Genetic and biochemical insights into the itaconate pathway of Ustilago maydis enable enhanced production

• Published in: Metabolic engineering 2016-11, Vol.38, p.427-435
• Main Authors: Geiser, Elena, Przybilla, Sandra K., Engel, Meike, Kleineberg, Wiebke, Büttner, Linda, Sarikaya, Eda, Hartog, Tim den, Klankermayer, Jürgen, Leitner, Walter, Bölker, Michael, Blank, Lars M., Wierckx, Nick
• Format: Article
• Language: English
• Subjects: 2-hydroxyparaconate; 4-Butyrolactone - analogs & derivatives; 4-Butyrolactone - metabolism; Aspergillus terreus; Biosynthetic Pathways - genetics; Cytochrome P450 Family 3 - genetics; Gene Expression Regulation, Fungal - genetics; Genetic Enhancement - methods; Itaconate; Metabolic engineering; Metabolic Engineering - methods; Metabolic Networks and Pathways - genetics; P450 monooxygenase; Secondary metabolites; Succinates - isolation & purification; Succinates - metabolism; Up-Regulation - genetics; Ustilago - classification; Ustilago - physiology; Ustilago maydis
• ISSN: 1096-7176; 1096-7184
• DOI: 10.1016/j.ymben.2016.10.006

The Ustilaginaceae family of smut fungi, especially Ustilago maydis, gained biotechnological interest over the last years, amongst others due to its ability to naturally produce the versatile bio-based building block itaconate. Along with itaconate, U. maydis also produces 2-hydroxyparaconate. The latter was proposed to be derived from itaconate, but the underlying biochemistry and associated genes were thus far unknown. Here, we confirm that 2-hydroxyparaconate is a secondary metabolite of U. maydis and propose an extension of U. maydis’ itaconate pathway from itaconate to 2-hydroxyparaconate. This conversion is catalyzed by the P450 monooxygenase Cyp3, encoded by cyp3, a gene, which is adjacent to the itaconate gene cluster of U. maydis. By deletion of cyp3 and simultaneous overexpression of the gene cluster regulator ria1, it was possible to generate an itaconate hyper producer strain, which produced up to 4.5–fold more itaconate in comparison to the wildtype without the by-product 2-hydroxyparaconate. By adjusting culture conditions in controlled pulsed fed-batch fermentations, a product to substrate yield of 67% of the theoretical maximum was achieved. In all, the titer, rate and yield of itaconate produced by U. maydis was considerably increased, thus contributing to the industrial application of this unicellular fungus for the biotechnological production of this valuable biomass derived chemical. •2-hydroxyparaconate is a secondary metabolite of Ustilago maydis.•The P450 monooxygenase Cyp3 catalyzes the conversion of itaconate to 2-hydroxyparaconate in U. maydis.•Itaconate production by U. maydis could be increased 4.5 fold by metabolic engineering.•Production of 63gL−1 itaconate in controlled fed-batch cultivation.