Elucidation of the zinc-dependent regulation in Amycolatopsis japonicum enabled the identification of the ethylenediamine-disuccinate ([S,S]-EDDS) genes.

Abstract

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.