From 2013.igem.org

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Our Project. Foundational Advance in Peptide Synthesis.

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.

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 [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

Our team developed a novel approach for creating customized peptides that overcomes the above mentioned limitations by engineering synthetic Non-Ribosomal Peptide Synthetases (NRPSs). NRPSs are organized in modules recognizing one specific amino acid substrate and catalyzing the formation of a peptide bond between the amino acid substrate and the nascent peptide chain. Notably, non-ribosomal peptide synthesis does not require mRNA to direct the sequence of amino acid monomers incorporated into the growing peptide and is therefore not limited to proteinogenic amino acids (reviewed in [7]).

Functionality and Labeling of NRPSs

In order to show the applicability of custom NRPSs for synthetic peptide production, we created a set of synthetic NRPSs composed of varying modules derived from the Bacillus brevis Tyrocidine NRPS pathway. We successfully produced a number of di- and tri-peptides composed of proteinogenic and non-proteinogenic amino acids in E. coli. In order to simplify the detection, purification and quality control of those peptides, we invented an in-vivo labeling procedure for non-ribosomal peptides by fusion of the corresponding, synthetic NRPSs to an engineered blue pigment module (IndC).

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. By quantitative dynamic modelling, we obtained a more detailed understanding on the synthesis efficiency of the different IndC variants in different contexts and investigated the influence of indigoidine production on bacterial growth.

Standardized NRPS-Assembly

In order to enable the synthetic biology community to easily engineer custom NRPSs producing user-defined peptides, we developed a standardized framework for the production of non-ribosomal peptides. The framework consists of (1) the NRPS Designer, a software tool for the in-silico design of user-defined NRPSs, (2) a platform for standardized cloning and expression of NRPSs in different bacterial hosts and (3) a quality control procedure for the validation of NRP production. We documented detailed instructions for applying our framework in BBF RFC 100.

Environmental Applications

To exemplify the infinite application possibilities of NRPSs, we developed an sustainable procedure for gold recycling from electronic waste using the non-ribosomal peptide Delftibactin produced by Delftia acidovorans, which is known to precipitate elemental gold from gold-ion solutions [8]. Again we applied mathematical modelling in order to quantitatively investigate the feasibility of this approach for an industrial-scale recycling of gold. Currently, we are working on transferring the complete delftibacin production pathway into E. coli in order to increase yield and lower costs of delftibactin production.

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

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.

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From 2013.igem.org

Latest revision as of 03:54, 29 October 2013

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