| dc.description.abstract |
Although they are only needed in small amounts, trace elements, e.g. zinc or iron, are still essential for all living organisms. To facilitate the uptake of those trace elements, bacteria are able to produce so-called metallophores, which are usually low molecular weight compounds. These naturally produced chelators (ion complexing agents) are secreted to extracellularly bind hardly soluble metal ions. When metal ions are complexed by metallophores, their solubility increases and thus their uptake improves. However, not only bacteria rely on chelators to increase the solubility and bioavailability of metal ions. The synthetic chelator ethylenediaminetetraacetic acid (EDTA) e.g. is one of the most abundantly used commercial complexing agents. Its field of application ranges from an ingredient in various washing agents, cosmetics or fertilizers to a use as additive in foods and medicinal products. Due to a combination of both, the abundant industrial usage as well as a poor biodegradability, the synthetic chelator EDTA has accumulated in rivers and lakes in the last decades. Since this poses a putative environmental threat, there is an ongoing effort to replace EDTA with a sustainable alternative with a higher biodegradability, while having similar chelating capacities. One of the compounds that is considered as a potential substitute is ethylenediaminedisuccinc acid (EDDS). EDDS is a structural isomer of EDTA and is naturally produced by various species of actinomycete bacteria of the genus Amycolatopsis, e.g. by Amycolatopsis japonicum. Although both compounds share similar chelating capacities, the [S,S]-isomer of EDDS is extremely-well biodegradable compared to EDTA, making it an efficient as well as a sustainable alternative to EDTA. Since the biosynthesis of [S,S]-EDDS in A. japonicum is zinc regulated, the metallophore is presumably used as zincophore in order to facilitate the uptake of zinc. However, even trace amounts of zinc, which occur ubiquitously in glass or steel fermenters, are able to inhibit the production of [S,S]-EDDS. Aiming at an economical biotechnological production of this compound, the production of [S,S]-EDDS in A. japonicum was optimized by metabolic engineering. Since both the biosynthesis genes as well as the molecular mechanisms of the zinc regulation had already been elucidated in this strain, the first step of the optimization process focused on the exchange of the native promoter of the [S,S]-EDDS biosynthesis genes with a strong, constitutive one. Hence, the zinc inhibition by the so called Zur-regulator was abolished and the productivity increased. Furthermore, additional copies of the biosynthesis genes were introduced and the supply of the precursor O-phosphoserine was optimized. This optimization process resulted in an increase of [S,S]-EDDS production in A. japonicum from 0.3 g/L to 3.0 g/L when minimal medium was used in small scale cultivation. Moreover, when tested in complex medium in bioreactors, the newly generated [S,S]-EDDS-producer strain reached a production titer of 9.8 g/L.
Another focus of this work lies on investigating the distribution of the potential to produce [S,S]-EDDS or EDDS-like compounds in actinomycetes and their functional role in the producer strains. A MultiGene-Blast analysis searching for EDDS-like biosynthesis proteins in actinomycetes genomes led to the discovery of a new biosynthesis gene cluster, which is present is various species from different actinomycete genera. Based on the high similarity of the genes in this cluster to the known genes of the EDDS biosynthesis as well as to another known gene of the viomycin-biosynthesis, the metallophore ethylendiaminesuccinic acid hydroxyarginine (EDHA) could be assigned as the putative gene cluster product. Although the compound EDHA had already been discovered, the corresponding gene cluster has not been described so far. Bioinformatic analysis of two EDHA cluster containing strains, Streptomyces scabies and Streptomyces sp. MA5143a, revealed a putative DNA binding motif of an IdeR-regulator in this gene cluster. These IdeR regulators are known to be iron dependent, DNA binding proteins, which regulate the expression of siderophore biosynthesis clusters in Gram-positive bacteria. Transcription of the EDHA biosynthesis genes in those two strains was analyzed by reverse transcriptase and polymerase chain reaction (RT-PCR) and was shown to be iron repressed. Additionally, in both strains iron inhibited EDHA production could be detected via high performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS). These experiments resulted in the conclusion, that the EDDS-like metallophore EDHA in contrast to EDDS presumably functions as siderophore to facilitate the iron uptake in the strains S. scabies und Streptomyces sp. MA5143a. |
en |
