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Abstract Background Currently, Aspergillus terreus is used for the industrial production of itaconic acid. Although, alternative feedstock use in fermentations is crucial for cost-efficient and sustainable itaconic acid production, their utilisation with A. terreus most often requires expensive pretreatment. Ustilaginacea are robust alternatives for itaconic acid production, evading the challenges, including the pretreatment of crude feedstocks regarding reduction of manganese concentration, that A. terreus poses. Results In this study, five different Ustilago strains were screened for their growth and production of itaconic acid on defined media. The most promising strains were then used to find a suitable alternative feedstock, based on the local food industry. U. cynodontis ITA Max pH, a highly engineered production strain, was selected to determine the biologically available nitrogen concentration in thick juice and molasses. Based on these findings, thick juice was chosen as feedstock to ensure the necessary nitrogen limitation for itaconic acid production. U. cynodontis ITA Max pH was further characterised regarding osmotolerance and product inhibition and a successful scale-up to a 2 L stirred tank reactor was accomplished. A titer of 106.4 gitaconic acid/L with a theoretical yield of 0.50 gitaconic acid/gsucrose and a space-time yield of 0.72 gitaconic acid/L/h was reached. Conclusions This study demonstrates the utilisation of alternative feedstocks to produce ITA with Ustilaginaceae, without drawbacks in either titer or yield, compared to glucose fermentations.

Background: Currently, Aspergillus terreus is used for the industrial production of itaconic acid. Although, alternative feedstock use in fermentations is crucial for cost-efficient and sustainable itaconic acid production, their utilisation with A. terreus most often requires expensive pretreatment. Ustilaginacea are robust alternatives for itaconic acid production, evading the challenges, including the pretreatment of crude feedstocks regarding reduction of manganese concentration, that A. terreus poses. Results: In this study, five different Ustilago strains were screened for their growth and production of itaconic acid on defined media. The most promising strains were then used to find a suitable alternative feedstock, based on the local food industry. U. cynodontis ITA Max pH, a highly engineered production strain, was selected to determine the biologically available nitrogen concentration in thick juice and molasses. Based on these findings, thick juice was chosen as feedstock to ensure the necessary nitrogen limitation for itaconic acid production. U. cynodontis ITA Max pH was further characterised regarding osmotolerance and product inhibition and a successful scale-up to a 2 L stirred tank reactor was accomplished. A titer of 106.4 gitaconic acid/L with a theoretical yield of 0.50 gitaconic acid/gsucrose and a space-time yield of 0.72 gitaconic acid/L/h was reached. Conclusions: This study demonstrates the utilisation of alternative feedstocks to produce ITA with Ustilaginaceae, without drawbacks in either titer or yield, compared to glucose fermentations. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Mannosylerythritol lipids are glycolipid biosurfactants with many interesting properties. Despite the general interest in those molecules and the need for a robust process, studies on their production in bioreactors are still scarce. In the current study, the fermentative production of MEL in a bioreactor with Moesziomyces aphidis was performed using a defined mineral salt medium. Several kinetic process parameters like substrate consumption rates and product formation rates were evaluated and subsequently enhanced by increasing the biomass concentration through an exponential fed-batch strategy. The fed-batch approaches resulted in two to three fold increased dry biomass concentrations of 10.9–15.5 g/L at the end of the growth phase, compared with 4.2 g/L in the batch process. Consequently, MEL formation rates were increased from 0.1 g/Lh up to around 0.4 g/Lh during the MEL production phase. Thus, a maximum concentration of up to 50.5 g/L MEL was obtained when oil was added in excess, but high concentrations of residual fatty acids were also present in the broth. By adjusting the oil feeding to biomass-specific hydrolysis and MEL production rates, a slightly lower MEL concentration of 34.3 g/L was obtained after 170 h, but at the same time a very pure crude lipid extract with more than 90% MEL and a much lower concentration of remaining fatty acids. With rapeseed oil as substrate, the ideal oil-to-biomass ratio for full substrate conversion was found to be around 10 goil/gbiomass. In addition, off-gas analysis and pH trends could be used to assess biomass growth and MEL production. Finally, kinetic models were developed and compared to the experimental data, allowing for a detailed prediction of the process behavior in future experiments.

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. [ABSTRACT FROM AUTHOR]

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.

Mannosylerythritol lipids (MELs) are glycolipid biosurfactants produced by fungi of the Ustilaginaceae family in the presence of hydrophobic carbon sources like plant oils. In the present study,we investigated the structural composition of MELs produced from castor oil using seven different microorganisms and compared them to MEL structures resulting fromother plant oils. Castor oil is an industrially relevant plant oil that presents as an alternative to currently employed edible plant oils like rapeseed or soybean oil. The main fatty acid in castor oil is the mono-hydroxylated ricinoleic acid, providing the possibility to produce novel MEL structures with interesting features. Analysis of the produced MELs from castor oil by different chromatographic and mass spectrometry techniques revealed that all seven microorganisms were generally able to integrate hydroxylated fatty acids into the MEL molecule, although at varying degrees. These novel MELs containing a hydroxy fatty acid (4-O-[2'-O-alka(e)noyl-3'-O-hydroxyalka(e)noyl-4'/6'-O-acetyl-β- D-mannopyranosyl]-erythritol) were more hydrophilic than conventional MEL and therefore showed a different elution behavior in chromatography. Large shares of novel hydroxy MELs (around 50% of total MELs) were found for the two MEL-B/C producing species Ustilago siamensis and Ustilago shanxiensis, but also for the MEL-A/B/C producer Moesziomyces aphidis (around 25%). In addition, triacylated hydroxylated MELs with a third long-chain fatty acid esterified to the free hydroxyl group of the hydroxy fatty acid were identified for some species. Overall, production of MEL from castor oil with the investigated organisms provided a complex mixture of various novel MEL structures that can be exploited for further research. [ABSTRACT FROM AUTHOR]

The biotrophic fungus Sporisorium reilianum causes destructive head smut disease in maize (Zea mays L.). To explore the pathogenicity arsenal of this fungus, we tracked its transcriptome changes during infection of the maize seedling mesocotyls of two near-isogenic lines, HZ4 and HZ4R, differing solely in the disease resistance gene ZmWAK. Parasitic growth of S. reilianum resulted in thousands of differentially expressed genes (DEGs) compared with growth in axenic culture. The protein synthesis and energy metabolism of S. reilianum were predominantly enriched with down-regulated DEGs, consistent with the arrested hyphal growth observed following colonization. Nutrition-related metabolic processes were enriched with both up- and down-regulated DEGs, which, together with activated transmembrane transport, reflected a potential transition in nutrition uptake of S. reilianum once it invaded maize. Notably, genes encoding secreted proteins of S. reilianum were mostly up-regulated during biotrophy. ZmWAK-mediated resistance to head smut disease reduced the number of DEGs of S. reilianum, particularly those related to the secretome. These observations deepen our understanding of the mechanisms underlying S. reilianum pathogenicity and ZmWAK-induced innate immunity. [ABSTRACT FROM AUTHOR]

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-ofprinciple 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. [ABSTRACT FROM AUTHOR]

Fungi of the Ustilaginaceae family are a promising source for many biotechnologically relevant products. Among these, mannosylerythritol lipid (MEL) biosurfactants have drawn a special interested over the last decades due to their manifold application possibilities. Nevertheless, there is still a knowledge gap regarding process engineering of MEL production. As an example, no reports on the use of a chemically defined culture medium have been published yet, although such a defined medium might be beneficial for scaling-up the production process toward industrial scale. Our aim therefore was to find a mineral medium that allows fast biomass growth and does not negatively affect the successive MEL production from plant oils. The results showed comparable growth performance between the newly evaluated mineral medium and the established yeast extract medium for all seven investigated Ustilaginaceae species. Final biomass concentrations and specific growth rates of 0.16-0.25 h–1 were similar for the two media. Oxygen demand was generally higher in the mineral medium than in the yeast extract medium. It was shown that high concentrations of vitamins and trace elements were necessary to support the growth. Increasing starting concentrations of the media by a factor of 10 resulted in proportionally increasing final biomass concentrations and up to 2.3-times higher maximum growth rates for all species. However, it could also lead to oxygen limitation and stagnant growth rates when too high medium concentrations were used, which was observed for Ustilago siamensis and Moesziomyces aphidis. Successive MEL production from rapeseed oil was effectively shown for 4 out of 7 organisms when the mineral medium was used for cell growth, and it was even enhanced for two organisms, M. aphidis and Pseudozyma hubeiensis pro tem., as compared to the established yeast extract medium. Conversion of rapeseed oil into MEL was generally improved when higher biomass concentrations were achieved during the initial growth phase, indicating a positive relationship between biomass concentration and MEL production. Overall, this is the first report on the use of a chemically defined mineral medium for the cell growth of Ustilaginaceae fungi and successive MEL production from rapeseed oil, as an alternative to the commonly employed yeast extract medium.

Ustilago maydis, a smut fungus, is an appealing model in fundamental research and an upcoming cell factory for industrial biotechnology. The genome of U. maydis has been sequenced and some synthesis pathways were biochemically described; however, the operation of the cellular metabolic network is not well-characterized. Thus, we conducted a comprehensive study to optimize the sample preparation procedure for metabolomics of U. maydis using GC-MS/MS. Due to the unique characteristics of U. maydis cell culture, two quenching solutions, different washing steps, eight extraction methods, and three derivatization conditions have been examined. The optimal method was then applied for stable isotope-assisted quantification of low molecular weight hydrophilic metabolites while U. maydis utilized different carbon sources including sucrose, glucose, and fructose. This study is the first report on a methodology for absolute quantification of intracellular metabolites in U. maydis central carbon metabolism such as sugars, sugar phosphates, organic acids, amino acids, and nucleotides. For biotechnological use, this method is crucial to exploit the full production potential of this fungus and can also be used to study other fungi of the family Ustilaginaceae.

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.

Mannosylerythritol lipids (MEL) are microbial glycolipid biosurfactants with great potential for application in cosmetics and household detergents. In current biotechnological processes, they are produced by basidiomycetous fungi, the Ustilaginaceae, as a complex mixture of different chemical structures. It was the aim of this paper to study the influence of producer organisms and substrates on the resulting MEL structures with a novel high-resolution HPTLC–MALDI-TOF method. Given the seven different microbes and four plant oils, our analysis revealed that the product concentrations varied strongly between organisms, while they were similar for the different substrates. Coconut oil presented an exception, since only one organism was able to synthesize MEL from this substrate in considerable yields. Analysis by GC-FID further showed that the chain length pattern of hydrophobic fatty acid side-chains was very specific for individual organisms, while substrates had only a minor influence on the chain length. Our novel HPTLC–MALDI-TOF combination method finally demonstrated the presence of multiple MEL sub-variants with differing acetylation and fatty acid chain lengths. It also revealed the production of a more hydrophilic biosurfactant mannosylmannitol lipid (MML) as a side-product in certain fungi. Overall, it was concluded that the pattern of produced biosurfactant structures are mainly governed by producer organisms rather than substrates. [ABSTRACT FROM AUTHOR]

A combined ecological, morphological, and molecular approach was used to examine 26 herbarium specimens and eight strains of Moesziomyces. The phylogenetic analysis resolved eight well-supported clades, of which three contained type specimens of known species of Moesziomyces. One clade contained two specimens that produced a teleomorph in the flowers of Echinochloa kimberleyensis in Australia. The name Moesziomyces kimberleyensis is proposed for this smut fungus. Another clade contained specimens that produced sori in the flowers of Leersia hexandra. The name Thecaphora globuligera (now Moesziomyces globuligerus) is available for this species, which is lectotypified. The teleomorph of Moesziomyces antarcticus, previously known only from Japan, is found for the first time in China, on Echinochloa crus-galli. [ABSTRACT FROM AUTHOR]

The basidiomycetous yeast Moesziomyces antarcticus (often cited as Pseudozyma antarctica), originally isolated from a sediment sample obtained from Lake Vanda in Antarctica, was asexually typified but closely related to the smut fungus Moesziomyces bullatus (Ustilaginales). We found a smut fungus on an ovary of barnyardgrass (Echinochloa crus-galli) in Japan, which had been identified as M. bullatus. The teliospores germinated and formed yeast-like colonies. Physiological and phylogenetic studies revealed that the characteristics of the yeast-like isolates coincided with those of "P. antarctica." We thus recognised the smut fungus as the teleomorph of M. antarcticus, and then emended the description of M. antarcticus based on the holomorph. The identified fungus could degrade certain biodegradable plastics and produce mannosylerythritol lipids (MELs) in similar qualities as the "P. antarctica" type strain. This discovery provides a significant bioresource, as genetically diverse M. antarcticus isolates could be obtained from the smut fungus. [ABSTRACT FROM AUTHOR]

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.

Host specialization is a key evolutionary process for the diversification and emergence of new pathogens. However, the molecular determinants of host range are poorly understood. Smut fungi are biotrophic pathogens that have distinct and narrow host ranges based on largely unknown genetic determinants. Hence, we aimed to expand comparative genomics analyses of smut fungi by including more species infecting different hosts and to define orphans and positively selected genes to gain further insights into the genetics basis of host specialization. We analyzed nine lineages of smut fungi isolated from eight crop and non-crop hosts: maize, barley, sugarcane, wheat, oats, Zizania latifolia (Manchurian rice), Echinochloa colona (a wild grass), and Persicaria sp. (a wild dicot plant). We assembled two new genomes: Ustilago hordei (strain Uhor01) isolated from oats and U. tritici (strain CBS 119.19) isolated from wheat. The smut genomes were of small sizes, ranging from 18.38 to 24.63 Mb. U. hordei species experienced genome expansions due to the proliferation of transposable elements and the amount of these elements varied among the two strains. Phylogenetic analysis confirmed that Ustilago is not a monophyletic genus and, furthermore, detected misclassification of the U. tritici specimen. The comparison between smut pathogens of crop and non-crop hosts did not reveal distinct signatures, suggesting that host domestication did not play a dominant role in shaping the evolution of smuts. We found that host specialization in smut fungi likely has a complex genetic basis: different functional categories were enriched in orphans and lineage-specific selected genes. The diversification and gain/loss of effector genes are probably the most important determinants of host specificity.

Ustilago esculenta, resembling a fungal endophyte in Zizania latifolia, inhibits the host plant flowering and induces the host stems to swell and form edible galls. It is well believed that when and how the fungus infects and proliferates in the host plants during the host development is of importance in the edible gall formation. Mitogen-activated protein kinases (MAPKs) have been found to play an important role in sensing environment cues and regulating infection. Two MAPK genes UeKpp2 and UeKpp6 from U. esculenta were cloned and suggested to be involved in the Fus3/Kss1 pathway by a phylogenetic analysis with the neighbor-joining method. Quantitative RT-PCR (qRT-PCR) analyses indicated that expression of UeKpp2 and UeKpp6 were induced during mating and infection processes, and their expression patterns displayed differentially under different carbon and nitrogen sources. In addition, subcellular localization of UeKpp2 or UeKpp6 fused with the reporter green fluoresce protein was observed by confocal laser scanning microscope, and yeast two-hybrid assays were carried out. Results showed that both UeKpp2 and UeKpp6 were located in cytoplasm and interacted with UePrf1, indicating their involvement in hyphal growth and host-pathogen regulation. Only UeKpp2 but not UeKpp6 interacted with the upstream MAPK kinase UeFuz7, implying an additional MAPK pathway, in which UeKpp6 involved, existed. [ABSTRACT FROM AUTHOR]

Leaf-stripe smuts on grasses are a highly polyphyletic group within Ustilaginomycotina, occurring in three genera, Tilletia, Urocystis, and Ustilago. Currently more than 12 Ustilago species inciting stripe smuts are recognised. The majority belong to the Ustilago striiformis-complex, with about 30 different taxa described from 165 different plant species. This study aims to assess whether host distinct-lineages can be observed amongst the Ustilago leaf-stripe smuts using nine different loci on a representative set. Phylogenetic reconstructions supported the monophyly of the Ustilago striiformiscomplex that causes leaf-stripe and the polyphyly of other leaf-stripe smuts within Ustilago. Furthermore, smut specimens from the same host genus generally clustered together in well-supported clades that often had available species names for these lineages. In addition to already-named lineages, three new lineages were observed, and described as new species on the basis of host specificity and molecular differences: namely Ustilago jagei sp. nov. on Agrostis stolonifera, U. kummeri sp. nov. on Bromus inermis, and U. neocopinata sp. nov. on Dactylis glomerata. [ABSTRACT FROM AUTHOR]

The plant cell wall is known to be the first barrier against plant pathogens. Detailed information about sugarcane cell wall-associated defense responses to infection by the causal agent of smut, Sporisorium scitamineum, is scarce. Herein, (immuno)histochemical analysis of two smut resistant and two susceptible sugarcane cultivars was conducted to understand host cell wall structural and compositional modifications in response to fungal infection. Results showed that the fungus grew on the surface and infected the outermost bud scale of both susceptible and resistant cultivars. The present findings also supported the existence of early (24 h after inoculation) and later (72-96 h after inoculation) inducible histopathological responses related to the cell wall modification in resistant cultivars. Lignin and phenolic compounds accumulated during early stages of infection. Later infection response was characterized by the formation of a protective barrier layer with lignin, cellulose and arabinoxylan in the cell walls. Overall, the results suggest possible induction of cell wall-modified responses in smut resistant cultivars to prevent initial entry of the fungus into the meristematic tissues. [ABSTRACT FROM AUTHOR]

Host specialization is a key evolutionary process for the diversification and emergence of new pathogens. However, the molecular determinants of host range are poorly understood. Smut fungi are biotrophic pathogens that have distinct and narrow host ranges based on largely unknown genetic determinants. Hence, we aimed to expand comparative genomics analyses of smut fungi by including more species infecting different hosts and to define orphans and positively selected genes to gain further insights into the genetics basis of host specialization. We analyzed nine lineages of smut fungi isolated from eight crop and non-crop hosts: maize, barley, sugarcane, wheat, oats, Zizania latifolia (Manchurian rice), Echinochloa colona (a wild grass), and Persicaria sp. (a wild dicot plant). We assembled two new genomes: Ustilago hordei (strain Uhor01) isolated from oats and U. tritici (strain CBS 119.19) isolated from wheat. The smut genomes were of small sizes, ranging from 18.38 to 24.63 Mb. U. hordei species experienced genome expansions due to the proliferation of transposable elements and the amount of these elements varied among the two strains. Phylogenetic analysis confirmed that Ustilago is not a monophyletic genus and, furthermore, detected misclassification of the U. tritici specimen. The comparison between smut pathogens of crop and non-crop hosts did not reveal distinct signatures, suggesting that host domestication did not play a dominant role in shaping the evolution of smuts. We found that host specialization in smut fungi likely has a complex genetic basis: different functional categories were enriched in orphans and lineage-specific selected genes. The diversification and gain/loss of effector genes are probably the most important determinants of host specificity. [ABSTRACT FROM AUTHOR]

The sugar alcohol d-arabitol is one of the Department of Energy's top twelve bio-based building block chemicals. In this study, we found that the oleaginous yeast Rhodosporidium toruloides IFO0880 produces d-arabitol during growth on xylose in nitrogen-rich medium. Efficient xylose utilization was a prerequisite for extracellular d-arabitol production. During growth in complex media, R. toruloides produced 22 ± 2, 32 ± 2, and 49 ± 2 g/L d-arabitol from 70, 105, and 150 g/L xylose, respectively. In addition, we found that R. toruloides could potentially be used for the co-production of lipids and d-arabitol from xylose. These results demonstrate that R. toruloides can be used to produce multiple value-added chemicals from xylose. [ABSTRACT FROM AUTHOR]

The use of yeasts in bioprocesses can be considered one of the most relevant strategies in industrial biotechnology, and their potential is recognized due to the ability of these microorganisms for production of diverse value-added compounds. Yeasts from Ustilaginaceae family have been highlighted in the last years as a promising source of industrial interesting compounds, including enzymes, sugars, lipids, organic acids, and biosurfactants. These compounds may exhibit various applications in pharmaceutical, cosmetic, food, medical, and environmental fields, increasing the scientific attention in the study of ustilaginomycetous for biotechnological purposes. In this mini-review, we provide a comprehensive overview about the biotechnological use of yeasts from Ustilaginaceae family to produce value-added compounds, focusing in recent trends, characteristics of processes currently developed, new opportunities, and potential applications. [ABSTRACT FROM AUTHOR]

The Ustilaginomycotina, often collectively referred to as smut fungi, represent one of the three subphyla of the Basidiomycota. Smut fungi predominantly parasitize Angiosperms, are globally distributed, and contain several economically important pathogens. The most species-rich family of the smut fungi is the Ustilaginaceae. To investigate the molecular phylogeny of smut fungi, most studies rely on nrDNA loci, such as ITS and LSU. Protein coding genes, like rpb1, rpb2, TeF1 a, atp6, and ß-tubulin, have been used in some studies. However, because of the huge diversity of smut fungi and the lack of dedicated primers, amplification of these loci has proven difficult for several groups. Thus, it was the aim of the current study to develop primers for new loci for the smut fungi with the focus on the largest family, the Ustilaginaceae. Here, the development and testing of new primers for nine loci based on protein-coding genes is reported ( myosin, map, rpl3, tif2, ssc1, ß-tubulin, sdh1, rpl4A and atp2). A list of various primer combinations for the amplification of the new loci is given, with the corresponding PCR conditions and the best combinations for several genera of the Ustilaginaceae and some other Ustilaginomycetes. We hope that the primers presented in this study will be useful in overcoming the limitations of currently-used loci in terms of phylogenetic resolution, especially with respect to resolving species complexes and providing a better resolution of the higher-level phylogenetic relationships of smut fungi. [ABSTRACT FROM AUTHOR]

Yeasts of the now unused asexually typified genus Pseudozyma belong to the smut fungi (Ustilaginales) and are mostly believed to be apathogenic asexual yeasts derived from smut fungi that have lost pathogenicity on plants. However, phylogenetic studies have shown that most Pseudozyma species are phylogenetically close to smut fungi parasitic to plants, suggesting that some of the species might represent adventitious isolations of the yeast morph of otherwise plant pathogenic smut fungi. However, there are some species, such as Moesziomyces aphidis (syn. Pseudozyma aphidis) that are isolated throughout the world and sometimes are also found in clinical samples and do not have a known plant pathogenic sexual morph. In this study, it is revealed by phylogenetic investigations that isolates of the biocontrol agent Moesziomyces aphidis are interspersed with M. bullatus sexual lineages, suggesting conspecificity. This raises doubts regarding the apathogenic nature of asexual morphs previously placed in Pseudozyma, but suggests that there might also be pathogenic sexual morph counterparts for those species known only from asexual morphs. The finding that several additional species currently only known from their yeast morphs are embedded within the genus Moesziomyces, suggests that the yeast morph might play a more dominant role in this genus as compared to other genera of Ustilaginaceae. In addition, phylogenetic reconstructions demonstrated that Moesziomyces bullatus has a narrow host range and that some previously described but not widely used species names should be applied for Moesziomyces on other host genera than Echinochloa. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Macalpinomyceswas established in 1977, with the type speciesM. eriachnesdescribed from a specimen collected in northern Australia on the grassEriachnesp. in 1855. Subsequently,M. eriachneshas been reported on more than 21 species ofEriachnein northern Australia. In this study, a polyphasic approach was employed to determine whetherM. eriachnesmasked cryptic diversity. On the basis of morphology, multilocus phylogeny, and coalescent methods of generalized mixed Yule-coalescent (GMYC) and Poisson tree processes (PTP) models, 26 specimens ofMacalpinomyceson 13 species ofEriachneheld in Australian herbaria were studied. Consequently, 10 new species ofMacalpinomycesthat satisfied the phylogenetic species recognition criteria are described. [ABSTRACT FROM PUBLISHER]

Ustilago is a polyphyletic genus of smut fungi found mainly on Poaceae. The development of a taxonomy that reflects phylogeny requires subdivision of Ustilago into smaller monophyletic genera. Several separate systematic analyses have determined that Macalpinomyces mackinlayi, M. tubiformis, Tolyposporella pachycarpa, Ustilago bouriquetii and U. maydis, occupy a unique phylogenetic position within the Ustilaginaceae. A previously introduced monotypic generic name typified by U. maydis, Mycosarcoma, is available to accommodate these species, which resolves one component of polyphyly for Ustilago s. lat. in Ustilaginaceae. An emended description of Mycosarcoma is provided to reflect the morphological synapomorphies of this monophyletic group. A specimen of Ustilago maydis that has had its genome sequenced is designated as a neotype for this species. Taxonomic stability will further be provided by a forthcoming proposal to conserve the name Uredo maydis over Lycoperdon zeae, which has priority by date, in order to preserve the well-known epithet maydis. [ABSTRACT FROM AUTHOR]

Villosiclava virens (Vv) is an ascomycete fungal pathogen that causes false smut disease in rice. Recent reports have revealed some interesting aspects of the enigmatic pathogen to address the question of why it specifically infects rice flowers and converts a grain into a false smut ball. Comparative and functional genomics have suggested specific adaptation of Vv in the colonization of rice flowers. Anatomical studies have disclosed that Vv specifically infects rice stamen filaments before heading and intercepts seed formation. In addition, Vv can occupy the whole inner space of a spikelet embracing all floral organs and activate the rice grain-filling network, presumably for nutrient acquisition to support the development of the false smut ball. This profile provides a general overview of the rice false smut pathogen, and summarizes advances in the Vv life cycle, genomics and genetics, and the molecular Vv-rice interaction. Current understandings of the Vv-rice pathosystem indicate that it is a unique and interesting system which can enrich the study of plant-pathogen interactions. Taxonomy: Ustilaginoidea virens is the anamorph form of the pathogen (Kingdom Fungi; Phylum Ascomycota; Class Ascomycetes; Subclass Incertae sedis; Order Incertae sedis; Family Incertae sedis; Genus Ustilaginoidea). The teleomorph form is Villosiclava virens (Kingdom Fungi; Phylum Ascomycota; Class Ascomycetes; Subclass Sordariomycetes; Order Hypocreales; Family Clavicipitaceae; Genus Villosiclava). Disease symptoms: The only visible symptom is the replacement of rice grains by ball-shaped fungal mycelia, namely false smut balls. When maturing, the false smut ball is covered with powdery chlamydospores, and the colour changes to yellowish, yellowish orange, green, olive green and, finally, to greenish black. Sclerotia are often formed on the false smut balls in autumn. Identification and detection: Vv conidia are round to elliptical, measuring 3-5 μm in diameter. Chlamydospores are ornamented with prominent irregularly curved spines, which are 200-500 nm in length. The sclerotia are black, horseshoe-shaped and irregular oblong or flat, ranging from 2 to 20 mm. Nested polymerase chain reaction (PCR) and quantitative PCR have been developed to specifically detect Vv presence in rice tissues and other biotic and abiotic samples in fields. Host range: Rice is the primary host for Vv. Natural infection by Vv has been found on several paddy field weeds, including Digitaria marginata, Panicum trypheron, Echinochloa crusgalli and Imperata cylindrica. However, the occurrence of infection in these potential alternative hosts is very rare. Life cycle: Vv infects rice spikelets at the late rice booting stage, and produces false smut balls covered with dark-green chlamydospores. Occasionally, sclerotia form on the surface of false smut balls in late autumn when the temperature fluctuates greatly between day and night. Both chlamydospores and sclerotia may serve as primary infection sources. Rainfall at the rice booting stage is a major environmental factor resulting in epidemics of rice false smut disease. Disease control: The use of fungicides is the major approach for the control of Vv. Several fungicides, such as cuproxat SC, copper oxychloride, tebuconazole, propiconazole, difenoconazole and validamycin, are often applied. However, the employment of resistant rice cultivars and genes has been limited, because of the poor understanding of rice resistance to Vv. Useful websites: Villosiclava virens genome sequence: [ABSTRACT FROM AUTHOR]

Crude glycerol (byproduct of biodiesel preparation) was utilised as the carbon source to produce lipid using oleaginous yeast Rhodosporidium toruloides 32489. Under the same conditions, lipid production with crude glycerol was higher than those produced with glucose and pure glycerol. The effects of 4 main impurities in crude glycerol (methyl oleate, sodium oleate, NaCl and methanol) on lipid production were investigated. Compared with utilising pure glycerol, addition of methyl oleate, sodium oleate, and NaCl impurities increased lipid production by 47.0%, 68.0% and 64.0%, respectively, while methanol decreased lipid production by 17.7%. However, when methanol was mixed with other impurities, its inhibition effect was alleviated due to the promoting effect of other impurities. Hence, crude glycerol could be used as a renewable and low-cost carbon source to replace pure glucose or glycerol for lipid preparation. [ABSTRACT FROM AUTHOR]

This study investigated the potential use of yeast Rhodosporidium toruloides for lipid production and removal of organics and nutrients while treating a mixture of distillery and domestic wastewater at low cost without sterilization. The highest specific biomass (24.1 × 10 −9 g L −1 cell −1 h −1 ), lipid yield (9.9 × 10 −9 g L −1 cell −1 h −1 ), and lipid content (8.54 × 10 −3 g lipid g −1 biomass h −1 ) were achieved on the 2nd day of cultivation. The organics and nutrients removal also reached the highest removal rates within 2 days, with the specific removal rates for COD (chemical oxygen demand), TN (total nitrogen), and TP (total phosphorus) at 39.5, 2.57, and 0.29 × 10 −9 mg cell −1 h −1 , respectively. The indigenous microorganisms slightly contributed to both lipid production and removal of organics and nutrients. The direct transesterification process via microwave irradiation from wet biomass was further optimized under the conditions of solvent to wet biomass rate 16 mL/g, methanol and chloroform mix rate 1:1, addition of catalyst H 2 SO 4 6%, and reaction time 10 min at the reaction temperature of 60 °C. The FAMEs (fatty acid methyl esters) composition and the possibility of biodiesel production from wet oleaginous yeast biomass by the direct transesterification were also assessed. [ABSTRACT FROM AUTHOR]

Background: In order to establish a cost-efficient biodiesel biorefinery, valorization of its main by-product, crude glycerol, is imperative. Recently, Ustilago trichophora TZ1 was found to efficiently produce malic acid from glycerol. By adaptive laboratory evolution and medium optimization, titer and rate could be improved significantly. Results: Here we report on the investigation of this strain in fed-batch bioreactors. With pH controlled at 6.5 (automatic NaOH addition), a titer of 142 ± 1 g L-1 produced at an overall rate of 0.54 ± 0.00 g L-1 h-1 was reached by optimizing the initial concentrations of ammonium and glycerol. Combining the potential of bioreactors and CaCO3 as buffer system, we were able to increase the overall production rate to 0.74 ± 0.06 g L-1 h-1 with a maximum production rate of 1.94 ± 0.32 g L-1 reaching a titer of 195 ± 15 g L-1. The initial purification strategy resulted in 90 % pure calcium malate as solid component. Notably, the fermentation is not influenced by an increased temperature of up to 37 °C, which reduces the energy required for cooling. However, direct acid production is not favored as at a lowered pH value of pH 4.5 the malic acid titer decreased to only 9 ± 1 g L-1. When using crude glycerol as substrate, only the product to substrate yield is decreased. The results are discussed in the context of valorizing glycerol with Ustilaginaceae. Conclusions: Combining these results reveals the potential of U. trichophora TZ1 to become an industrially applicable production host for malic acid from biodiesel-derived glycerol, thus making the overall biodiesel production process economically and ecologically more feasible. [ABSTRACT FROM AUTHOR]

A chemoenzymatic strategy was developed for ( S )-duloxetine production employing carbonyl reductases from newly isolated Rhodosporidium toruloides into the enantiodetermining step. Amongst the ten most permissive enzymes identified, cloned, and overexpressed in Escherichia coli , Rt SCR9 exhibited excellent activity and enantioselectivity. Using co-expressed E. coli harboring both Rt SCR9 and glucose dehydrogenase, ( S )-3-(dimethylamino)-1-(2-thienyl)-1-propanol 3a was fabricated with so far the highest substrate loading (1000 mM) in a space-time yield per gram of biomass (DCW) of 22.9 mmol L −1 h −1 g DCW −1 at a 200-g scale. The subsequent synthetic steps from Rt SCR9-catalyzed ( S )- 3a were further performed, affording ( S )-duloxetine with 60.2% overall yield from 2-acethylthiophene in >98.5% ee . [ABSTRACT FROM AUTHOR]

Background: The large surplus of crude glycerol, as main low-value waste stream in biodiesel production, has led to the investigation of new possibilities for the production of value-added chemicals from this feedstock. New and efficient (bio-) catalysts are needed that are able to convert glycerol to versatile chemical building blocks. This would contribute to further develop away from a mainly petroleum based, to a sustainable, bio-based industry. One promising group of discussed building block chemicals are dicarbonic acids. Results: Here, we report the efficient synthesis of malate from glycerol using Ustilago trichophora RK089, which was identified in a screening of 74 Ustilaginaceae. For economically feasible production that can compete with existing processes, a high productivity is required. By adaptive laboratory evolution, the growth and production rate were increased by 2.5- and 6.6-fold, respectively. Further medium optimization increased the final titer, yield, and overall production rate to 196 g L-1, 0.82 gmal g-1 gly, and 0.39 g L-1 h-1, respectively. Conclusions: This titer is the highest reported for microbial malate production, making U. trichophora TZ1 a promising microbial production host for malate from crude glycerol, especially since it is not genetically engineered. Since this production process starts from an industrial waste stream as substrate and yields an interesting platform chemical, which can be used to replace petro-chemicals, it greatly contributes to a sustainable bio-economy. [ABSTRACT FROM AUTHOR]

The optimal medium pH to produce biomass and fatty acids by the red yeast Rhodosporidium toruloides NCYC 921 is 4.0, and to produce carotenoids is 5.0. Based on this difference, a dual-stage pH control fed-batch cultivation strategy for the enhancement of lipids and carotenoids production by this yeast was studied. The results showed that when the yeast growth phase was conducted at pH 4.0, and the products accumulation phase was conducted at pH 5.0, biomass, total fatty acid and total carotenoid productivities were significantly improved comparing with the yeast fed batch cultivations carried out at fixed medium pH (4 or 5). Under dual-stage pH control conditions, the biomass, carotenoids and lipids productivities attained 2.35 g/L h, 0.29 g/L h and 0.40 g/L h, respectively. It was also observed that the oxygen played a major role in the yeast carotenoid production. [ABSTRACT FROM AUTHOR]

The smut Ustilago maydis, a ubiquitous pest of corn, is highly adapted to its host to parasitize on its organic carbon sources. We have identified a hexose transporter, Hxt1, as important for fungal development during both the saprophytic and the pathogenic stage of the fungus., Hxt1 was characterized as a high-affinity transporter for glucose, fructose, and mannose; ∆hxt1 strains show significantly reduced growth on these substrates, setting Hxt1 as the main hexose transporter during saprophytic growth., After plant infection, ∆hxt1 strains show decreased symptom development. However, expression of a Hxt1 protein with a mutation leading to constitutively active signaling in the yeast glucose sensors Snf3p and Rgt2p results in completely apathogenic strains. Fungal development is stalled immediately after plant penetration, implying a dual function of Hxt1 as transporter and sensor., As glucose sensors are only known for yeasts, 'transceptor' as Hxt1 may constitute a general mechanism for sensing of glucose in fungi. In U. maydis, Hxt1 links a nutrient-dependent environmental signal to the developmental program during pathogenic development. [ABSTRACT FROM AUTHOR]

Microbial oil is drawing increasing interest all over the world as an alternative non-food feedstock for biodiesel production. Rhodosporidium toruloides has been considered as a promising candidate to convert glycerol into microbial oils due to its good substrate adaptability, high intracellular lipid content, and the potential of co-production of some pigments. Interestingly, we have also found that sodium lignosulphonate (SL) could significantly enhance the cell growth and lipid accumulation of R. toruloides . In the present work, we investigated the possible mechanisms for the enhancement of lipid production by SL. It was found that SL could not be utilized by R. toruloides as a carbon source. No apparent changes on the morphology of cell membranes and the fatty acid profiles of microbial lipids were found when SL was added. Further experiments indicated that the volumetric oxygen transfer coefficient was increased by nearly 15% on average by adding 4 g/L SL, which might be the main mechanism for the improvement of lipid production. [ABSTRACT FROM AUTHOR]

Ustilago maydis is a fungus infecting aerial parts of maize to form smutted galls. Due to its interest as a genetic tool in plant pathology, we evaluated its ability to penetrate into plant roots. The fungus can penetrate between epidermic root cells, forming inter and intracellular pseudohyphae. Root infection didn't provoke gall formation on the maize lines tested, and targeted PCR detection showed that U. maydis , unlike the other maize smut fungus Sporisorium reilianum , has a weak aptitude to grow from the roots up to the aerial part of maize. We also observed that U. maydis can infect Medicago truncatula hairy roots as an alternative host. This plant species is a model host to study root symbiosis, and this pathosystem can provide new insights on root–microbe interactions. Considering that U. maydis could be a soil fungus, we tested its responsiveness to GR24, a strigolactone analogue. Strigolactones are root exuded molecules which activate mitochondrial metabolism of arbuscular mycorrhizal (AM) fungi. Physiologic and molecular analysis revealed that GR24 also increases cell respiration of U. maydis . This result points out that strigolactones could have an incidence on several rhizospheric microbes. These data provide evidences that the biotrophic pathogen U. maydis has to be considered for studying root infection. [ABSTRACT FROM AUTHOR]