Engineering the morphology and metabolism of Ustilago to expand the process window for itaconic acid production
Aachen / Apprimus Verlag (2019) [Book, Dissertation / PhD Thesis]
Page(s): 1 Online-Ressource (XIII, 159 Seiten) : Illustrationen, Diagramme
Itaconic acid is a versatile building block in the polymer industry due to its two functional groups. Radical polymerization of the methylene group and/or esterification of the carboxylic acid with a wide range of co-monomers enable a rapidly expanding application range. Since 1950, Aspergillus terreus is used for industrial production of itaconate. However, despite the long history and experience, itaconate production in A. terreus remains challenging and above all the required control of the morphology leads to high production costs, which causes a relatively low market volume of itaconate despite its chemical potential. Due to good scientific fundamentals and robustness, Ustilaginaceae promises alternative hosts that offer new possibilities to achieve more efficient itaconate production.The overall aim of this thesis was to achieve efficient itaconate production from glucose with Ustilaginaceae. In a screening of several Ustilaginaceae, genetic equipment for itaconate production could be determined for all tested strains. In addition to Ustilago maydis, which is well studied in the context of itaconate production, Ustilago cynodontis was chosen mainly due to its tolerance to low pH. Comparative analysis of the mitochondrial and extracellular transporters involved in itaconate and (S)-2-hydroxyparaconate biosynthesis by U. maydis, and A. terreus elucidated that the mitochondrial transporter of A. terreus (MttA) enabled a more efficient itaconate production in U. maydis and U. cynodontis. Itaconate production could be further improved in both strains by metabolic engineering using CRISPR/Cas9 and FLP/FRT systems for marker-free deletion of the itaconate oxidase (Δcyp3), knock-in of the strong and constitutive promoter Petef upstream of the regulator-encoding gene ria in U. maydis or by overexpression of this regulator in U. cynodontis. Thus, production could be enhanced 4.2-fold in U. maydis and 6.5-fold in U. cynodontis compared to corresponding wildtype strains. In order to ensure robust and non-filamentous cells growing in a yeast-like manner under certain process-relevant conditions, both strains were modified in a morphological engineering approach. The gene fuz7, which is part of the Ras/mitogen-activated protein kinase (MAPK) pathway and plays an important role in conjugation tube formation and filamentous growth, was therefore deleted in both strains. The obtained yeast-like-growing strains open up a range of possibilities in the field of process development. Thus, for the first time, itaconate production in a bioreactor with the otherwise strong filamentously growing U. cynodontis could be realized. By optimizing the pH value, different feeding strategies and repeated-batch systems titer up to 83 g L-1, overall yields up to 0.45 gITA gGLC-1 and maximum productivities up to 1.4 g L-1 h-1 could be reached. In U. maydis, fermentations in combination with in situ product removal using calcium carbonate precipitation resulted in a titer of 220 g L-1 itaconate, which is so far the highest reported value for microbially produced itaconate. In conclusion, by an integrated approach of metabolic and morphological engineering, coupled with process development, the efficiency of itaconate production with U. maydis and U. cynodontis could be significantly enhanced. The production strains engineered in this thesis enable new process engineering strategies and ensure stable unicellular growth, thereby likely contributing to the future expansion of the fields of application of itaconate as an important bio-based building block in the near future.
Hosseinpour Tehrani, Hamed
Blank, Lars Mathias