Itaconic acid production by co-feeding of Ustilago maydis: A combined approach of experimental data, design of experiments, and metabolic modeling.

Itaconic acid is a platform chemical with a range of applications in polymer synthesis and is also discussed for biofuel production. While produced in industry from glucose or sucrose, co-feeding of glucose and acetate was recently discussed to increase itaconic acid production by the smut fungus Ustilago maydis. In this study, we investigate the optimal co-feeding conditions by interlocking experimental and computational methods. Flux balance analysis indicates that acetate improves the itaconic acid yield up to a share of 40% acetate on a carbon molar basis. A design of experiment results in the maximum yield of 0.14 itaconic acid per carbon source from 100 g L - 1 $\,\text{g L}{}^{-1}$ glucose and 12 g L - 1 $\,\text{g L}{}^{-1}$ acetate. The yield is improved by around 22% when compared to feeding of glucose as sole carbon source. To further improve the yield, gene deletion targets are discussed that were identified using the metabolic optimization tool OptKnock. The study contributes ideas to reduce land use for biotechnology by incorporating acetate as co-substrate, a C2-carbon source that is potentially derived from carbon dioxide.

Renewable carbon sources to biochemicals and -fuels: contributions of the smut fungi Ustilaginaceae.

The global demand for food, fuels, and chemicals increases annually. Using renewable C-sources (i.e. biomass, CO2, and organic waste) is a prerequisite for a future free of fossil carbon. The smut fungi Ustilaginaceae naturally produce a versatile spectrum of valuable products, such as organic acids, polyols, and glycolipids, applicable in the food, energy, chemistry, and pharmaceutical sector. Combined with the use of alternative (co-)substrates (e.g. acetate, butanediol, formate, and glycerol), these microorganisms offer excellent potential for industrial biotechnology, thereby overcoming central challenges humankind faces, including CO2 release and land use. Here, we provide insight into fundamental production capacities, present genetic modifications that improve the biotechnical application, and review recent high-performance engineering of Ustilaginaceae toward relevant platform chemicals.

Improved Itaconate Production with Ustilago cynodontis via Co-Metabolism of CO2-Derived Formate.

In recent years, it was shown that itaconic acid can be produced from glucose with Ustilago strains at up to maximum theoretical yield. The use of acetate and formate as co-feedstocks can boost the efficiency of itaconate production with Ustilaginaceae wild-type strains by reducing the glucose amount and thus the agricultural land required for the biotechnological production of this chemical. Metabolically engineered strains (U. cynodontis Δfuz7 Δcyp3 ↑Pria1 and U. cynodontis Δfuz7 Δcyp3 PetefmttA ↑Pria1) were applied in itaconate production, obtaining a titer of 56.1 g L-1 and a yield of 0.55 gitaconate per gsubstrate. Both improved titer and yield (increase of 5.2 g L-1 and 0.04 gitaconate per gsubstrate, respectively) were achieved when using sodium formate as an auxiliary substrate. By applying the design-of-experiments (DoE) methodology, cultivation parameters (glucose, sodium formate and ammonium chloride concentrations) were optimized, resulting in two empirical models predicting itaconate titer and yield for U. cynodontis Δfuz7 Δcyp3 PetefmttA ↑Pria1. Thereby, an almost doubled itaconate titer of 138 g L-1 was obtained and a yield of 0.62 gitaconate per gsubstrate was reached during confirmation experiments corresponding to 86% of the theoretical maximum. In order to close the carbon cycle by production of the co-feed via a "power-to-X" route, the biphasic Ru-catalysed hydrogenation of CO2 to formate could be integrated into the bioprocess directly using the obtained aqueous solution of formates as co-feedstock without any purification steps, demonstrating the (bio)compatibility of the two processes.

Ustilago maydis Metabolic Characterization and Growth Quantification with a Genome-Scale Metabolic Model.

Ustilago maydis is an important plant pathogen that causes corn smut disease and serves as an effective biotechnological production host. The lack of a comprehensive metabolic overview hinders a full understanding of the organism's environmental adaptation and a full use of its metabolic potential. Here, we report the first genome-scale metabolic model (GSMM) of Ustilago maydis (iUma22) for the simulation of metabolic activities. iUma22 was reconstructed from sequencing and annotation using PathwayTools, and the biomass equation was derived from literature values and from the codon composition. The final model contains over 25% annotated genes (6909) in the sequenced genome. Substrate utilization was corrected by BIOLOG phenotype arrays, and exponential batch cultivations were used to test growth predictions. The growth data revealed a decrease in glucose uptake rate with rising glucose concentration. A pangenome of four different U. maydis strains highlighted missing metabolic pathways in iUma22. The new model allows for studies of metabolic adaptations to different environmental niches as well as for biotechnological applications.

Seventeen Ustilaginaceae High-Quality Genome Sequences Allow Phylogenomic Analysis and Provide Insights into Secondary Metabolite Synthesis.

The family of Ustilaginaceae belongs to the order of Basidiomycetes. Despite their plant pathogenicity causing, e.g., corn smut disease, they are also known as natural producers of value-added chemicals such as extracellular glycolipids, organic acids, and polyols. Here, we present 17 high-quality draft genome sequences (N50 > 1 Mb) combining third-generation nanopore and second-generation Illumina sequencing. The data were analyzed with taxonomical genome-based bioinformatics methods such as Percentage of Conserved Proteins (POCP), Average Nucleotide Identity (ANI), and Average Amino Acid Identity (AAI) analyses indicating that a reclassification of the Ustilaginaceae family might be required. Further, conserved core genes were determined to calculate a phylogenomic core genome tree of the Ustilaginaceae that also supported the results of the other phylogenomic analysis. In addition, to genomic comparisons, secondary metabolite clusters (e.g., itaconic acid, mannosylerythritol lipids, and ustilagic acid) of biotechnological interest were analyzed, whereas the sheer number of clusters did not differ much between species.

Ustilaginaceae Biocatalyst for Co-Metabolism of CO2-Derived Substrates toward Carbon-Neutral Itaconate Production.

The family Ustilaginaceae (belonging to the smut fungi) are known for their plant pathogenicity. Despite the fact that these plant diseases cause agricultural yield reduction, smut fungi attracted special attention in the field of industrial biotechnology. Ustilaginaceae show a versatile product spectrum such as organic acids (e.g., itaconate, malate, succinate), polyols (e.g., erythritol, mannitol), and extracellular glycolipids, which are considered value-added chemicals with potential applications in the pharmaceutical, food, and chemical industries. This study focused on itaconate as a platform chemical for the production of resins, plastics, adhesives, and biofuels. During this work, 72 different Ustilaginaceae strains from 36 species were investigated for their ability to (co-) consume the CO2-derived substrates acetate and formate, potentially contributing toward a carbon-neutral itaconate production. The fungal growth and product spectrum with special interest in itaconate was characterized. Ustilago maydis MB215 and Ustilago rabenhorstiana NBRC 8995 were identified as promising candidates for acetate metabolization whereas Ustilago cynodontis NBRC 7530 was identified as a potential production host using formate as a co-substrate enhancing the itaconate production. Selected strains with the best itaconate production were characterized in more detail in controlled-batch bioreactor experiments confirming the co-substrate utilization. Thus, a proof-of-principle study was performed resulting in the identification and characterization of three promising Ustilaginaceae biocatalyst candidates for carbon-neutral itaconate production contributing to the biotechnological relevance of Ustilaginaceae.