title: Genetic and biochemical insights into the itaconate pathway of Ustilago maydis enable enhanced production.
abstract: 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.
authors.affiliation: iAMB - Institute of Applied Microbiology, ABBt - Aachen Biology and Biotechnology, RWTH Aachen University, Worringerweg 1, D-52074 Aachen, Germany; BioSC, c/o Forschungszentrum Jülich, D-52425 Jülich, Germany.
authors.forename: Elena
authors.initials: E
authors.lastname: Geiser
authors.affiliation: iAMB - Institute of Applied Microbiology, ABBt - Aachen Biology and Biotechnology, RWTH Aachen University, Worringerweg 1, D-52074 Aachen, Germany.
authors.forename: Sandra K
authors.initials: SK
authors.lastname: Przybilla
authors.affiliation: iAMB - Institute of Applied Microbiology, ABBt - Aachen Biology and Biotechnology, RWTH Aachen University, Worringerweg 1, D-52074 Aachen, Germany.
authors.forename: Meike
authors.initials: M
authors.lastname: Engel
authors.affiliation: iAMB - Institute of Applied Microbiology, ABBt - Aachen Biology and Biotechnology, RWTH Aachen University, Worringerweg 1, D-52074 Aachen, Germany; BioSC, c/o Forschungszentrum Jülich, D-52425 Jülich, Germany.
authors.forename: Wiebke
authors.initials: W
authors.lastname: Kleineberg
authors.affiliation: Department of Biology, Philipps-University Marburg, Karl-von-Frisch-Straße 8, D-35032 Marburg, Germany.
authors.forename: Linda
authors.initials: L
authors.lastname: Büttner
authors.affiliation: iAMB - Institute of Applied Microbiology, ABBt - Aachen Biology and Biotechnology, RWTH Aachen University, Worringerweg 1, D-52074 Aachen, Germany.
authors.forename: Eda
authors.initials: E
authors.lastname: Sarikaya
authors.affiliation: Institut für Technische und Makromolekulare Chemie (ITMC), RWTH Aachen University, Worringerweg 2, D-52074 Aachen, Germany.
authors.forename: Tim den
authors.initials: TD
authors.lastname: Hartog
authors.affiliation: Institut für Technische und Makromolekulare Chemie (ITMC), RWTH Aachen University, Worringerweg 2, D-52074 Aachen, Germany.
authors.forename: Jürgen
authors.initials: J
authors.lastname: Klankermayer
authors.affiliation: Institut für Technische und Makromolekulare Chemie (ITMC), RWTH Aachen University, Worringerweg 2, D-52074 Aachen, Germany.
authors.forename: Walter
authors.initials: W
authors.lastname: Leitner
authors.affiliation: Department of Biology, Philipps-University Marburg, Karl-von-Frisch-Straße 8, D-35032 Marburg, Germany; LOEWE Center for Synthetic Microbiology (SYNMIKRO), D-35032 Marburg, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Bölker
authors.affiliation: iAMB - Institute of Applied Microbiology, ABBt - Aachen Biology and Biotechnology, RWTH Aachen University, Worringerweg 1, D-52074 Aachen, Germany. Electronic address: lars.blank@rwth-aachen.de.
authors.forename: Lars M
authors.initials: LM
authors.lastname: Blank
authors.affiliation: iAMB - Institute of Applied Microbiology, ABBt - Aachen Biology and Biotechnology, RWTH Aachen University, Worringerweg 1, D-52074 Aachen, Germany.
authors.forename: Nick
authors.initials: N
authors.lastname: Wierckx
mesh_terms: D015107:4-Butyrolactone / Q000031:analogs & derivatives* / Q000378:metabolism; D053898:Biosynthetic Pathways / Q000235:genetics*; D000072469:Cytochrome P450 Family 3 / Q000235:genetics*; D015966:Gene Expression Regulation, Fungal / Q000235:genetics; D024861:Genetic Enhancement / Q000379:methods*; D060847:Metabolic Engineering / Q000379:methods*; D053858:Metabolic Networks and Pathways / Q000235:genetics; D013386:Succinates / Q000302:isolation & purification / Q000378:metabolism*; D015854:Up-Regulation / Q000235:genetics; D014588:Ustilago / Q000145:classification / Q000502:physiology*
chemical_list: D013386:Succinates; C571316:paraconic acid; D000072469:Cytochrome P450 Family 3; D015107:4-Butyrolactone; C005229:itaconic acid
keywords: 2-hydroxyparaconate; Aspergillus terreus; Itaconate; Metabolic engineering; P450 monooxygenase; Secondary metabolites; Ustilago maydis