Charakterisierung des trans-Aconitat-Metabolismus und dessen Regulierung in Ustilago maydis

The unsaturated carboxylic acid trans-aconitate is an isomer of cis-aconitate, an intermediate of the citric acid cycle, which is synthesized by the aconitase. It is known that trans-aconitate is produced by plants containing sugar and also by bacteria as a toxic substance, since it acts as an inhibitor of aconitase and fumarase. In Ustilago maydis a gene cluster was characterized in the course of this work which allows the fungus to use trans-aconitate as the sole carbon source and also counteracts the toxicity of this metabolite. This gene cluster contains four genes that are regulated together. Two code for transporters that are located in the plasma membrane and in the mitochondria. The characterization of mutants and HPLC measurements of the trans-aconitate degradation showed that the transporter Aip1 located in the cell membrane is necessary to import trans-aconitate into the cells. The mitochondrial transporter Mtt2 is also necessary for the growth of the cells on trans-aconitate as the sole carbon source. In addition an aconitate-delta-isomerase Adi2 and a transcription factor Ram1 are encoded in the cluster. The isomerase Adi2 gave the cluster its name because it is very similar to the isomerase Adi1, which is necessary for itaconic acid biosynthesis in U. maydis (Geiser et al., 2016). Adi2 was identified due to its similarity to Adi1 (Przybilla, 2014). Genetic analyzes in combination with HPLC measurements showed that Adi2 is used for the isomerization of trans-aconitate to cis-aconitate and is also necessary to remove toxic trans-aconitate from the cytosol. The transcription factor Ram1 belongs to the family of Zn(II)2-Cys6 transcription factors and is responsible for the regulation of the Adi2 gene cluster. This family of transcription factors is able to bind conserved DNA motifs using a binuclear zinc cluster. A DNA motif was postulated for the binding of Ram1 to the promoter region of the individual cluster genes. The activation domain of Ram1 is stimulated by the presence of trans-aconitate, which means that Ram1 is only able to initiate transcription of the cluster genes when trans-aconitate is present. The exact mechanism of the effect of trans-aconitate on the transcription factor has not been fully elucidated yet. However, experiments with chimeric transcription factors suggest that binding to DNA is probably not directly influenced by trans-aconitate. In addition, it was shown that glucose can inhibit the expression of the genes of the Adi2 gene cluster independently of Ram1.