In the currently favored biotechnological production process of ITA Aspergillus terreus is used. The conversion of cis-aconitate into ITA is catalyzed by the enzyme cis-aconitate decarboxylase (CAD). The sequence of CAD from A. terreus has already been published (Kanamasa et al. 2008).
The basidiomycetous fungus Ustilago maydis is an alternative candidate for ITA production. By nature, it is able to produce a high amount of ITA under certain conditions.
As a first step in order to obtain a U. maydis strain fit for efficient and cost-effective industrial application a putative cis-aconitate decarboxylase gene from U. maydis (cadUM) was overexpressed. This gene was under the control of a strong constitutive otef promotor. Transformants were screened for increased ITA production, proofs of which were found in all of them. Three transformants showed significantly higher ITA production when compared with the wild type demonstrating that cadUM is indeed one of the key genes involved in the ITA synthesis pathway.
Yet another approach was chosen to prove this hypothesis. To this end, the same gene (cadUM) will be cloned into suitable hosts, which are not able to produce ITA naturally.