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

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Results

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