The interplay between transport and metabolism in fungal itaconic acid production.

Besides enzymatic conversions, many eukaryotic metabolic pathways also involve transport proteins that shuttle molecules between subcellular compartments, or into the extracellular space. Fungal itaconate production involves two such transport steps, involving an itaconate transport protein (Itp), and a mitochondrial tricarboxylate transporter (Mtt). The filamentous ascomycete Aspergillus terreus and the unicellular basidiomycete Ustilago maydis both produce itaconate, but do so via very different molecular pathways, and under very different cultivation conditions. In contrast, the transport proteins of these two strains are assumed to have a similar function. This study aims to investigate the roles of both the extracellular and mitochondrial transporters from these two organisms by expressing them in the corresponding U. maydis knockouts and monitoring the extracellular product concentrations. Both transporters from A. terreus complemented their corresponding U. maydis knockouts in mediating itaconate production. Surprisingly, complementation with At_MfsA from A. terreus led to a partial switch from itaconate to (S)-2-hydroxyparaconate secretion. Apparently, the export protein from A. terreus has a higher affinity for (S)-2-hydroxyparaconate than for itaconate, even though this species is classically regarded as an itaconate producer. Complementation with At_MttA increased itaconate production by 2.3-fold compared to complementation with Um_Mtt1, indicating that the mitochondrial carrier from A. terreus supports a higher metabolic flux of itaconic acid precursors than its U. maydis counterpart. The biochemical implications of these differences are discussed in the context of the biotechnological application in U. maydis and A. terreus for the production of itaconate and (S)-2-hydroxyparaconate.

Genetic and biochemical insights into the itaconate pathway of Ustilago maydis enable enhanced production.

The Ustilaginaceae family of smut fungi, especially Ustilago maydis, gained biotechnological interest over the last years, amongst others due to its ability to naturally produce the versatile bio-based building block itaconate. Along with itaconate, U. maydis also produces 2-hydroxyparaconate. The latter was proposed to be derived from itaconate, but the underlying biochemistry and associated genes were thus far unknown. Here, we confirm that 2-hydroxyparaconate is a secondary metabolite of U. maydis and propose an extension of U. maydis' itaconate pathway from itaconate to 2-hydroxyparaconate. This conversion is catalyzed by the P450 monooxygenase Cyp3, encoded by cyp3, a gene, which is adjacent to the itaconate gene cluster of U. maydis. By deletion of cyp3 and simultaneous overexpression of the gene cluster regulator ria1, it was possible to generate an itaconate hyper producer strain, which produced up to 4.5-fold more itaconate in comparison to the wildtype without the by-product 2-hydroxyparaconate. By adjusting culture conditions in controlled pulsed fed-batch fermentations, a product to substrate yield of 67% of the theoretical maximum was achieved. In all, the titer, rate and yield of itaconate produced by U. maydis was considerably increased, thus contributing to the industrial application of this unicellular fungus for the biotechnological production of this valuable biomass derived chemical.

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

UNLABELLED: 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 ß-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.

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

BACKGROUND: The 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. RESULTS: In 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. CONCLUSIONS: The 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.

Evolutionary freedom in the regulation of the conserved itaconate cluster by Ria1 in related Ustilaginaceae.

BACKGROUND: Itaconate is getting growing biotechnological significance, due to its use as a platform compound for the production of bio-based polymers, chemicals, and novel fuels. Currently, Aspergillus terreus is used for its industrial production. The Ustilaginaceae family of smut fungi, especially Ustilago maydis, has gained biotechnological interest, due to its ability to naturally produce this dicarboxylic acid. The unicellular, non-filamentous growth form makes these fungi promising alternative candidates for itaconate production. Itaconate production was also observed in other Ustilaginaceae species such as U. cynodontis, U. xerochloae, and U. vetiveriae. The investigated species and strains varied in a range of 0-8 g L-1 itaconate. The genes responsible for itaconate biosynthesis are not known for these strains and therefore not characterized to explain this variability. RESULTS: Itaconate production of 13 strains from 7 species known as itaconate producers among the family Ustilaginaceae were further characterized. The sequences of the gene cluster for itaconate synthesis were analyzed by a complete genome sequencing and comparison to the annotated itaconate cluster of U. maydis. Additionally, the phylogenetic relationship and inter-species transferability of the itaconate cluster transcription factor Ria1 was investigated in detail. Doing so, itaconate production could be activated or enhanced by overexpression of Ria1 originating from a related species, showing their narrow phylogenetic relatedness. CONCLUSION: Itaconate production by Ustilaginaceae species can be considerably increased by changing gene cluster regulation by overexpression of the Ria1 protein, thus contributing to the industrial application of these fungi for the biotechnological production of this valuable biomass derived chemical.

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 Y IA = 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 ( b T - C 8 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.

Efficient itaconic acid production from glycerol with Ustilago vetiveriae TZ1.

BACKGROUND: The family of Ustilaginaceae is known for their capability to naturally produce industrially valuable chemicals from different carbon sources. Recently, several Ustilaginaceae were reported to produce organic acids from glycerol, which is the main side stream in biodiesel production. RESULTS: In this study, we present Ustilago vetiveriae as new production organism for itaconate synthesis from glycerol. In a screening of 126 Ustilaginaceae, this organism reached one of the highest titers for itaconate combined with a high-glycerol uptake rate. By adaptive laboratory evolution, the production characteristics of this strain could be improved. Further medium optimization with the best single colony, U. vetiveriae TZ1, in 24-deep well plates resulted in a maximal itaconate titer of 34.7 ± 2.5 g L-1 produced at a rate of 0.09 ± 0.01 g L-1 h-1 from 196 g L-1 glycerol. Simultaneously, this strain produced 46.2 ± 1.4 g L-1 malate at a rate of 0.12 ± 0.00 g L-1 h-1. Due to product inhibition, the itaconate titer in NaOH-titrated bioreactor cultivations was lower (24 g L-1). Notably, an acidic pH value of 5.5 resulted in decreased itaconate production, however, completely abolishing malate production. Overexpression of ria1 or mtt1, encoding a transcriptional regulator and mitochondrial transporter, respectively, from the itaconate cluster of U. maydis resulted in a 2.0-fold (ria1) and 1.5-fold (mtt1) higher itaconate titer in comparison to the wild-type strain, simultaneously reducing malate production by 75 and 41%, respectively. CONCLUSIONS: The observed production properties of U. vetiveriae TZ1 make this strain a promising candidate for microbial itaconate production. The outcome of the overexpression experiments, which resulted in reduced malate production in favor of an increased itaconate titer, clearly strengthens its potential for industrial itaconate production from glycerol as major side stream of biodiesel production.

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 P etef 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 P etefssu2 and U. trichophora TZ1 P etefmdh2 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.

Promoters from the itaconate cluster of Ustilago maydis are induced by nitrogen depletion.

BACKGROUND: Ustilago maydis is known for its natural potential to produce a broad range of valuable chemicals, such as itaconate, from both industrial carbon waste streams and renewable biomass. Production of itaconate, and many other secondary metabolites, is induced by nitrogen limitation in U. maydis. The clustered genes responsible for itaconate production have recently been identified, enabling the development of new expression tools that are compatible with biotechnological processes. RESULTS: Here we report on the investigation of two of the native promoters, P tad1 and P mtt1 , from the itaconate cluster of U. maydis MB215. For both promoters the specific activation upon nitrogen limitation, which is known to be the trigger for itaconate production in Ustilago, could be demonstrated by gfp expression. The promoters cover a broad range of expression levels, especially when combined with the possibility to create single- and multicopy construct integration events. In addition, these reporter constructs enable a functional characterization of gene induction patterns associated with itaconate production. CONCLUSIONS: The promoters are well suited to induce gene expression in response to nitrogen limitation, coupled to the itaconate production phase, which contributes towards the further improvement of organic acid production with Ustilago.

Draft Genome Sequences of Itaconate-Producing Ustilaginaceae.

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.

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.

Prospecting the biodiversity of the fungal family Ustilaginaceae for the production of value-added chemicals.

BACKGROUND: Ustilaginaceae (belonging to the smut fungi) are commonly known for their plant pathogenicity. Although these microbes lead to yield reduction of cereal production, they can also have an economically positive side. Ustilaginaceae naturally produce a versatile range of value-added chemicals with potential applications in the food, pharmaceutical, and chemical industry. RESULTS: In this study 68 Ustilaginaceae of 13 species were screened for the production of organic acids, polyols, and glycolipids from glucose to characterize their biodiversity and identify potential novel strains for biocatalysis of these valuable chemicals. Ustilago cynodontis, Ustilago maydis, Ustilago avenae, and Sporisorium exsertum were identified as promising production organisms for itaconate, malate, succinate, and erythritol, respectively. The influence of buffer concentration (pH) on acid production was investigated. Selected strains with best itaconate and malate production were characterized in more detail in bioreactor experiments obtaining total acid concentrations of up to 47 ± 1 g L-1. CONCLUSION: The identification and detailed characterization of these producers of valuable chemicals highlights the potential of these unicellular smut fungi for industrial applications and is a further step towards the biotechnological utilization of Ustilaginaceae.