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ABSTRACT For over a century, the filamentous Ascomycete fungus Aspergillus niger has played a pivotal role in the industrial production of citric acid. A critical fermentation parameter that sustains high-yield citric acid accumulation is the suboptimal concentration of manganese(II) ions in the culture broth at the early stages of the process. However, the requirement for this deficiency has not been investigated on a functional genomics level. In this study, we compared the transcriptome of the citric acid hyper-producer A. niger NRRL2270 strain grown under citric acid-producing conditions in 6-L scale bioreactors at Mn2+ ion-deficient (5 ppb) and Mn2+ ion-sufficient (100 ppb) conditions at three early time points of cultivation. Of the 11,846 genes in the genome, 963 genes (8.1% of the total) were identified as significantly differentially expressed under these conditions. Disproportionately high number of differentially regulated genes encode predicted extracellular and membrane proteins. The most abundant gene group that was upregulated in Mn2+ ion deficiency condition encodes enzymes acting on polysaccharides. In contrast, six clusters of genes encoding secondary metabolites showed downregulation under manganese deficiency. Mn2+ deficiency also triggers upregulation of the cexA gene, which encodes the citrate exporter. We provide functional evidence that the upregulation of cexA is caused by the intracellular accumulation of citrate or acetyl-CoA and is a major factor in triggering citrate overflow.IMPORTANCECitric acid is produced on industrial scale by batch fermentation of the filamentous fungus Aspergillus niger. High-yield citric acid production requires a low (

Aspergillus niger is an important filamentous fungus used for the industrial production of citric acid. One of the most important factors that affect citric acid production is the concentration of manganese(II) ions present in the culture broth. Under manganese(II)-limiting conditions, the fungus develops a pellet-like morphology that is crucial for high citric acid accumulation. The impact of manganese(II) ions on the transcription of the major citrate exporter encoding gene cexA was studied under manganese(II)-deficient and -sufficient conditions. Furthermore, citric acid production was analyzed in overexpression mutant strains of cexA in the presence and absence of manganese(II) ions, and the influence of CexA on fungal morphology was investigated by microscopy. Transcriptional upregulation of cexA in the absence of manganese(II) ions was observed and, by decoupling cexA expression from the native promoter system, it was possible to secrete more citric acid even in the presence of manganese. This effect was shown for both an inducible and a constitutive overexpression of cexA. Furthermore, it was found that the presence of CexA influences fungal morphology and promotes a more branched phenotype. According to this study, manganese(II) ions suppress transcription of the citrate exporter cexA in Aspergillus niger, causing citric acid secretion to decrease.

Alternative oxidase (Aox) is a terminal oxidase operating in branched electron transport. The activity correlates positively with overflow metabolisms in certain Aspergilli, converting intracellular glucose by the shortest possible path into organic acids, like citrate or itaconate. Aox is nearly ubiquitous in fungi, but aox gene multiplicity is rare. Nevertheless, within the family of the Aspergillaceae and among its various species of industrial relevance—Aspergillus niger, A. oryzae, A. terreus, Penicillium rubens—paralogous aox genes coexist. Paralogous genes generally arise from duplication and are inherited vertically. Here, we provide evidence of four independent duplication events along the lineage that resulted in aox paralogues (aoxB) in contemporary Aspergillus and Penicillium taxa. In some species, three aox genes are co-expressed. The origin of the A. niger paralogue is different than that of the A. terreus paralogue, but all paralogous clades ultimately arise from ubiquitous aoxA parent genes. We found different patterns of uncorrelated gene losses reflected in the Aspergillus pedigree, albeit the original aoxA orthologues persist everywhere and are never replaced. The loss of acquired paralogues co-determines the contemporary aox gene content of individual species. In Aspergillus calidoustus, the two more ancient paralogues have, in effect, been replaced by two aoxB genes of distinct origins.

Summary Filamentous fungi produce a wide variety of enzymes in order to efficiently degrade plant cell wall polysaccharides. The production of these enzymes is controlled by transcriptional regulators, which also control the catabolic pathways that convert the released monosaccharides. Two transcriptional regulators, GalX and GalR, control d‐galactose utilization in the model filamentous fungus Aspergillus nidulans, while the arabinanolytic regulator AraR regulates l‐arabinose catabolism. d‐Galactose and l‐arabinose are commonly found together in polysaccharides, such as arabinogalactan, xylan and rhamnogalacturonan I. Therefore, the catabolic pathways that convert d‐galactose and l‐arabinose are often also likely to be active simultaneously. In this study, we investigated the interaction between GalX, GalR and AraR in d‐galactose and l‐arabinose catabolism. For this, we generated single, double and triple mutants of the three regulators, and analysed their growth and enzyme and gene expression profiles. Our results clearly demonstrated that GalX, GalR and AraR co‐regulate d‐galactose catabolism in A. nidulans. GalX has a prominent role on the regulation of genes of d‐galactose oxido‐reductive pathway, while AraR can compensate for the absence of GalR and/or GalX.

Grape production worldwide is increasingly threatened by grapevine trunk diseases (GTDs). No grapevine cultivar is known to be entirely resistant to GTDs, but susceptibility varies greatly. To quantify these differences, four Hungarian grape germplasm collections containing 305 different cultivars were surveyed to determine the ratios of GTDs based on symptom expression and the proportion of plant loss within all GTD symptoms. The cultivars of monophyletic Vitis vinifera L. origin were amongst the most sensitive ones, and their sensitivity was significantly (p < 0.01) higher than that of the interspecific (hybrid) cultivars assessed, which are defined by the presence of Vitis species other than V. vinifera (e.g., V. labrusca L., V. rupestris Scheele, and V. amurensis Rupr.) in their pedigree. We conclude that the ancestral diversity of grapes confers a higher degree of resilience against GTDs.

High-yield citric acid production by the filamentous Ascomycete fungus Aspergillus niger requires a combination of extreme nutritional conditions, of which maintaining a low manganese (II) ion concentration (

Alternative oxidase is a terminal oxidase in the branched mitochondrial electron transport chain of most fungi including Aspergillus niger (subgenus Circumdati, section Nigri). A second, paralogous aox gene (aoxB) is extant in some A. niger isolates but also present in two divergent species of the subgenus Nidulantes—A. calidoustus and A. implicatus—as well as in Penicillium swiecickii. Black aspergilli are cosmopolitan opportunistic fungi that can cause diverse mycoses and acute aspergillosis in immunocompromised individuals. Amongst the approximately 75 genome-sequenced A. niger strains, aoxB features considerable sequence variation. Five mutations were identified that rationally affect transcription or function or terminally modify the gene product. One mutant allele that occurs in CBS 513.88 and A. niger neotype strain CBS 554.65 involves a chromosomal deletion that removes exon 1 and intron 1 from aoxB. Another aoxB allele results from retrotransposon integration. Three other alleles result from point mutations: a missense mutation of the start codon, a frameshift, and a nonsense mutation. A. niger strain ATCC 1015 has a full-length aoxB gene. The A. niger sensu stricto complex can thus be subdivided into six taxa according to extant aoxB allele, which may facilitate rapid and accurate identification of individual species

Abstract Background Citric acid, a commodity product of industrial biotechnology, is produced by fermentation of the filamentous fungus Aspergillus niger. A requirement for high-yield citric acid production is keeping the concentration of Mn2+ ions in the medium at or below 5 µg L−1. Understanding manganese metabolism in A. niger is therefore of critical importance to citric acid production. To this end, we investigated transport of Mn2+ ions in A. niger NRRL2270. Results we identified an A. niger gene (dmtA; NRRL3_07789), predicted to encode a transmembrane protein, with high sequence identity to the yeast manganese transporters Smf1p and Smf2p. Deletion of dmtA in A. niger eliminated the intake of Mn2+ at low (5 µg L−1) external Mn2+ concentration, and reduced the intake of Mn2+ at high (> 100 µg L−1) external Mn2+ concentration. Compared to the parent strain, overexpression of dmtA increased Mn2+ intake at both low and high external Mn2+ concentrations. Cultivation of the parent strain under Mn2+ ions limitation conditions (5 µg L−1) reduced germination and led to the formation of stubby, swollen hyphae that formed compact pellets. Deletion of dmtA caused defects in germination and hyphal morphology even in the presence of 100 µg L−1 Mn2+, while overexpression of dmtA led to enhanced germination and normal hyphal morphology at limiting Mn2+ concentration. Growth of both the parent and the deletion strains under citric acid producing conditions resulted in molar yields (Yp/s) of citric acid of > 0.8, although the deletion strain produced ~ 30% less biomass. This yield was reduced only by 20% in the presence of 100 µg L−1 Mn2+, whereas production by the parent strain was reduced by 60%. The Yp/s of the overexpressing strain was 17% of that of the parent strain, irrespective of the concentrations of external Mn2+. Conclusions Our results demonstrate that dmtA is physiologically important in the transport of Mn2+ ions in A. niger, and manipulation of its expression modulates citric acid overflow.

Walnut (Juglans regia L.) production is a developing sector in Hungarian horticulture, where preharvest fruit rot increasingly causes economic losses. Previously, fungi from the Diaporthaceae and Botryosphaeriaceae families were isolated with a high incidence from rotten fruits. Here, we identify these pathogens from different plant parts (twigs, buds, and shoots) in orchards in the north-east of Hungary, and test their pathogenicity on immature nuts. Diaporthe eres Nitschke and Diplodia seriata De Notaris were identified in isolates from different symptomatic and asymptomatic plant parts based on their ITS (nuclear ribosomal internal transcribed spacer) and tef1 (translation elongation factor EF-1-alpha gene) DNA sequences. Purified monocultures caused rot of immature nuts following in vitro inoculation. Our results suggest that D. eres Nitschke and D. seriata De Notaris lingering on buds and overwintering woody parts may affect the seasonal nuts through wound infection. Infection by Diaporthe and Botryosphaeriaceae species present on woody plant parts, as well as on/in buds has been reported in Mediterranean countries. This is the first report of such stepwise aetiology from a region with continental weather. Climate change, associated weather patterns and the limitations of fungicide use in the European Union, among other factors, could be responsible for the increasing number of infections and economic damage caused by these pathogens.

In the first part of this two-piece publication, the isolation, identification and in vitro characterization of ten endophytic Trichoderma isolates were reported. Here we report the ability of two different mixes of some of these isolates (Trichoderma simmonsii, Trichoderma orientale and Trichoderma gamsii as well as of Trichoderma afroharzianum and T. simmonsii) to colonize and stimulate the growth of grapevines. Two commercial vineyards about 400 km away from the site of isolation were used as experimental fields, from which the strains of three Trichoderma species were re-isolated up to four years after rootstock soaking treatment with conidiospores, performed before planting. The treatments decreased the overall percentage of lost plants of about 30%, although a low number of lost plants (about 5%) were observed also in the control plot. For all cultivars and clones, the Trichoderma treatments significantly increased both the bud burst ratio and bud burst vigor index. In addition, the grape must parameters such as the Brix degrees, as well as the extract, the D-glucose and the D-fructose concentrations all appeared to be improved, suggesting a potentially higher ethanol content of the produced wine. We conclude that grapevine-endophytic Trichoderma isolates promote plant growth, which could be a useful feature for sustainable agriculture in general and integrated plant production in particular.

The effects of the interplay of copper(II) and manganese(II) ions on growth, morphology and itaconic acid formation was investigated in a high-producing strain of Aspergillus terreus (NRRL1960), using carbon sources metabolized either mainly via glycolysis (D-glucose, D-fructose) or primarily via the pentose phosphate shunt (D-xylose, L-arabinose). Limiting Mn2+ concentration in the culture broth is indispensable to obtain high itaconic acid yields, while in the presence of higher Mn2+ concentrations yield decreases and biomass formation is favored. However, this low yield in the presence of high Mn2+ ion concentrations can be mitigated by increasing the Cu2+ concentration in the medium when D-glucose or D-fructose is the growth substrate, whereas this effect was at best modest during growth on D-xylose or L-arabinose. A. terreus displays a high tolerance to Cu2+ which decreased when Mn2+ availability became increasingly limiting. Under such conditions biomass formation on D-glucose or D-fructose could be sustained at concentrations up to 250 mg L–1 Cu2+, while on D-xylose- or L-arabinose biomass formation was completely inhibited at 100 mg L–1. High (>75%) specific molar itaconic acid yields always coincided with an “overflow-associated” morphology, characterized by small compact pellets (

Introns are usually non-coding sequences interrupting open reading frames in pre-mRNAs [D1,2]. Stwintrons are nested spliceosomal introns, where an internal intron splits a second donor sequence into two consecutive splicing reactions leading to mature mRNA. In Hypoxylon sp. CO27-5, 36 highly sequence-similar [D1,2] stwintrons are extant (sister stwintrons). An additional 81 [D1,2] sequence-unrelated stwintrons are described here. Most of them are located at conserved gene positions rooted deep in the Hypoxylaceae. Absence of exonic sequence bias at the exon–stwintron junctions and a very similar phase distribution were noted for both groups. The presence of an underlying sequence symmetry in all 117 stwintrons was striking. This symmetry, more pronounced near the termini of most of the full-length sister stwintrons, may lead to a secondary structure that brings into close proximity the most distal splice sites, the donor of the internal and the acceptor of the external intron. The Hypoxylon stwintrons were overwhelmingly excised by consecutive splicing reactions precisely removing the whole intervening sequence, whereas one excision involving the distal splice sites led to a frameshift. Alternative (mis)splicing took place for both sister and uniquely occurring stwintrons. The extraordinary symmetry of the sister stwintrons thus seems dispensable for the infrequent, direct utilisation of the distal splice sites.

This paper reports on the identification and in vitro characterization of several Trichoderma strains isolated from the Tokaj Wine Region in North-East Hungary. Ten isolates were analyzed and found to consist of six individual species—T. gamsii, T. orientale, T. simmonsii, T. afroharzianum, T. atrobrunneum and T. harzianum sensu stricto. The growth potential of the strains was assessed at a range of temperatures. We also report here on the in vitro biocontrol properties and fungicide tolerance of the most promising strains.

Spliceosomal introns are pervasive in eukaryotes. Intron gains and losses have occurred throughout evolution, but the origin of new introns is unclear. Stwintrons are complex intervening sequences where one of the sequence elements (5′-donor, lariat branch point element or 3′-acceptor) necessary for excision of a U2 intron (external intron) is itself interrupted by a second (internal) U2 intron. In Hypoxylaceae, a family of endophytic fungi, we uncovered scores of donor-disrupted stwintrons with striking sequence similarity among themselves and also with canonical introns. Intron–exon structure comparisons suggest that these stwintrons have proliferated within diverging taxa but also give rise to proliferating canonical introns in some genomes. The proliferated (stw)introns have integrated seamlessly at novel gene positions. The recently proliferated (stw)introns appear to originate from a conserved ancestral stwintron characterised by terminal inverted repeats (45–55 nucleotides), a highly symmetrical structure that may allow the formation of a double-stranded intron RNA molecule. No short tandem duplications flank the putatively inserted intervening sequences, which excludes a DNA transposition-based mechanism of proliferation. It is tempting to suggest that this highly symmetrical structure may have a role in intron proliferation by (an)other mechanism(s).

Organic acid accumulation is probably the best-known example of primary metabolic overflow. Both bacteria and fungi are capable of producing various organic acids in large amounts under certain conditions, but in terms of productivity-and consequently, of commercial importance-fungal platforms are unparalleled. For high product yield, chemical composition of the growth medium is crucial in providing the necessary conditions, of which the concentrations of four of the first-row transition metal elements, manganese (Mn2+), iron (Fe2+), copper (Cu2+) and zinc (Zn2+) stand out. In this paper we critically review the biological roles of these ions, the possible biochemical and physiological consequences of their influence on the accumulation of the most important mono-, di- and tricarboxylic as well as sugar acids by fungi, and the metal ion-related aspects of submerged organic acid fermentations, including the necessary instrumental analytics. Since producing conditions are associated with a cell physiology that differs strongly to what is observed under “standard” growth conditions, here we consider papers and patents only in which organic acid accumulation levels achieved at least 60% of the theoretical maximum yield, and the actual trace metal ion concentrations were verified.

Abstract Background In the primary transcript of nuclear genes, coding sequences—exons—usually alternate with non-coding sequences—introns. In the evolution of spliceosomal intron–exon structure, extant intron positions can be abandoned and new intron positions can be occupied. Spliceosomal twin introns (“stwintrons”) are unconventional intervening sequences where a standard “internal” intron interrupts a canonical splicing motif of a second, “external” intron. The availability of genome sequences of more than a thousand species of fungi provides a unique opportunity to study spliceosomal intron evolution throughout a whole kingdom by means of molecular phylogenetics. Results A new stwintron was encountered in Aspergillus nidulans and Aspergillus niger. It is present across three classes of Leotiomyceta in the transcript of a well-conserved gene encoding a putative lipase (lipS). It occupies the same position as a standard intron in the orthologue gene in species of the early divergent classes of the Pezizomycetes and the Orbiliomycetes, suggesting that an internal intron has appeared within a pre-extant intron. On the other hand, the stwintron has been lost from certain taxa in Leotiomycetes and Eurotiomycetes at several occasions, most likely by a mechanism involving reverse transcription and homologous recombination. Another ancient stwintron present across whole Pezizomycotina orders—in the transcript of the bifunctional biotin biosynthesis gene bioDA—occurs at the same position as a standard intron in many species of non-Dikarya. Nevertheless, also the bioDA stwintron has disappeared from certain lineages within the taxa where it occurs, i.e., Sordariomycetes and Botryosphaeriales. Intriguingly, only the internal intron was lost from the Sordariomycetes bioDA stwintron at all but one occasion, leaving a standard intron in the same position, while where the putative lipase stwintron was lost, no intronic sequences remain. Conclusions Molecular phylogeny of the peptide product was used to monitor the existence and fate of a stwintron in the transcripts of two neatly defined fungal genes, encoding well conserved proteins. Both defining events—stwintron emergence and loss—can be explained with extant models for intron insertion and loss. We thus demonstrate that stwintrons can serve as model systems to study spliceosomal intron evolution.

Itaconic acid is used as a bio-based, renewable building block in the polymer industry. It is produced by submerged fermentations of the filamentous fungus Aspergillus terreus from molasses or starch, but research over the efficient utilization of non-food, lignocellulosic plant biomass is soaring. The objective of this study was to test whether the application of two key cultivation parameters for obtaining itaconic acid from D-glucose in high yields – Mn2+ ion deficiency and high concentration of the carbon source – would also occur on D-xylose, the principal monomer of lignocellulose. To this end, a carbon and energy balance for itaconic acid formation was established, which is 0.83 moles/mole D-xylose. The effect of Mn2+ ions on itaconic acid formation was indeed similar to that on D-glucose and maximal yields were obtained below 3 μg L–1 Mn2+ ions, which were, however, only 0.63 moles of itaconic acid per mole D-xylose. In contrast to the case on D-glucose, increasing D-xylose concentration over 50 g L–1 did not change the above yield. By-products such as xylitol and α-ketoglutarate were found, but in total they remained below 2% of the concentration of D-xylose. Mass balance of the fermentation with 110 g L–1D-xylose revealed that >95% of the carbon from D-xylose was accounted as biomass, itaconic acid, and the carbon dioxide released in the last step of itaconic acid biosynthesis. Our data show that the efficiency of biomass formation is the critical parameter for itaconic acid yield from D-xylose under otherwise optimal conditions.

Aspergillus nidulans has one gene for alternative oxidase (EC 1.10.3.11). To investigate the relationship between this mitochondrial terminal oxidase and the formation of the mycotoxin sterigmatocystin, the encoding aodA gene was both deleted and overexpressed. Relative to the wild-type, the cyanide-resistant fraction of respiration in the late stationary stage—when sterigmatocystin production occurs—doubled in the overexpressing mutant carrying three aodA gene copies, but decreased to 10% in the deletant. Essentially identical results were obtained regardless whether the cultures were illuminated or protected from light. In contrast, sterigmatocystin yield in the aodA deletant was about half of that in the control when grown in the dark, while aodA overexpression resulted in up to 70% more sterigmatocystin formed, the yield increasing with alternative oxidase activity. Results were quite different when cultures were illuminated: under those conditions, sterigmatocystin volumetric yields were considerably lower, and statistically unvarying, regardless of the presence, absence, or the copy number of aodA. We conclude that the copy number of aodA, and hence, the balance between alternative- and cytochrome C-mediated respiration, appears to correlate with sterigmatocystin production in A. nidulans, albeit only in the absence of light.

This review summarises the current knowledge on the biochemical and physiological events that directly or indirectly alter the engagement of the cyanide-resistant alternative respiratory pathway in the cephalosporin C producer filamentous fungus Acremonium chrysogenum. Particular emphasis is placed on the role this activity plays in the overproduction of antibiotic, and also on the critical fermentation technology background that supports its operation.

Trichoderma reesei is the industrial producer of cellulases and hemicellulases for biorefinery processes. Their expression is obligatorily dependent on the function of the protein methyltransferase LAE1. The Aspergillus nidulans orthologue of LAE1--LaeA--is part of the VELVET protein complex consisting of LaeA, VeA and VelB that regulates secondary metabolism and sexual as well as asexual reproduction. Here we have therefore investigated the function of VEL1, the T. reesei orthologue of A. nidulans VeA. Deletion of the T. reesei vel1 locus causes a complete and light-independent loss of conidiation, and impairs formation of perithecia. Deletion of vel1 also alters hyphal morphology towards hyperbranching and formation of thicker filaments, and with consequently reduced growth rates. Growth on lactose as a sole carbon source, however, is even more strongly reduced and growth on cellulose as a sole carbon source eliminated. Consistent with these findings, deletion of vel1 completely impaired the expression of cellulases, xylanases and the cellulase regulator XYR1 on lactose as a cellulase inducing carbon source, but also in resting mycelia with sophorose as inducer. Our data show that in T. reesei VEL1 controls sexual and asexual development, and this effect is independent of light. VEL1 is also essential for cellulase gene expression, which is consistent with the assumption that their regulation by LAE1 occurs by the VELVET complex.

Seed contamination with polyketide mycotoxins such as sterigmatocystin (ST) produced by Aspergilli is a worldwide issue. The ST biosynthetic pathway is well-characterized in A. nidulans, but regulatory aspects related to the carbon source are still enigmatic. This is particularly true for lactose, inasmuch as some ST production mutant strains still synthesize ST on lactose but not on other carbon substrates. Here, kinetic data revealed that on d-glucose, ST forms only after the sugar is depleted from the medium, while on lactose, ST appears when most of the carbon source is still available. Biomass-specified ST production on lactose was significantly higher than on d-glucose, suggesting that ST formation may either be mediated by a carbon catabolite regulatory mechanism, or induced by low specific growth rates attainable on lactose. These hypotheses were tested by d-glucose limited chemostat-type continuous fermentations. No ST formed at a high growth rate, while a low growth rate led to the formation of 0.4 mg·L−1 ST. Similar results were obtained with a CreA mutant strain. We concluded that low specific growth rates may be the primary cause of mid-growth ST formation on lactose in A. nidulans, and that carbon utilization rates likely play a general regulatory role during biosynthesis.

The article cites a study of galactidol as the actual inducers of β-galactosidase formation during growth on D-galactose in hypocrea jecorina. It investigates the ability of hypocrea jecorina to grow on lactose strongly depends on the formation of an extracellular glycoside hydrolase family. It highlights the induction of D-galactose and galactitol that found strain deleted in the galactokinase-encoding gene, responsible for D-galactose catabolism.