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Team:Heidelberg/Project
From 2013.igem.org
Project
Notebook
Human Practice
Our Project. Non-Ribosomal Peptide Synthesis.
Highlights
- Novel approach for creating customized peptides
- Demonstration of NRPS modularity
- Engineering of entirely synthetic NRPS domains
- Blue pigment tag for in-vivo labeling of synthetic peptides
- 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 peptides. To 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- and energy-efficient way through the heterologous expression of the NRPS pathway of Delftia acidovorans that naturally enables precipitation of gold ions from solution.
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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 [1] 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.
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.
Recombinant peptide synthesis invented in the 1980s was advertised as an alternative to chemical peptide synthesis, as it is easily scalable once the production is up and running. However, this approach is mostly restricted to peptides composed of proteinogenic amino acids, thus limiting the number of available amino acid building blocks and thereby narrowing the applicability of this approach.
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.
Recombinant peptide synthesis invented in the 1980s was advertised as an alternative to chemical peptide synthesis, as it is easily scalable once the production is up and running. However, this approach is mostly restricted to peptides composed of proteinogenic amino acids, thus limiting the number of available amino acid building blocks and thereby narrowing the applicability of this approach.
Introduction of Non-Ribosomal Peptide Synthesis
Applications and Usage 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 applied a novel cloning approach termed HiCT, which enables a cost-efficient and rapid assembly of synthetic NRPS module libraries BBF RFC 99.
Remarkably, we were able to engineer a library of IndC modules exhibiting varying blue pigment production efficacies in combination with different NRPS activating PPTases. Notably, a subset of the so-derived 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.
NRPSDesigner: NRPS for Everybody
The importance of the environment
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.
1. Merrifield RB (1963) Solid Phase Peptide Synthesis. I. The Synthesis of a Tetrapeptide. J. Am. Chem. Soc. 85 (14): 2149–2154.
1. Mootz HD, Schwarzer D, Marahiel MA (2000) Construction of hybrid peptide synthetases by module and domain fusions. Proc Natl Acad Sci USA 97: 5848–5853.
2. 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.
