Itaconic acid production by ustilago maydis

  • Itakonsäureproduktion von Ustilago maydis

Geiser, Elena; Blank, Lars M. (Thesis advisor); Bölker, Michael (Thesis advisor)

1. Aufl.. - Aachen : Apprimus-Verl. (2015, 2015)
Book, Dissertation / PhD Thesis

In: Applied microbiology 1
Page(s)/Article-Nr.: XIII, 131 S. : Ill., graph. Darst.

Zugl.: Aachen, Techn. Hochsch., Diss., 2015

Abstract

The overall goal of this thesis was to establish Ustilago maydis as an alternative whole cell biocatalyst for the biosynthesis of itaconate from renewable substrates, such as cellulose or hemicellulose. Since the production of valuable and biotechnologically relevant chemicals by microorganisms is strongly influenced by the composition of the medium, its optimization is an integral part of biotechnological process development. Therefore, a new medium composition (modified Tabuchi medium) for itaconate production by Ustilaginaceae was developed. This modified Tabuchi medium consists of 50 g L-1 glucose, 0.8 g L-1 NH4Cl, 0.2 g L-1 MgSO4•7H2O, 0.01 g L-1 FeSO4•7H2O, 0.5 g L-1 KH2PO4, 1 mL L-1 vitamin solution, 10 mL L 1 trace element solution and as buffer 19.5 g L-1 2-(N-morpholino)ethanesulfonic acid (MES). The modified medium circumvents the disadvantages of the standard Tabuchi medium, such as low reproducibility and complex/insoluble medium components, and therefore allows reliable physiological experiments as well as high throughput screenings (HTS).To identify potential novel natural biocatalysts for possible platform chemicals, such as itaconate, malate, or succinate, the biodiversity of the fungal family Ustilaginaceae (68 Ustilaginaceae of 13 species) was prospected in this medium via HTS, showing the production of a versatile range of value-added chemicals. Additionally, the influence of buffer concentration (pH) on acid production was investigated. Based on this screening, the most suitable itaconate producer, U. maydis MB215, was selected and characterized in more detail in bioreactor experiments obtaining total acid concentrations (itaconate, malate, and succinate) of up to 35 ± 4 g L-1.In order to combine the utilization of raw biomass components such as cellulose and hemicellulose with the production of valuable platform chemicals such as itaconate, the xylan degradation ability of this strain was investigated. Thereby, the endo-1,4-beta xylanase UmXyn11A (um06350.1) of U. maydis responsible for xylan degradation was identified, characterized, and expressed heterologously.Furthermore, the clustered genes, um05074 (cyp3), um05076 (tad1), um11777 (itp1), um11778 (adi1), um05079 (mtt1), and um05080 (ria1), encoding the proteins responsible for itaconate production and possibly its degradation in U. maydis MB215 were identified. Based on this information a novel biosynthesis pathway for itaconate was established. This knowledge was used to enhance U. maydis’ itaconate production twofold by overexpressing the transcription factor which regulates the gene expression of the itaconate cluster. With the identification of the itaconate biosynthesis pathway, this thesis lays the foundation for further optimization of this pathway and is therefore a first step towards industrial application of U. maydis as a member of the highly interesting group of Ustilaginaceae.

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