Team:Heidelberg/Project
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- | + | Peptides represent an astonishingly diverse class of molecules comprising highly versatile functions ranging from signaling, detoxifying and antibiotic function to enzymatic activity in nature. The invention of solid-phase synthesis of small peptides pioneered by Robert Bruce Merrifield in 1963 <span class="citation">[1]</span> sparked the hope to exploit and use those functions for our own purposes. This set the basis for an entirely novel research field termed “synthetic peptide chemistry” associated with big hopes to now being able to engineer novel antibiotics and develop peptides for solving environmental issues. <br> | |
Although solid-phase peptide synthesis could be successfully standardized and automated, an important limitation was never overcome: the method is too expensive to be applied for industry-scale production of synthetic peptides <span class="citation">[2]</span>. <br> | Although solid-phase peptide synthesis could be successfully standardized and automated, an important limitation was never overcome: the method is too expensive to be applied for industry-scale production of synthetic peptides <span class="citation">[2]</span>. <br> | ||
<b>Recombinant peptide synthesis</b> invented in the 1980s was advertised as an alternative to chemical peptide synthesis <span class="citation">[3,4]</span>, as it is easily scalable once the production is up and running (reviewed in <span class="citation">[5]</span>). However, this approach is mostly restricted to peptides composed of <b>proteinogenic amino acids</b> <span class="citation">[6]</span>, thus limiting the number of available amino acid building blocks and thereby narrowing the applicability of this approach. | <b>Recombinant peptide synthesis</b> invented in the 1980s was advertised as an alternative to chemical peptide synthesis <span class="citation">[3,4]</span>, as it is easily scalable once the production is up and running (reviewed in <span class="citation">[5]</span>). However, this approach is mostly restricted to peptides composed of <b>proteinogenic amino acids</b> <span class="citation">[6]</span>, thus limiting the number of available amino acid building blocks and thereby narrowing the applicability of this approach. |
Revision as of 01:56, 29 October 2013
![](https://static.igem.org/mediawiki/2013/a/a6/Heidelberg_go_up2.png)
![](https://static.igem.org/mediawiki/2013/a/ae/Heidelberg_tour2_btn.png)
Our Project. Foundational Advance in Peptide Synthesis.
![](https://static.igem.org/mediawiki/2013/5/58/Heidelberg_GRAPHICAL_ABSTRACT.png)
Highlights
- Novel approach for creating customized peptides
- Blue pigment tag for in-vivo labeling of synthetic peptides
- Demonstration of NRPS modularity
- Engineering of entirely synthetic NRPS domains
- Software for in-silico design of custom NRPSs
- Sustainable and efficient gold recycling from electronic waste using Delftibactin
Abstract
Several secondary metabolites, such as commonly used antibiotics, pigments and detoxifying enzymes, are synthesized by non-ribosomal peptide synthetases (NRPSs). These enzymes beautifully reflect one of the fundamental principles of synthetic biology, as they are remarkably modular. We will assemble new NRPSs by combining individual domains and modules of different origin, thus setting the basis for novel and customized synthesis of non-ribosomal peptidesTo make the use of NRPSs amenable to a wider community, we will devise a new software-tool, called NRPS Designer, which predicts the optimal modular composition of synthetic NRPSs for production of any desired peptide and outputs a cloning strategy based on Gibson assembly. As an application relevant to society, we will engineer Escherichia coli to recycle gold from electronic waste in a cost-efficient and environmentally friendly way through the heterologous expression of the NRPS pathway of Delftia acidovorans that naturally enables precipitation of gold ions from solution.
Although solid-phase peptide synthesis could be successfully standardized and automated, an important limitation was never overcome: the method is too expensive to be applied for industry-scale production of synthetic peptides [2].
Recombinant peptide synthesis invented in the 1980s was advertised as an alternative to chemical peptide synthesis [3,4], as it is easily scalable once the production is up and running (reviewed in [5]). However, this approach is mostly restricted to peptides composed of proteinogenic amino acids [6], thus limiting the number of available amino acid building blocks and thereby narrowing the applicability of this approach.
Non-Ribosomal Peptide Synthesis
![](https://static.igem.org/mediawiki/2013/d/de/Heidelberg_NRPS_overview.png)
Functionality and Labeling of NRPSs
Furthermore, we developed a procedure for improving the functionality of NRPS modules by shuffling single domains derived from different species and by engineering entirely synthetic domains. As proof of concept, we shuffled domains within the unimodular blue pigment NRPS IndC or introduced synthetic domains derived from consensus sequences across different species. To this end, we devised a novel cloning strategy termed HiCT (BBF RFC 99), which enables a cost-efficient and rapid assembly of synthetic NRPS module libraries.
We were able to engineer a library of IndC modules exhibiting varying blue pigment production efficacies in combination with different NRPS activating PPTases. Remarkebly, a subset of these synthetic IndC variants showed a broader PPTase specificity compared to their natural counterpart.
Furthermore, we identified the location of domain borders and optimized linker regions used for introduction of synthetic domains.
![](https://static.igem.org/mediawiki/2013/b/b8/Heidelberg_applications.png)
Standardized NRPS-Assembly
Environmental Applications
We believe, that our project demonstrates the power of non-ribosomal peptide synthesis and that our standardized framework will enable the synthetic biology community to use this power to address many of the challenges of our century.
![](https://static.igem.org/mediawiki/2013/7/70/Heidelberg_goldrecovery.png)
1. Merrifield RB (1963) Solid Phase Peptide Synthesis. I. The Synthesis of a Tetrapeptide. J. Am. Chem. Soc. 85 (14): 2149–2154
2. Marahiel MA, Stachelhaus T, Mootz HD (1997)Modular Peptide Synthetases Involved in Nonribosomal Peptide Synthesis. Chem Rev. 97(7):2651-2674
3. Löwenadler B, Jansson B, Paleus S, Holmgren E, Nilsson B, Moks T, Palm G, Josephson S, Philipson L, Uhlén M (1987) A gene fusion system for generating antibodies against short peptides. Gene 58(1):87-97
4. Nilsson B, Moks T, Jansson B, Abrahmsén L, Elmblad A, Holmgren E, Henrichson C, Jones TA, Uhlén M. (1987) A synthetic IgG-binding domain based on staphylococcal protein A. Protein Eng. 1(2):107-13
5. Bommarius B, Jenssen H, Elliott M, Kindrachuk J, Pasupuleti M, Gieren H, Jaeger KE, Hancock RE, Kalman D. (2010) Cost-effective expression and purification of antimicrobial and host defense peptides in Escherichia coli. Peptides 31(11):1957-65
6. Lu Y, Freeland S. (2006) On the evolution of the standard amino-acid alphabet. Genome Biol.7(1):102
7. Andersson L, Blomberg L, Flegel M, Lepsa L, Nilsson B, Verlander M (2000) Large-scale synthesis of peptides. Peptide Science 55(3):227–250
8. Johnston CW, Wyatt MA, Li X, Ibrahim A, Shuster J, et al. (2013) Gold biomineralization by a metallophore from a gold-associated microbe. Nature chemical biology 9: 241–243.