Spohn Marius, Wohlleben Wolfgang, Stegmann Evi
Interfaculty Institute of Microbiology and Infection Medicine Tuebingen, Microbiology/Biotechnology, University of Tuebingen, 72076, Tuebingen, Germany.
Partner Site Tuebingen, German Centre for Infection Research (DZIF), Tuebingen, Germany.
Environ Microbiol. 2016 Apr;18(4):1249-63. doi: 10.1111/1462-2920.13159. Epub 2016 Jan 18.
The actinomycete Amycolatopsis japonicum produces the complexing agent ethylenediamine-disuccinate ([S,S]-EDDS), which is an isomer of the widely industrially applied ethylenediamine-tetraacetate (EDTA). In contrast to EDTA, [S,S]-EDDS is readily biodegradable and is therefore an alternative with a favourable environmental profile. Biotechnological production of [S,S]-EDDS, however, is not currently possible because its biosynthesis is inhibited by low-micromolar zinc concentrations. Here we illustrate the development of a new strategy for identifying a biosynthetic pathway that is based on the elucidation of transcriptional regulation and the screening for binding sites of the respective regulator that controls the [S,S]-EDDS biosynthesis genes. To achieve this, we identified the zinc uptake regulator Zur in A. japonicum and showed that it mediates the repression of the zinc uptake system ZnuABCAj . The Zur-binding motif, recognized by the zinc-bound Zur protein in the upstream region of znuABCAj , was used to screen the genome, leading to the identification of the aes genes. Transcriptional analysis and shift assays reveal specific zinc-responsive regulation of the aes genes by Zur, and gene inactivation shows their involvement in [S,S]-EDDS biosynthesis. Zur-mediated zinc repression of the [S,S]-EDDS biosynthesis genes is abolished in a Δzur mutant, which offers now the opportunity to develop a biotechnological process.
放线菌日本拟无枝酸菌可产生络合剂乙二胺二琥珀酸（[S,S]-EDDS），它是工业上广泛应用的乙二胺四乙酸（EDTA）的一种异构体。与EDTA不同，[S,S]-EDDS易于生物降解，因此是一种具有良好环境特性的替代品。然而，目前还无法通过生物技术生产[SS]-EDDS，因为其生物合成会受到低微摩尔浓度锌的抑制。在这里，我们阐述了一种新策略的开发过程，该策略基于对转录调控的阐明以及对控制[S,S]-EDDS生物合成基因的相应调节因子结合位点的筛选。为实现这一目标，我们在日本拟无枝酸菌中鉴定出锌摄取调节因子Zur，并表明它介导了锌摄取系统ZnuABCAj的抑制作用。在znuABCAj上游区域被锌结合的Zur蛋白识别的Zur结合基序，被用于筛选基因组，从而鉴定出aes基因。转录分析和转移试验揭示了Zur对aes基因的特异性锌响应调节，基因失活表明它们参与了[S,S]-EDDS的生物合成。在Δzur突变体中，Zur介导的对[S,S]-EDDS生物合成基因的锌抑制作用被消除，这为开发生物技术工艺提供了契机。
