Elena Geiser

Influence of carbon and nitrogen concentration on itaconic acid production by the smut fungus Ustilago maydis

Itaconic acid is a valuable platform compound for the production of bio‐based polymers, chemicals, and fuels. Ustilago maydis is a promising host for the production of itaconic acid from biomass‐derived substrates due to its unicellular growth pattern and its potential to utilize biomass‐derived sugar monomers and polymers. The potential of U. maydis for industrial itaconate production was assessed in pH‐controlled batch fermentations with varying medium compositions. Using 200 g/L glucose and 75 mM ammonium, 44.5 g/L of itaconate was produced at a maximum rate of 0.74 g L−1 h−1. By decreasing the substrate concentrations to 50 g/L glucose and 30 mM ammonium, a yield of 0.34 g/g (47 mol%) could be achieved. Itaconate production from xylose was also feasible. These results indicate that high itaconic acid titers can be achieved with U. maydis. However, further optimization of the biocatalyst itself through metabolic engineering is still needed in order to achieve an economically feasible process, which can be used to advance the development of a bio‐based economy.

Ustilago maydis produces itaconic acid via the unusual intermediate trans‐aconitate

Itaconic acid is an important biomass‐derived chemical building block but has also recently been identified as a metabolite produced in mammals, which has antimicrobial activity. The biosynthetic pathway of itaconic acid has been elucidated in the ascomycetous fungus Aspergillus terreus and in human macrophages. In both organisms itaconic acid is generated by decarboxylation of the tricarboxylic acid (TCA) cycle intermediate cis‐aconitate. Here, we show that the basidiomycetous fungus Ustilago maydis uses an alternative pathway and produces itaconic acid via trans‐aconitate, the thermodynamically favoured isomer of cis‐aconitate. We have identified a gene cluster that contains all genes involved in itaconic acid formation. Trans‐aconitate is generated from cis‐aconitate by a cytosolic aconitate‐Δ‐isomerase (Adi1) that belongs to the PrpF family of proteins involved in bacterial propionate degradation. Decarboxylation of trans‐aconitate is catalyzed by a novel enzyme, trans‐aconitate decarboxylase (Tad1). Tad1 displays significant sequence similarity with bacterial 3‐carboxy‐cis,cis‐muconate lactonizing enzymes (CMLE). This suggests that U. maydis has evolved an alternative biosynthetic pathway for itaconate production using the toxic intermediate trans‐aconitate. Overexpression of a pathway‐specific transcription factor (Ria1) or a mitochondrial tricarboxylic acid transporter (Mtt1) resulted in a twofold increase in itaconate yield. Therefore, our findings offer new strategies for biotechnological production of this valuable biomass‐derived chemical.

Identification of an endo-1,4-beta-xylanase of Ustilago maydis.

BackgroundThe utilization of raw biomass components such as cellulose or hemicellulose for the production of valuable chemicals has attracted considerable research interest in recent years. One promising approach is the application of microorganisms that naturally convert biomass constituents into value added chemicals. One of these organisms – Ustilago maydis – can grow on xylan, the second most abundant polysaccharide in nature, while at the same time it produces chemicals of biotechnological interest.ResultsIn this study, we present the identification of an endo-1,4-beta xylanase responsible for xylan degradation. Xylanase activity of U. maydis cells was indirectly detected by the quantification of released reducing sugars and could be confirmed by visualizing oligosaccharides as degradation products of xylan by thin layer chromatography. A putative endo-1,4-beta-xylanase, encoded by um06350.1, was identified in the supernatant of xylan-grown cells. To confirm the activity, we displayed the putative xylanase on the surface of the xylanase negative Saccharomyces cerevisiae EBY100. The presented enzyme converted xylan to xylotriose, similar to the source organism U. maydis.ConclusionsThe xylan degradation ability together with its unicellular and yeast-like growth makes U. maydis MB215 a promising candidate for the production of valuable chemicals such as itaconic acid or glycolipids from lignocellulosic biomass. Therefore, the characterization of the endo-1,4-beta-xylanase, encoded by um06350.1, is a further step towards the biotechnological application of U. maydis and its enzymes.

Metabolic engineering of Ustilago trichophora TZ1 for improved malic acid production

Ustilago trichophora RK089 has been found recently as a good natural malic acid producer from glycerol. This strain has previously undergone adaptive laboratory evolution for enhanced substrate uptake rate resulting in the strain U. trichophora TZ1. Medium optimization and investigation of process parameters enabled titers and rates that are able to compete with those of organisms overexpressing major parts of the underlying metabolic pathways. Metabolic engineering can likely further increase the efficiency of malate production by this organism, provided that basic genetic tools and methods can be established for this rarely used and relatively obscure species. Here we investigate and adapt existing molecular tools from U. maydis for use in U. trichophora. Selection markers from U. maydis that confer carboxin, hygromycin, nourseothricin, and phleomycin resistance are applicable in U. trichophora. A plasmid was constructed containing the ip-locus of U. trichophora RK089, resulting in site-specific integration into the genome. Using this plasmid, overexpression of pyruvate carboxylase, two malate dehydrogenases (mdh1, mdh2), and two malate transporters (ssu1, ssu2) was possible in U. trichophora TZ1 under control of the strong Petef promoter. Overexpression of mdh1, mdh2, ssu1, and ssu2 increased the product (malate) to substrate (glycerol) yield by up to 54% in shake flasks reaching a titer of up to 120 g L−1. In bioreactor cultivations of U. trichophora TZ1 Petefssu2 and U. trichophora TZ1 Petefmdh2 a drastically lowered biomass formation and glycerol uptake rate resulted in 29% (Ssu1) and 38% (Mdh2) higher specific production rates and 38% (Ssu1) and 46% (Mdh2) increased yields compared to the reference strain U. trichophora TZ1. Investigation of the product spectrum resulted in an 87% closed carbon balance with 134 g L−1 malate and biomass (73 g L−1), succinate (20 g L−1), CO2 (17 g L−1), and α-ketoglutarate (8 g L−1) as main by-products. These results open up a wide range of possibilities for further optimization, especially combinatorial metabolic engineering to increase the flux from pyruvate to malic acid and to reduce by-product formation.

Activating Intrinsic Carbohydrate-Active Enzymes of the Smut Fungus Ustilago maydis for the Degradation of Plant Cell Wall Components

ABSTRACT The microbial conversion of plant biomass to valuable products in a consolidated bioprocess could greatly increase the ecologic and economic impact of a biorefinery. Current strategies for hydrolyzing plant material mostly rely on the external application of carbohydrate-active enzymes (CAZymes). Alternatively, production organisms can be engineered to secrete CAZymes to reduce the reliance on externally added enzymes. Plant-pathogenic fungi have a vast repertoire of hydrolytic enzymes to sustain their lifestyle, but expression of the corresponding genes is usually highly regulated and restricted to the pathogenic phase. Here, we present a new strategy in using the biotrophic smut fungus Ustilago maydis for the degradation of plant cell wall components by activating its intrinsic enzyme potential during axenic growth. This fungal model organism is fully equipped with hydrolytic enzymes, and moreover, it naturally produces value-added substances, such as organic acids and biosurfactants. To achieve the deregulated expression of hydrolytic enzymes during the industrially relevant yeast-like growth in axenic culture, the native promoters of the respective genes were replaced by constitutively active synthetic promoters. This led to an enhanced conversion of xylan, cellobiose, and carboxymethyl cellulose to fermentable sugars. Moreover, a combination of strains with activated endoglucanase and β-glucanase increased the release of glucose from carboxymethyl cellulose and regenerated amorphous cellulose, suggesting that mixed cultivations could be a means for degrading more complex substrates in the future. In summary, this proof of principle demonstrates the potential applicability of activating the expression of native CAZymes from phytopathogens in a biocatalytic process. IMPORTANCE This study describes basic experiments that aim at the degradation of plant cell wall components by the smut fungus Ustilago maydis. As a plant pathogen, this fungus contains a set of lignocellulose-degrading enzymes that may be suited for biomass degradation. However, its hydrolytic enzymes are specifically expressed only during plant infection. Here, we provide the proof of principle that these intrinsic enzymes can be synthetically activated during the industrially relevant yeast-like growth. The fungus is known to naturally synthesize valuable compounds, such as itaconate or glycolipids. Therefore, it could be suited for use in a consolidated bioprocess in which more complex and natural substrates are simultaneously converted to fermentable sugars and to value-added compounds in the future.

Integrated process development of a reactive extraction concept for itaconic acid and application to a real fermentation broth

Itaconic acid (IA) has a high potential to be used as a bio‐based platform chemical and its biocatalytic production via fermentation has significantly improved within the last decade. Additionally downstream processing using reactive extraction (RE) was described, potentially enabling a more efficient sustainable bioprocess producing IA. The bottleneck to overcome is the connection of up‐ and downstream processing, caused by lack of biocompatibility of the RE systems and direct application to fermentation broth. Within this study, a biocompatible RE system for IA is defined (pH dependency, extraction mechanism) and used for direct application to a fermentation broth. By optimizing the biocatalyst, the production medium, and the extraction system in an integrated approach, it was possible to define critical parameters that enabled a tuning of the overall bioprocess. With an extraction yield of YIA = 0.80 ± 0.03, IA could be produced as sole carboxylic acid ( b IA ,0 aq = 0.490 mol/kgaq) using a RE system consisting of ethyl oleate as organic solvent and tri‐n‐octylamine as extractant ( bT-C8 org = 0.6 mol/kgorg). This work is a proof of concept and demonstrates that by joint consideration of up‐ and downstream processing, optimized bioprocesses can be developed.

Draft Genome Sequences of Itaconate-Producing Ustilaginaceae

ABSTRACT Some smut fungi of the family Ustilaginaceae produce itaconate from glucose. De novo genome sequencing of nine itaconate-producing Ustilaginaceae revealed genome sizes between 19 and 25 Mbp. Comparison to the itaconate cluster of U. maydis MB215 revealed all essential genes for itaconate production contributing to metabolic engineering for improving itaconate production.