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@PHDTHESIS{Ernst:1032167,
      author       = {Ernst, Philipp},
      title        = {{E}xploring the process window for production of itaconic,
                      2 hydroxyparaconic, and itatartaric acid with engineered
                      {U}stilago strains},
      volume       = {293},
      school       = {Düsseldorf},
      type         = {Dissertation},
      address      = {Jülich},
      publisher    = {Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag},
      reportid     = {FZJ-2024-06042},
      isbn         = {978-3-95806-825-4},
      series       = {Schriften des Forschungszentrums Jülich Reihe
                      Schlüsseltechnologien / Key Technologies},
      pages        = {x, 145},
      year         = {2025},
      note         = {Dissertation, Düsseldorf, 2024},
      abstract     = {To combat the current challenges of overpopulation, global
                      warming and the limited availability of fossil resources,
                      the linear petrochemical-based industry needs to be replaced
                      by a more sustainable bioeconomy. Therefore, economic
                      production of bio-based platform chemicals such as itaconic
                      acid is an emerging research topic. Itaconic acid is a
                      versatile monomer in the polymer industry and has also high
                      relevance in the medical and pharmaceutical sectors due to
                      its anti-microbial and anti-inflammatory properties. Up to
                      now, itaconic acid is commercially produced by the fungus
                      Aspergillus terreus, but its filamentous morphology poses
                      major limitations on bioprocess technology developments and
                      elevates production costs. Thus, current efforts are
                      focusing on the dimorphic basidiomycete Ustilago as an
                      alternative, natural itaconic acid producer, which offers
                      several advantages including a stable yeast-like morphology,
                      robustness and biosafety. In previous studies, Ustilago
                      maydis und Ustilago cynodontis have already been deeply
                      engineered to optimize itaconate production. In frame of
                      this thesis, established modifications from two different
                      itaconatehyperproducing U. maydis strains were consolidated
                      into one strain named U. maydis K14. This strain 1) features
                      stable yeast-like growth due to deletion of fuz7 involved in
                      filamentous development, 2) produces less byproducts due to
                      deletion of competing pathways (ΔMEL, ΔUA, Δdgat,
                      Δcyp3), and 3) circumvents enzymatic bottlenecks by
                      overproduction of the itaconate cluster regulator Ria1 and
                      the mitochondrial cis-aconitate transporter MttA from A.
                      terreus. A lower osmotolerance of U. maydis K14 as a side
                      effect of this engineering was counteracted by a continuous
                      glucose feeding strategy in high and low cell-density
                      fed-batch fermentations. With the latter strategy, high
                      product titers with the maximum theoretical
                      substrate-to-product yield of 0.72 ± 0.02 gITA gGLC-1
                      during the production phase were obtained, thereby mastering
                      one of the main challenges during fungal itaconate
                      production. However, improving economics is not just about
                      optimizing individual parameters such as yield, titer and
                      productivity, but also about minimizing main cost drivers
                      such as base and acid consumption during fermentation and
                      downstream processing, respectively. Using the previously
                      engineered and naturally acid-tolerant U. cynodontis ITA MAX
                      pH (Δfuz7 Δcyp3 PetefmttA Pria1ria1), the process window
                      of itaconate production with regard to pH was systematically
                      explored in continuous fed-batch fermentations aiming at a
                      rational analysis of operational costs. A subsequent
                      techno-economic analysis exposed that a production pH of 3.6
                      provided the best trade-off between yield, titer and
                      productivity on the one hand, and the use of base and acid
                      and associated salt waste production on the other hand.
                      While such process optimizations are usually carried out
                      using the conventional feedstock glucose, long-term
                      solutions for bio-based production processes envisage the
                      usage of unprocessed, low-cost feedstocks in order to
                      further reduce production costs and meet the circular
                      bioeconomy concept. In this context, this thesis revealed
                      the natural production of the amylolytic enzymes
                      glucoamylase and α-glucosidase by U. cynodontis ITA MAX pH,
                      enabling the utilization of starch as feedstock for
                      itaconate production. Production was optimized by
                      overexpression of an α-amylase gene otherwise not expressed
                      under the applied conditions. In addition to itaconate,
                      Ustilago species produce the two itaconate derivatives
                      2-hydroxyparaconate and itatartarate, which are potential
                      novel anti-microbial drug candidates. To restore
                      2-hydroxyparaconate and itatartarate production in U.
                      cynodontis ITA MAX pH, the itaconate-oxidizing P450
                      monooxygenase gene cyp3 was overexpressed under a
                      constitutive promotor, yielding a product mixture of
                      itaconate, 2-hydroxyparaconate and itatartarate. Derivatives
                      specificity was increased by using glycerol as alternative
                      carbon source, exchanging the native itaconate transporter
                      Itp1 with the one from A. terreus (MfsA), and low pH
                      conditions. In batch fermentations on glycerol, this strain
                      was able to produce 2-hydroxyparaconate and itatartarate
                      with 100 ± 0.0 $\%$ derivatives specificity, allowing
                      subsequent purification of both, not yet commercially
                      available products for structural and biochemical
                      characterization. In conclusion, this thesis demonstrates
                      that an integrated approach of strain and process
                      engineering can provide major advances for optimizing
                      economic feasibility of itaconate, 2-hydroxyparaconate and
                      itatartarate production with Ustilago species in a
                      biorefinery context, thereby enabling an expanded production
                      of bio-based building blocks of industrial and potentially
                      also pharmaceutical relevance.},
      cin          = {IBG-1},
      cid          = {I:(DE-Juel1)IBG-1-20101118},
      pnm          = {2171 - Biological and environmental resources for
                      sustainable use (POF4-217)},
      pid          = {G:(DE-HGF)POF4-2171},
      typ          = {PUB:(DE-HGF)3 / PUB:(DE-HGF)11},
      urn          = {urn:nbn:de:0001-2506171149098.453479022595},
      doi          = {10.34734/FZJ-2024-06042},
      url          = {https://juser.fz-juelich.de/record/1032167},
}