Introduction

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 aim to produce gold. In the old days the fabled Philosopher’s Stone also stood for knowledge, rejuvenation and health. Nowadays, gold is still of great importance for us as it is needed for most electronic devices. However, gold supplies become more and more depleted. One reason for this is the very inefficient recycling of gold from electronic waste: every year 1.9-2.4 tons of gold [] go to waste as the recovery efficiency is very limited with around 28 %. [] Mining for new gold, however, has a devastating environmental impact. Therefore, a way to increase the gold recovery yield from electronic waste is urgently needed. . In this project we want to show one of the versatile applications of non-ribosomal peptide synthetases (NRPSs) and how they could help to solve the dilemma of gold recycling. Our project was inspired by the bacterium Delftia acidovorans, which is known for generating a non-ribosomal peptide (NRP) called delftibactin[citation needed]. As these bacteria grow in environments that contain toxic gold ions they use delftibactin to chelate and dispose of these ions. The efficient large-scale production of this NRP in E.coli could revolutionize the recovery of gold from electronic waste and therefore help to slow down the environmental problems associated with gold mining and the increasing amounts of electronic waste. One feature of NRPSs is that they are able to utilize and introduce not only the common 21 amino acids into peptides, but also numerous modified amino acids. NRPSs cover a second pathway especially for their own survival. These huge protein protein complexes are characteristically for specific small peptides. As mentioned above, delftibactin is an NRP. It is produced by a hybrid NRPS/PKS system. Therefore, it is produced in a non-ribosomal fashion through huge protein complexes termed non-ribosomal peptide synthetases (NRPSs). In their recent publication Johnston et al. (Ref.) predicted that the enzymes responsible for producing delftibactin iare encoded on a single gene cluster, hereafter referred to as Del-cluster (cluster of genes [File with 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 a PKS. The rest of the enzymes 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:

1. DelA: MbtH-like protein, most likely required for efficient delftibactin synthesis[Pmid21826462]
2. DelB: thioesterase
3. DelC: 4'-phosphopanteinyl transferase: required for maturation of ACP/PCP subunits
4. DelD: taurine dioxygenase
5. DelL: Ornithine N-monooxygenase
6. DelP: N5-hydroxyornithine formyltransferase

We aimed to introduce the large Del-cluster for the production of delftibactin into the commonly used, easy-to-culture model organism E. coli. The native bacterium already contains many components needed for the functionality of non-ribosomal-peptide synthetases. Nevertheless we introduce almost the complete Del-cluster into E.coli except DelC (native pptase) because sfp covers this function. As DelF is a polyketide synthase (PKS) it requires the substrate methylmalonyl-CoA which E. coli is not nativly producing. The SPH1 strain (D.acidovorans) contains a methylmalonyl-CoA synthesis pathway and also B. subtilis has the Sfp phosphopanteinyl transferase, which is able to activate a wide variety of ACP domains including those from S. cerevisiae [Pmid9484229]. Thus it could be used as host for the delftibactin synthesis pathway, possibly also eliminating the need to clone DelC. As promoters are only predicted[Pmid22747501] in Daci_4750 (DelK) and Daci_4760 (DelA) and the cluster is transcribed starting with Daci_4760, it can be assumed that the whole ca. 40 kb are transcribed as whole to a polycistronic mRNA. With this knowledge we started our experiments and planning different strategies.

Experiments

Our aim is to express delftibactin in E.coli. 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.

1. Methylmalonyl-CoA, ppTase & permeability device
2. DelH
3. DelA-P - The rest of the genes of the Del-cluster
Basic Strategy will be described in the following paragraphs. For further detailed experiments you can visit our LabJournal[Link to labjournal].

1. Our first aim was to achieve a genomic integration of the genes that encode for components of the Methylmalonyl-CoA pathway into E.coli. 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 Bacillus subtilis in the Biobrick Backbone pSB3C5 and the permeability device (BBa_I746200) for the outer membrane of E.coli 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 9467 bp, which was inserted in DH10ß and BL21DE3 via electroporation.
2. 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 E.coli 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 E.coli has no need to express and mutate DelH. Finally DelH is going to be inserted in DH10ß and BL21 via electroporation.
3. DelA-P (the rest of the genes of the Del-cluster) [File:Del cluster.gb] was amplified with different primer combinations out of D.acidovorans, and a plasmid was created containing these genes on the pSB4K5 Backbone with lacI promotor and without mRFP. The plasmid size is .... and was transformed into DH10ß and BL21(DE3) via electroporation.

All three plasmid were then electroporated together into BL21 and are able to export delftibactin which reduces soluble gold-ions out of the solution when present in the media.

Results

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