Team:Heidelberg/Project

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                         <h1><span style="font-size:180%;color:#FFCC00;">Project Description.</span><span class="text-muted" style="font-family:Arial, sans-serif; font-size:90%"> Non-ribosomal Synthesis.</span></h1>  
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                         <h1><span style="font-size:200%;color:#333333;">Our Project.</span><span class="text-muted" style="font-family:Arial, sans-serif; font-size:110%"> Foundational Advance in Peptide Synthesis.</span></h1>
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                         <h2>Highlights</h2>
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                                <li>Transfer of the whole delftibactin NRPS pathway from <i>D. acidovorans</i> into <i>E. coli</i></li>
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                            <b>
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                                <li>Novel approach for transfering a whole NRPS pathway more than 50 kb in size from one bacterial species into another</li>
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<li>Novel approach for creating customized peptides
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                                <li>Optimization of the Gibson Cloning Strategy for the creation of large plasmids (over 30 kb in size) with high GC content </li>
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<li>Blue pigment tag for <i>in-vivo</i> labeling of synthetic peptides
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                                <li>Precipitation of pure gold from electronic waste using delftibactin </li>
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<li>Demonstration of NRPS modularity
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<li>Engineering of entirely synthetic NRPS domains
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<li>Software for <i>in-silico</i> design of custom NRPSs
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<li>Sustainable and efficient gold recycling from electronic waste using Delftibactin
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                         <h2>Abstract</h2>
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                            Efficient recycling of gold from electronic waste using recombinant delftibactin<br><br>
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Several secondary metabolites, such as commonly used antibiotics, pigments and detoxifying enzymes, are synthesized by <b>non-ribosomal peptide synthetases (NRPSs)</b>. These enzymes beautifully reflect one of the fundamental principles of synthetic biology, as they are <b>remarkably modular</b>. We will <b>assemble new NRPSs by combining individual domains and modules of different origin</b>, thus setting the basis for novel and <b>customized synthesis of non-ribosomal peptides</b>To make the use of NRPSs amenable to a wider community, we will devise a new software-tool, called <b>NRPS Designer</b>, 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 <i>Escherichia coli</i> to <b>recycle gold from electronic waste in a cost-efficient and environmentally friendly</b> way through the heterologous expression of the NRPS pathway of <i>Delftia acidovorans</i> that naturally enables precipitation of gold ions from solution.
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Undoubtedly, <a href='#gold'>gold</a> is one of the most precious materials on earth. Besides its common use in art and jewelry, gold is also an essential component of our modern computers and cell-phones. Due to the fast turn-over of today’s high-tech equipment, millions of tons of electronic waste accumulate each year containing tons of this valuable metal. The main approach nowadays to recycle gold from electronic waste is by electrolysis. Unfortunately, this is a highly inefficient and expensive procedure, preventing most of the gold from being recovered.<br><br>
 
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Earlier this year, a publication in Nature Chemical Biology reported the existence of a non-ribosomal peptide – delftibactin - which has the astonishing property to specifically precipitate elemental gold from gold-ion containing solutions. Naturally, delftibactin is produced by <i>Delftia acidovorans</i>, an extremophile bacterium, which secretes delftibactin to complexate and dispose of toxic gold ions present in its environment. Although the exact delftibactin production pathway is not known, bioinformatic predictions claim a non-ribosomal peptide synthesis pathway encoded on a large, 59 kb gene cluster (the del-cluster) to be responsible for delftibactin production. <br><br>
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In this subproject we want to demonstrate that the natural secondary metabolite delftibactin can be efficiently produced in <i>E. coli</i> and used for the recycling of gold from electronic waste. To this end, we developed a cloning strategy based on an optimized Gibson Assembly protocol, enabling the cloning of large, GC-rich genomic regions onto regular low-copy plasmids. We thereby engineered three different plasmids (about 70 kb in total size) enabling the expression of the predicted del-cluster from regular <i>E. coli</i> promoters along with the methylmalonyl-CoA pathway providing the basic delftibactin building blocks and a NRPS activating PPTase, Sfp from Bacillus subtilis. <br><br>
 
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We want to show that these large constructs can be potentially inserted and expressed by <i>E. coli</i> with the promising perspective that delftibactin could readily be used as an efficient way of gold recycling from electronic waste.
 
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                    <h2>Introduction</h2>
 
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                        The quest for a magical substance to generate gold from inferior metals stirred the imagination of generations. However, this substance, the Philosopher’s Stone, stands for more than just the desire to produce gold. In the old days, the fabled Philosopher’s Stone also represented wisdom, rejuvenation and health. Nowadays, gold is still of great importance for us as it is needed for most of our electronic devices.
 
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In 2007, more than two tons of gold were discarded hidden in electronic waste in Germany. Most of the precious element end up on waste disposal sites as only a minor fraction of 28 % of the gold is recycled <bib id="chancerel2010perrine"/><bib id="KauffmanZEIT2011"/>  also due to the small amounts per devide. Since our planet's gold supplies are limited, the metal is more and more depleted and the value of gold continously reaches all-time highs. In order to satisfy our society's need for gold, we have to develop heavy mining techniques involving strong acids, causing devastating impact on human and environment <bib id="pmid15369321"/><bib id="pmid14561078"/><bib id="pmid17540445"/>.
 
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Besides economical usage of the resource gold, one way to reduce global demands for gold is elevation of gold recovery <bib id="PMC3715747"/>. Intriguingly, nature itself offers a structure that has been reported to efficiently extract pure gold from solutions containing gold ions. This fascinating molecule is called Delftibactin and is in fact a small peptide secreted by a metal-tolerant bacterium called <i>Delftia acidovorans</i>.
 
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This extremophile has the incredible ability to withstand toxic amounts of gold ions in contaminated soil <http://bacmap.wishartlab.com/organisms/606><http://www.dsmz.de/catalogues/details/culture/DSM-14801.html?tx_dsmzresources_pi5[returnPid]=304><http://www.dsmz.de/catalogues/details/culture/DSM-39.html>. What is the special feature of Delfibactin enabling precipitation of gold that efficiently? If one could culture these bacteria and produce Delftibactin in large scales, could one potentially recover gold from electronic waste in a cost- and energy-efficient way?
 
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But what is the special feature of Delfibactin to precipitate gold that efficiently?
 
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Delftibactin is no ordinary peptide but a non-ribosomal peptide (NRP) <bib id="23377039"/><bib id="20153164"/>.
 
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The efficient and non-polutative large-scale production of this NRP in <i>E. coli</i> could revolutionize the recovery of gold from electronic waste and additionally highlight the plethora of versatile applications for non-ribosomal peptide synthetases (NRPSs). The most sriking feature of these non-ribosomal synthetases is their ablity to incorporate far more than the 21 common amino acids into peptides. They make use of numerous modified and even non-proteinogenic amino acids <bib id="PMC2944527"/> to assembly peptides of diverse functions.
 
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Delftibactin is a NRP produced by a hybrid NRPS/ polyketide synthase (PKS) system. In their recent publication, Johnston ''et al.'' <bib id="pmid23377039"/> predicted that the enzymes responsible for producing delftibactin are encoded on a single gene cluster, hereafter referred to as Del cluster. It comprises 59 kbp encoding for 21 genes. DelE, DelF, DelG and DelH constitute the hybrid NRPS/ PKS system producing delftibactin, with DelE, DelG and DelH being NRPS and DelF the PKS. The remaining enzymes involved in the Delftibactin synthesis pathway are required for NPRS/ PKS maturation or post-synthesis modification of Delftibactin. The predicted activities of the assumed proteins are: :</p>
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  <li>DelA: MbtH-like protein, most likely required for efficient delftibactin synthesis[Pmid21826462]</li>
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  <li>DelB: thioesterase </li>
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  <li>DelC: 4'-phosphopanteinyl transferase: required for maturation of ACP/PCP subunits </li>
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We aimed to introduce the large Del cluster into the commonly used, easy-to-culture model organism <i>E. coli</i> to produce Delftibactin. This target bacterium already possesses many components needed for the functionality of non-ribosomal-peptide synthetases. Nevertheless, we introduce nearly the entire Del cluster into <i>E. coli</i> except for DelC (native PPTase). This function is covered by the sfp phosphopanteinyl transferaseintroduced from <i>Bacillus subtilis</i>. As DelF is a PKS, it requires methylmalonyl-CoA as substrate, which is not produced by <i>E. coli</i>. Therefore, the MMCoA synthesis pathway from <i>B. subtilus</i> which is able to activate a wide variety of PKSs including those from <i>Saccharomyces cerevisiae</i> <bib id="pmid9484229"/> was transferred, too.
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The resulting engineered <i>E. coli</i> could be used as host for the delftibactin synthesis pathway, possibly also eliminating the need to introduce DelC. As promoters of the Del cluster were only predicted <bib id="pmid22747501"/> for Daci_4750 (DelK) and Daci_4760 (DelA) and the cluster is transcribed starting with Daci_4760, we assumed that the entire sequence stretch of approximately 40 kbp is transcribed as single polycistronic mRNA.
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Facing these challenges, we decided to approach the project straight forward by cultivation of <i>D. acidovorans</i> and the isolation of native Delftibactin to reproduce the findings of ohnston <i>et al.</i> <bib id="pmid23377039"/>. </p>
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                    <h2>Experiments</h2>
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                        Our aim is to express delftibactin in <i>E. coli</i>. This will be achieved by introducing three different plasmids which contain parts of the delftibactin-cluster [File:Del cluster.gb] ,a Methylmalonyl-CoA pathway, a Pptase which replaces the DelC-function and a permeability device for the export of the desired NRP.</p>
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<img src="https://2013.igem.org/File:Heidelberg_Delftibactin_Intro.png"> </img>
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<ol>
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<li>Methylmalonyl-CoA, ppTase & permeability device</li>
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<li>DelH</li>  
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<li>DelA-P - The rest of the genes of the Del-cluster</li>  
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Basic Strategy will be described in the following paragraphs. For further detailed experiments you can visit our LabJournal [Link to labjournal].
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<li> Our first aim was to achieve a genomic integration of the genes that encode for components of the Methylmalonyl-CoA  pathway into <i>E. coli</i>. The presence of this pathway is required for the production of NRPs.  Because the genomic integration turned out to be more challenging then expected a new strategy was developed. Therefore, two plasmids were created (pIK2) containing MethylmalonylCoA amplified from Streptomyces coeliolor and a ppTase amplified from <i>Bacillus subtilis</i> in the Biobrick Backbone pSB3C5 and the permeability device (BBa_I746200) for the outer membrane of <i>E. coli</i> was inserted in another plasmid (pIK1). Team Cambridge revealed in 2007 that Bba_I746200 is toxic. It was itherefore inserted into pIK2 between the two terminators driven by a weak promoter (BBa_J23114) and a weak RBS (Bba_B0030), yielding pIK8 with a total size of 9,467 bp, which was inserted in DH10ß and BL21DE3 via electroporation.</li>
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<br>  
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<li>
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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>
-
As the gene encoding DelH alone has a size of 18 kb we decided to clone and introduce this huge gene on a separate plasmid. The first restriction enzyme strategy was problematic because of DelH amplification and the low yield in the ligation. A new GibsonAssembly-strategy was performed and DelH amplified in smaller pieces. It seemed to appear the same problem of as in the pIK1 that <i>E. coli</i> is selecting out the mutated DelH-constructs or is activly mutating it for toxic reasons. A plasmid was designed with the same low copy promotor as in the pIK8 and a low copy RBS [BBa_]. Another shot was a plasmid without promotor so that <i>E. coli</i> has no need to express and mutate DelH. Finally DelH is going to be inserted in <i>E. coli</i> DH10ß and BL21 via electroporation.</li>  
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                            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>
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<li>
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                            <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.
-
DelA-P (the rest of the genes of the Del-cluster) [File:Del cluster.gb] was amplified with different primer combinations out of <i>D.acidovorans</i>, and a plasmid was created containing these genes on the pSB4K5 Backbone with lacI promotor and without mRFP. The was transformed into DH10ß and BL21(DE3) via electroporation.</li>
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All three plasmid were then electroporated together into <i>E. coli</i> BL21 and are able to export delftibactin which reduces soluble gold-ions out of the solution when present in the media.  
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                    <h2>Results</h2>
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                  <h2>Non-Ribosomal Peptide Synthesis</h2>
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                            Our team developed a novel approach for creating customized peptides that overcomes the above mentioned limitations by engineering synthetic <b>Non-Ribosomal Peptide Synthetases (NRPSs)</b>. 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 <b>not limited</b> to proteinogenic amino acids (reviewed in <span class="citation">[7]</span>).
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                                    <img src="https://static.igem.org/mediawiki/2013/d/de/Heidelberg_NRPS_overview.png" style="width:100%; padding:1%" />
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                    <h2>Functionality and Labeling of NRPSs</h2>
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                    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 <i>Bacillus brevis</i> <a href="https://2013.igem.org/Team:Heidelberg/Project/Tyrocidine">Tyrocidine NRPS</a> pathway. We successfully produced a number of di- and tri-peptides composed of proteinogenic and non-proteinogenic amino acids in <i>E.&nbsp;coli</i>. In order to simplify the detection, purification and quality control of those peptides, <b>we invented an <i>in-vivo</i> labeling procedure</b> for non-ribosomal peptides by fusion of the corresponding, synthetic NRPSs to an <a href="https://2013.igem.org/Team:Heidelberg/Project/Indigoidine">engineered <b>blue pigment</b> module (IndC)</a>.
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                    <br><br>
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                    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 (<a href="/Team:Heidelberg/RFCs#rfc99">BBF RFC 99</a>), which enables a cost-efficient and rapid assembly of synthetic NRPS module libraries. <br>
 +
                    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. <br>
 +
                    Furthermore, we identified the location of domain borders and optimized linker regions used for introduction of synthetic domains. By <a href="https://2013.igem.org/Team:Heidelberg/Modelling/Ind_Production">quantitative dynamic modelling</a>, 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.
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                    <h2>Standardized NRPS-Assembly</h2>
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                    <div class="row">
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                                <div class="col-md-8" style="text-align:justify">
 +
                        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 <a href="http://igem2013.bioquant.uni-heidelberg.de/NRPSDesigner/">NRPS Designer</a>, a software tool for the <i>in-silico</i> 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 <a href="/Team:Heidelberg/RFCs">BBF RFC 100</a>.
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                        <br><br>
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                                      <img src="https://static.igem.org/mediawiki/2013/f/f1/Heidelberg_bn_round_rfc100_half.png" style="width:100%; padding:1%" />
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                        <h2>Environmental Applications</h2>
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To exemplify the infinite application possibilities of NRPSs, we developed an sustainable procedure for <a href="https://2013.igem.org/Team:Heidelberg/Project/Delftibactin">gold recycling from electronic waste</a> using the non-ribosomal peptide Delftibactin produced by <i>Delftia acidovorans</i>, which is known to precipitate elemental gold from gold-ion solutions <span class="citation">[8]</span>. Again we applied mathematical modelling in order to <a href="https://2013.igem.org/Team:Heidelberg/Modelling/Gold_Recovery">quantitatively investigate the feasibility</a> of this approach for an industrial-scale recycling of gold. Currently, we are working on transferring the complete delftibacin production pathway into <i>E. coli</i> in order to increase yield and lower costs of delftibactin production.
 +
<br><br>
 +
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.
 +
<br><br><br>
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</div>
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                                <div class="col-md-4">
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                                    <img src="https://static.igem.org/mediawiki/2013/7/70/Heidelberg_goldrecovery.png" style="width:100%; padding:1%" />
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</div>
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<div class="col-sm-12 jumbotron references" style="margin-top:5%">
 +
<p>1. Merrifield RB (1963) Solid Phase Peptide Synthesis. I. The Synthesis of a Tetrapeptide. J. Am. Chem. Soc. 85 (14): 2149–2154</p>
 +
<p>2. Marahiel MA, Stachelhaus T, Mootz HD (1997)Modular Peptide Synthetases Involved in Nonribosomal Peptide Synthesis. Chem Rev. 97(7):2651-2674</p>
 +
<p>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</p>
 +
<p>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</p>
 +
<p>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</p>
 +
<p>6. Lu Y, Freeland S. (2006) On the evolution of the standard amino-acid alphabet. Genome Biol.7(1):102
 +
<p>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</p>
 +
<p>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.</p>
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Latest revision as of 03:54, 29 October 2013

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.

Thanks to

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