Investigation on [S,S]-EDDS biosynthesis and its applications

Abstract

Zinc is a crucial trace element for bacteria and plays an essential role in several physiological processes, including DNA replication and metabolism. Bacteria have evolved various mechanisms to acquire zinc from the environment, such as the production of zincophores. One example is the zincophore [S,S]-EDDS produced by Amycolatopsis japonicum. [S,S]-EDDS is an isomer of ethylenediamine tetraacetate (EDTA), which is widely used in industry. However, unlike EDTA, [S,S]-EDDS is biodegradable, making it a promising alternative with a favorable environmental profile. Besides its application in various industrial processes, [S,S]- EDDS is also interesting for agriculture. These two aspects were investigated in the present work. (i) [S,S]-EDDS has demonstrated efficacy as a Fe2+ and Zn2+ fertilizer, but its short degradation time limits its long-term impact. To overcome this limitation, a promising approach is to use the [S,S]-EDDS producer A. japonicum as a biofertilizer. Continuous production of [S,S]-EDDS would avoid the need for multiple applications of [S,S]-EDDS. The experiments carried out with Phaseolus vulgaris cv. Black pole in calcareous soil showed that the use of A. japonicum as a biofertilizer, however, did not lead to any improvement in plant growth or microelement concentration. (ii) For the use of [S,S]-EDDS as a chelating agent in various industrial applications, the yield of [S,S]-EDDS in A. japonicum needs to be improved. A prerequisite for this is the understanding of the biosynthesis of [S,S]-EDDS. Using a wide range of methods (biochemistry, genetics, and labeling studies) it was confirmed that L-aspartic acid and oxaloacetic acid are precursors for [S,S]-EDDS biosynthesis. In addition, ((S)-2-amino-2-carboxyethyl)-L-aspartic acid (ACEAA) was identified for the first time as a biosynthetic intermediate. Furthermore, the results strongly indicated that the previously proposed precursor, L-diaminopropionic acid (L-DAPP), is not involved in the biosynthesis, suggesting a novel biosynthetic pathway for [S,S]-EDDS. Genetic strategies were used to redirect the metabolic flux towards the desired metabolites to further optimize [S,S]-EDDS yields. To prioritize target genes for metabolic engineering, the bioinformatic tool Secondary Metabolite Transcriptomic Pipeline (Sema-Trap) was used for RNA-Seq-based transcriptome analysis. This identified bldCAj, lacIAj and gltsAj as target genes for engineering in A. japonicum. Overexpression of these genes resulted in a 3-fold increase in [S,S]-EDDS production compared to A. japonicum wild type. Taken together, these findings provide the potential for further progress in optimizing [S,S]-EDDS production.

Die Dissertation ist gesperrt bis zum 20. Juni 2025 !