Engineering the morphology and metabolism of Ustilago to expand the process window for itaconic acid production

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

Engineering the morphology and metabolism of pH tolerant Ustilago cynodontis for efficient itaconic acid production

Besides Aspergillus terreus and Ustilago maydis, Ustilago cynodontis is also known as a natural itaconate producer. U. cynodontis was reported as one of the best itaconate producing species in the family of the Ustilaginaceae, featuring a relatively high pH tolerance in comparison to other smut fungi. However, in contrast to U. maydis, it readily displays filamentous growth under sub-optimal growth conditions. In this study, U. cynodontis is established as efficient pH-tolerant itaconic acid producer through a combination of morphological and metabolic engineering. Deletions of the genes ras2, fuz7, and ubc3 abolished the filamentous growth of U. cynodontis, leading to a stable yeast-like growth under a range of stress-inducing conditions. The yeast-like morphology was also maintained in a pulsed fed batch production of 21 g L−1 itaconic acid and 9.3 g L−1 (S)-2-hydroxyparaconate at a pH of 3.8. The genetic and metabolic basis of itaconic acid production in U. cynodontis was characterized through comparative genomics and gene deletion studies. A hyper-producer strain was metabolically engineered using this knowledge resulting in a 6.5-fold improvement of titer

Process engineering of pH tolerant Ustilago cynodontis for efficient itaconic acid production

BackgroundUstilago cynodontis ranks among the relatively unknown itaconate production organisms. In comparison to the well-known and established organisms like Aspergillus terreus and Ustilago maydis, genetic engineering and first optimizations for itaconate production were only recently developed for U. cynodontis, enabling metabolic and morphological engineering of this acid-tolerant organism for efficient itaconate production. These engineered strains were so far mostly characterized in small scale shaken cultures.ResultsIn pH-controlled fed-batch experiments an optimum pH of 3.6 could be determined for itaconate production in the morphology-engineered U. cynodontis Δfuz7. With U. cynodontis ∆fuz7r ∆cyp3r PetefmttA Pria1ria1, optimized for itaconate production through the deletion of an itaconate oxidase and overexpression of rate-limiting production steps, titers up to 82.9 ± 0.8 g L−1 were reached in a high-density pulsed fed-batch fermentation at this pH. The use of a constant glucose feed controlled by in-line glucose analysis increased the yield in the production phase to 0.61 gITA gGLC−1, which is 84% of the maximum theoretical pathway yield. Productivity could be improved to a maximum of 1.44 g L−1 h−1 and cell recycling was achieved by repeated-batch application.ConclusionsHere, we characterize engineered U. cynodontis strains in controlled bioreactors and optimize the fermentation process for itaconate production. The results obtained are discussed in a biotechnological context and show the great potential of U. cynodontis as an itaconate producing host