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| Home > Publications database > Exploring the process window for production of itaconic, 2 hydroxyparaconic, and itatartaric acid with engineered Ustilago strains |
| Home > Publications database > Exploring the process window for production of itaconic, 2 hydroxyparaconic, and itatartaric acid with engineered Ustilago strains |
| Book/Dissertation / PhD Thesis | FZJ-2024-06042 |
2025
Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag
Jülich
ISBN: 978-3-95806-825-4
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Please use a persistent id in citations: urn:nbn:de:0001-2506171149098.453479022595 doi:10.34734/FZJ-2024-06042
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.
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