications of designed polypeptide assemblies
| Code |
Science |
Field |
Subfield |
| 4.06.00 |
Biotechnical sciences |
Biotechnology |
|
| Code |
Science |
Field |
| T490 |
Technological sciences |
Biotechnology |
| Code |
Science |
Field |
| 2.09 |
Engineering and Technology |
Industrial biotechnology |
synthetic polypeptide scaffold, self-assembly, coiled-coil, beta-sheet, metabolic engineering, enzyme catalysis
Researchers (11)
| no. |
Code |
Name and surname |
Research area |
Role |
Period |
No. of publicationsNo. of publications |
| 1. |
14360 |
PhD Mojca Benčina |
Biotechnology |
Researcher |
2014 - 2017 |
392 |
| 2. |
37380 |
PhD Karen Butina Ogorelec |
Microbiology and immunology |
Technical associate |
2015 |
34 |
| 3. |
33301 |
Tibor Doles |
Biochemistry and molecular biology |
Technical associate |
2014 |
41 |
| 4. |
32254 |
PhD Rok Gaber |
Biotechnology |
Researcher |
2014 - 2015 |
52 |
| 5. |
17915 |
PhD Helena Gradišar |
Biotechnology |
Head |
2014 - 2017 |
130 |
| 6. |
06628 |
PhD Roman Jerala |
Biochemistry and molecular biology |
Researcher |
2014 - 2017 |
1,190 |
| 7. |
34252 |
Tina Lebar |
Biochemistry and molecular biology |
Technical associate |
2014 - 2017 |
67 |
| 8. |
17917 |
PhD Andreja Majerle |
Biotechnology |
Researcher |
2014 - 2017 |
92 |
| 9. |
23939 |
PhD Martina Mohorčič |
Biotechnology |
Researcher |
2014 - 2017 |
30 |
| 10. |
38275 |
Anja Perčič |
|
Technical associate |
2016 |
0 |
| 11. |
39364 |
Anže Verbič |
Biochemistry and molecular biology |
Technical associate |
2017 |
9 |
Organisations (2)
Abstract
Molecular self-assembly as the main organizing principle of biological systems can be applied as a strategy for the construction of artificial nanostructures. In self-assembly the desired structure is encoded by interactions among the building elements. Synthetic polypeptide scaffolds can be constructed from modular polypeptide building blocks such as coiled coils and b-sheets, whose folding and specificity are well understood. Design, production and functionalization of new synthetic nanoscaffolds offer outstanding opportunities as a biotechnological platform.
Biosynthesis of metabolic products usually requires multiple catalytic steps that are within cells accomplished by several enzymes operating consecutively on the substrate and reaction intermediates. Metabolic engineering based on the principles of synthetic biology has become an attractive alternative to natural routes of chemical synthesis or the existing industrial production methods. Assembly of biosynthetic enzymes into the organized multienzyme complexes via specific interactions or via protein scaffold has been also utilized by nature to improve the efficiency of biosynthetic reactions such as fatty acids or carbon fixation. We and others (Conrado et al., Nucl.Acids.Res.2012; Dueber et al., Nat. Biotech. 2009) have successfully repurposed nucleic acids and protein domains to improve biosynthesis by recruiting enzymes into the monomeric soluble complexes.
The central innovative idea of this project is to design and produce defined nanoscale protein aggregates which will achieve high local concentration of biosynthetic enzymes that should further improve the biosynthetic yield and stabilize the enzymes. This proposal aims to apply our expertise in the design of polypeptide nanostructures on the requirements of the biotechnological industry to increase the production yield and reduce the cost of production of selected chemicals. We expect that this project will significantly contribute to the exploitation of the potentials of designed polypeptide nanoscaffold technology for the improvement of metabolic product biosynthesis in genetically engineered microbial cells. Modularized building blocks will be rationally combined to provide control of the spatial positioning of biosynthetic enzymes in designed nanoassemblies for the metabolite production of the selected biosynthetic pathway.
The aim of the project is to advance and exploit the potentials of the designed polypeptide nanomaterial technology for the improvement of biosynthesis. The building blocks for polypeptide scaffolds will be produced in E. coli, where the polypeptide building blocks can be prepared cost effectively at the industrial scale. Two different types of polypeptide building blocks, composed either of concatenated coiled-coil-forming segments or b-sheet-forming segments, will be tested for the biosynthetic scaffold formation. Orthogonal coiled-coil forming peptide pairs that have been designed, characterized and successfully employed in our previous experiments for construction of nanoscale tetrahedron (Gradišar et al., Nat. Chem. Biol. 2013) will be used. For b-sheet-based scaffolds we will select amyloid-beta peptides of varying length, chain direction and stability of β-sheets-based fibrils. Self-assembly of building blocks will be monitored by biochemical and biophysical methods such as dynamic light scattering, transmission electron- and atomic force microscopy. The proposed biosynthetic strategy will be investigated both in vitro and in vivo. Synthetic coiled-coil-based and cross-beta-based scaffolds will be functionalized for the specific biosynthetic pathway. The efficiency will be tested using resveratrol and mevalonate biosynthetic pathways, in agreement and in collaboration with a co-funding industrial partner. This approach represents a completely new application of bionanomaterials as the enzymatically functionalized polypeptide scaffolds offer an exceptional biotechnological potentia
Significance for science
The project coincides with priorities of the Horizon 2020, the EU Framework Programme, that has a strong focus on developing European industrial capabilities in Key Enabling Tehnologies (KETs), and covers the areas of Nanotechnologies, Advanced materials and Biotechnology. The relevance of the project is first of all in the evaluation of the potential of the innovative technological platform for the designed polypeptide nanoassemblies that serve as scaffolds for the efficient biosynthesis of metabolic products. We developed the innovative strategies for functionalization of the polypeptide nanoscaffolds which is applicable in various fileds such as biocatalysis, chemical engineering, medicine, pharmacy, etc. Scaffolding of the biosynthetic enzymes involved in selected biosynthetic pathway is one of the strategies to increase production of desired compound. We applied interaction domains, that form designed coiled coils or beta-sheets, to bring the enzymes into proximity and increased the production yield and also the product purity. We demonstrated that clustering of the enzymes through formation of coiled coils and beta-sheets increase the production of selected compounds, resveratrol and mevalonate, in bacteria and yeast. Results of the project brought new scientific knowledge and will be interesting for biotechnological industry as well as for a broader scientific audience. A successful result would represent an important step in the development of new technological platforms for exploatation of application advances. Additionally, research provides new application of experimental methods, their adaptation and optimization for analysis of new bionanoassemblies.
Significance for the country
During the last years the members of project group achieved excellent success in a competition of research projects among the most eminent academic competitors and thus contributed to the promotion of natural science to the general audience as well as promotion of Slovenia as a country with good science and education standards. The members of the research group promoted the public awareness of science via interviews for mass media. The aquired knowledge was available to experts as well as a wider audience via lectures and scientific publications. The highest relevance of the project for the economy and the society is in the development of a highly specialized knowledge that is available for different applications in biocatalysis, biotechnology, chemistry and medicine for solving real problems. This is also a strategically very important area, since the bionanotechnology is fast-developing field of science with great potentials for industrial applications with high added value. New discoveries and knowledge in this project may finally lead to nanoassemblies with different, predetermined functionalities required in different fields of application. Examples of application of such materials with new properties are, besides in biocatalysis, also in the pharmacy, where the nanoassemblies based on proteins would enable a new route for drug delivery by means of biocompatible molecules. The proposed technology could increase the competitiveness of (Slovenian) biotech companies and eventually results in establishing new spin-off companies, which in turn may lead to new jobs for the biotechnologically educated staff. In addition to economic performance, new materials, implemented in the appropriate products, may serve to improve the quality of life. The innovative technologies and results were disseminated via publishing in internationally well established scientific journals, via presentations on conferences; both are important for relevance of the research and for the recognition of the research group and the country.
Most important scientific results
Annual report
2014,
2015,
final report
Most important socioeconomically and culturally relevant results
Annual report
2014,
2015,
final report
