Fusarium: more than a node or a foot-shaped basal cell.

Recent publications have argued that there are potentially serious consequences for researchers in recognising distinct genera in the terminal fusarioid clade of the family Nectriaceae. Thus, an alternate hypothesis, namely a very broad concept of the genus Fusarium was proposed. In doing so, however, a significant body of data that supports distinct genera in Nectriaceae based on morphology, biology, and phylogeny is disregarded. A DNA phylogeny based on 19 orthologous protein-coding genes was presented to support a very broad concept of Fusarium at the F1 node in Nectriaceae. Here, we demonstrate that re-analyses of this dataset show that all 19 genes support the F3 node that represents Fusarium sensu stricto as defined by F. sambucinum (sexual morph synonym Gibberella pulicaris). The backbone of the phylogeny is resolved by the concatenated alignment, but only six of the 19 genes fully support the F1 node, representing the broad circumscription of Fusarium. Furthermore, a re-analysis of the concatenated dataset revealed alternate topologies in different phylogenetic algorithms, highlighting the deep divergence and unresolved placement of various Nectriaceae lineages proposed as members of Fusarium. Species of Fusarium s. str. are characterised by Gibberella sexual morphs, asexual morphs with thin- or thick-walled macroconidia that have variously shaped apical and basal cells, and trichothecene mycotoxin production, which separates them from other fusarioid genera. Here we show that the Wollenweber concept of Fusarium presently accounts for 20 segregate genera with clear-cut synapomorphic traits, and that fusarioid macroconidia represent a character that has been gained or lost multiple times throughout Nectriaceae. Thus, the very broad circumscription of Fusarium is blurry and without apparent synapomorphies, and does not include all genera with fusarium-like macroconidia, which are spread throughout Nectriaceae (e.g., Cosmosporella, Macroconia, Microcera). In this study four new genera are introduced, along with 18 new species and 16 new combinations. These names convey information about relationships, morphology, and ecological preference that would otherwise be lost in a broader definition of Fusarium. To assist users to correctly identify fusarioid genera and species, we introduce a new online identification database, Fusarioid-ID, accessible at www.fusarium.org. The database comprises partial sequences from multiple genes commonly used to identify fusarioid taxa (act1, CaM, his3, rpb1, rpb2, tef1, tub2, ITS, and LSU). In this paper, we also present a nomenclator of names that have been introduced in Fusarium up to January 2021 as well as their current status, types, and diagnostic DNA barcode data. In this study, researchers from 46 countries, representing taxonomists, plant pathologists, medical mycologists, quarantine officials, regulatory agencies, and students, strongly support the application and use of a more precisely delimited Fusarium (= Gibberella) concept to accommodate taxa from the robust monophyletic node F3 on the basis of a well-defined and unique combination of morphological and biochemical features. This F3 node includes, among others, species of the F. fujikuroi, F. incarnatum-equiseti, F. oxysporum, and F. sambucinum species complexes, but not species of Bisifusarium [F. dimerum species complex (SC)], Cyanonectria (F. buxicola SC), Geejayessia (F. staphyleae SC), Neocosmospora (F. solani SC) or Rectifusarium (F. ventricosum SC). The present study represents the first step to generating a new online monograph of Fusarium and allied fusarioid genera (www.fusarium.org).

Aspergillus niger uses the peroxisomal CoA-dependent ß-oxidative genes to degrade the hydroxycinnamic acids caffeic acid, ferulic acid, and p-coumaric acid.

Aromatic compounds are important molecules which are widely applied in many industries and are mainly produced from nonrenewable sources. Renewable sources such as plant biomass are interesting alternatives for the production of aromatic compounds. Ferulic acid and p-coumaric acid, a precursor for vanillin and p-vinyl phenol, respectively, can be released from plant biomass by the fungus Aspergillus niger. The degradation of hydroxycinnamic acids such as caffeic acid, ferulic acid, and p-coumaric acid has been observed in many fungi. In A. niger, multiple metabolic pathways were suggested for the degradation of hydroxycinnamic acids. However, no genes were identified for these hydroxycinnamic acid metabolic pathways. In this study, several pathway genes were identified using whole-genome transcriptomic data of A. niger grown on different hydroxycinnamic acids. The genes are involved in the CoA-dependent ß-oxidative pathway in fungi. This pathway is well known for the degradation of fatty acids, but not for hydroxycinnamic acids. However, in plants, it has been shown that hydroxycinnamic acids are degraded through this pathway. We identified genes encoding hydroxycinnamate-CoA synthase (hcsA), multifunctional ß-oxidation hydratase/dehydrogenase (foxA), 3-ketoacyl CoA thiolase (katA), and four thioesterases (theA-D) of A. niger, which were highly induced by all three tested hydroxycinnamic acids. Deletion mutants revealed that these genes were indeed involved in the degradation of several hydroxycinnamic acids. In addition, foxA and theB are also involved in the degradation of fatty acids. HcsA, FoxA, and KatA contained a peroxisomal targeting signal and are therefore predicted to be localized in peroxisomes. KEY POINTS: • Metabolism of hydroxycinnamic acid was investigated in Aspergillus niger • Using transcriptome data, multiple CoA-dependent ß-oxidative genes were identified. • Both foxA and theB are involved in hydroxycinnamate but also fatty acid metabolism.

Evolutionary adaptation of Aspergillus niger for increased ferulic acid tolerance.

AIMS: To create an Aspergillus niger mutant with increased tolerance against ferulic acid using evolutionary adaptation. METHODS AND RESULTS: Evolutionary adaptation of A. niger N402 was performed by consecutive growth on increasing concentrations of ferulic acid in the presence of 25 mmol l-1 d-fructose, starting from 0·5 mmol l-1 and ending with 5 mmol l-1 ferulic acid. The A. niger mutant obtained after six months, named Fa6, showed increased ferulic acid tolerance compared to the parent. In addition, Fa6 has increased ferulic acid consumption and a higher conversion rate, suggesting that the mutation affects aromatic metabolism of this species. Transcriptome analysis of the evolutionary mutant on ferulic acid revealed a distinct gene expression profile compared to the wild type. Further analysis of this mutant and the parent strain provided the first experimental confirmation that A. niger converts coniferyl alcohol to ferulic acid. CONCLUSIONS: The evolutionary adaptive A. niger mutant Fa6 has beneficial mutations that increase the tolerance, conversion rate and uptake of ferulic acid. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates that evolutionary adaptation is a powerful tool to modify micro-organisms towards increased tolerance to harsh conditions, which is beneficial for various industrial applications.

Genomic and exoproteomic diversity in plant biomass degradation approaches among Aspergilli.

We classified the genes encoding carbohydrate-active enzymes (CAZymes) in 17 sequenced genomes representing 16 evolutionarily diverse Aspergillus species. We performed a phylogenetic analysis of the encoding enzymes, along with experimentally characterized CAZymes, to assign molecular function to the Aspergilli CAZyme families and subfamilies. Genome content analysis revealed that the numbers of CAZy genes per CAZy family related to plant biomass degradation follow closely the taxonomic distance between the species. On the other hand, growth analysis showed almost no correlation between the number of CAZyme genes and the efficiency in polysaccharide utilization. The exception is A. clavatus where a reduced number of pectinolytic enzymes can be correlated with poor growth on pectin. To gain detailed information on the enzymes used by Aspergilli to breakdown complex biomass, we conducted exoproteome analysis by mass spectrometry. These results showed that Aspergilli produce many different enzymes mixtures in the presence of sugar beet pulp and wheat bran. Despite the diverse enzyme mixtures produced, species of section Nigri, A. aculeatus, A. nidulans and A. terreus, produce mixtures of enzymes with activities that are capable of digesting all the major polysaccharides in the available substrates, suggesting that they are capable of degrading all the polysaccharides present simultaneously. For the other Aspergilli, typically the enzymes produced are targeted to a subset of polysaccharides present, suggesting that they can digest only a subset of polysaccharides at a given time.

Duplications and losses of genes encoding known elements of the stress defence system of the Aspergilli contribute to the evolution of these filamentous fungi but do not directly influence their environmental stress tolerance.

The contribution of stress protein duplication and deletion events to the evolution of the Aspergilli was studied. We performed a large-scale homology analysis of stress proteins and generated and analysed three stress defence system models based on Saccharomyces cerevisiae, Schizosaccharomyces pombe and Aspergillus nidulans. Although both yeast-based and A. nidulans-based models were suitable to trace evolutionary changes, the A. nidulans-based model performed better in mapping stress protein radiations. The strong Mantel correlation found between the positions of species in the phylogenetic tree on the one hand and either in the A. nidulans-based or S. cerevisiae-based models on the other hand demonstrated that stress protein expansions and reductions contributed significantly to the evolution of the Aspergilli. Interestingly, stress tolerance attributes correlated well with the number of orthologs only for a few stress proteins. Notable examples are Ftr1 iron permease and Fet3 ferro-O2-oxidoreductase, elements of the reductive iron assimilation pathway, in the S. cerevisiae-based model, as well as MpkC, a HogA-like mitogen activated protein kinase in the A. nidulans-based model. In the case of the iron assimilation proteins, the number of orthologs showed a positive correlation with H2O2-induced stress tolerance while the number of MpkC orthologs correlated positively with Congo Red induced cell wall stress, sorbitol induced osmotic stress and H2O2 induced oxidative stress tolerances. For most stress proteins, changes in the number of orthologs did not correlate well with any stress tolerance attributes. As a consequence, stress tolerance patterns of the studied Aspergilli did not correlate with either the sets of stress response proteins in general or with the phylogeny of the species studied. These observations suggest that stress protein duplication and deletion events significantly contributed to the evolution of stress tolerance attributes of Aspergilli. In contrast, there are other processes, which may counterbalance the effects of stress gene duplications or deletions including (i) alterations in the structures of stress proteins leading to changes in their biological activities, (ii) varying biosynthesis of stress proteins, (iii) rewiring stress response regulatory networks or even (iv) acquiring new stress response genes by horizontal gene transfer. All these multilevel changes are indispensable for the successful adaptation of filamentous fungi to altering environmental conditions, especially when these organisms are entering new ecological niches.

The gold-standard genome of Aspergillus niger NRRL 3 enables a detailed view of the diversity of sugar catabolism in fungi.

The fungal kingdom is too large to be discovered exclusively by classical genetics. The access to omics data opens a new opportunity to study the diversity within the fungal kingdom and how adaptation to new environments shapes fungal metabolism. Genomes are the foundation of modern science but their quality is crucial when analysing omics data. In this study, we demonstrate how one gold-standard genome can improve functional prediction across closely related species to be able to identify key enzymes, reactions and pathways with the focus on primary carbon metabolism. Based on this approach we identified alternative genes encoding various steps of the different sugar catabolic pathways, and as such provided leads for functional studies into this topic. We also revealed significant diversity with respect to genome content, although this did not always correlate to the ability of the species to use the corresponding sugar as a carbon source.

Mixed colonies of Aspergillus niger and Aspergillus oryzae cooperatively degrading wheat bran.

In both natural and man-made environments, microorganisms live in mixed populations, while in laboratory conditions monocultures are mainly used. Microbial interactions are often described as antagonistic, but can also be neutral or cooperative, and are generally associated with a metabolic change of each partner and cause a change in the pattern of produced bioactive molecules. A. niger and A. oryzae are two filamentous fungi widely used in industry to produce various enzymes (e.g. pectinases, amylases) and metabolites (e.g. citric acid). The co-cultivation of these two fungi in wheat bran showed an equal distribution of the two strains forming mixed colonies with a broad range of carbohydrate active enzymes produced. This stable mixed microbial system seems suitable for subsequent commercial processes such as enzyme production. XlnR knock-out strains for both aspergilli were used to study the influence of plant cell wall degrading enzyme production on the fitness of the mixed culture. Microscopic observation correlated with quantitative PCR and proteomic data suggest that the XlnR Knock-out strain benefit from the release of sugars by the wild type strain to support its growth.

Volatile compound profiling of Turkish Divle Cave cheese during production and ripening.

The formation of volatile compounds in Turkish Divle Cave cheese produced in 3 different dairy farms was determined during production and ripening, revealing 110 compounds including acids, alcohols, ketones, esters, and terpenes. The presence and concentration of these volatile compounds varied between specific phases of the production and the 120-d ripening process. Smaller differences were also detected between cheeses produced at different farms. Carboxylic acids were established as a major class at the end of ripening. The relative amounts of acids and ketones increased until d 90 of ripening, whereas alcohols increased for the first 30d and tailed off during the remaining part of the ripening process. The level of esters increased gradually until the end of ripening. Butanoic, acetic, and valeric acids, 2-butanol, 2-butanone, 2-heptanone, ethyl butanoate, α-pinene, and toluene were the most abundant compounds, likely contributing to the characteristic aroma of this traditional cheese.

Novel Receptor Specificity of Avian Gammacoronaviruses That Cause Enteritis.

UNLABELLED: Viruses exploit molecules on the target membrane as receptors for attachment and entry into host cells. Thus, receptor expression patterns can define viral tissue tropism and might to some extent predict the susceptibility of a host to a particular virus. Previously, others and we have shown that respiratory pathogens of the genus Gammacoronavirus, including chicken infectious bronchitis virus (IBV), require specific α2,3-linked sialylated glycans for attachment and entry. Here, we studied determinants of binding of enterotropic avian gammacoronaviruses, including turkey coronavirus (TCoV), guineafowl coronavirus (GfCoV), and quail coronavirus (QCoV), which are evolutionarily distant from respiratory avian coronaviruses based on the viral attachment protein spike (S1). We profiled the binding of recombinantly expressed S1 proteins of TCoV, GfCoV, and QCoV to tissues of their respective hosts. Protein histochemistry showed that the tissue binding specificity of S1 proteins of turkey, quail, and guineafowl CoVs was limited to intestinal tissues of each particular host, in accordance with the reported pathogenicity of these viruses in vivo. Glycan array analyses revealed that, in contrast to the S1 protein of IBV, S1 proteins of enteric gammacoronaviruses recognize a unique set of nonsialylated type 2 poly-N-acetyl-lactosamines. Lectin histochemistry as well as tissue binding patterns of TCoV S1 further indicated that these complex N-glycans are prominently expressed on the intestinal tract of various avian species. In conclusion, our data demonstrate not only that enteric gammacoronaviruses recognize a novel glycan receptor but also that enterotropism may be correlated with the high specificity of spike proteins for such glycans expressed in the intestines of the avian host. IMPORTANCE: Avian coronaviruses are economically important viruses for the poultry industry. While infectious bronchitis virus (IBV), a respiratory pathogen of chickens, is rather well known, other viruses of the genus Gammacoronavirus, including those causing enteric disease, are hardly studied. In turkey, guineafowl, and quail, coronaviruses have been reported to be the major causative agent of enteric diseases. Specifically, turkey coronavirus outbreaks have been reported in North America, Europe, and Australia for several decades. Recently, a gammacoronavirus was isolated from guineafowl with fulminating disease. To date, it is not clear why these avian coronaviruses are enteropathogenic, whereas other closely related avian coronaviruses like IBV cause respiratory disease. A comprehensive understanding of the tropism and pathogenicity of these viruses explained by their receptor specificity and receptor expression on tissues was therefore needed. Here, we identify a novel glycan receptor for enteric avian coronaviruses, which will further support the development of vaccines.

Resolving the polyphyletic nature of Pyricularia (Pyriculariaceae).

Species of Pyricularia (magnaporthe-like sexual morphs) are responsible for major diseases on grasses. Pyricularia oryzae (sexual morph Magnaporthe oryzae) is responsible for the major disease of rice called rice blast disease, and foliar diseases of wheat and millet, while Pyricularia grisea (sexual morph Magnaporthe grisea) is responsible for foliar diseases of Digitaria. Magnaporthe salvinii, M. poae and M. rhizophila produce asexual spores that differ from those of Pyricularia sensu stricto that has pyriform, 2-septate conidia produced on conidiophores with sympodial proliferation. Magnaporthe salvinii was recently allocated to Nakataea, while M. poae and M. rhizophila were placed in Magnaporthiopsis. To clarify the taxonomic relationships among species that are magnaporthe- or pyricularia-like in morphology, we analysed phylogenetic relationships among isolates representing a wide range of host plants by using partial DNA sequences of multiple genes such as LSU, ITS, RPB1, actin and calmodulin. Species of Pyricularia s. str. belong to a monophyletic clade that includes all P. oryzae/P. grisea isolates tested, defining the Pyriculariaceae, which is sister to the Ophioceraceae, representing two novel families. These clades are clearly distinct from species belonging to the Gaeumannomyces pro parte/Magnaporthiopsis/Nakataea generic complex that are monophyletic and define the Magnaporthaceae. A few magnaporthe- and pyricularia-like species are unrelated to Magnaporthaceae and Pyriculariaceae. Pyricularia oryzae/P. grisea isolates cluster into two related clades. Host plants such as Eleusine, Oryza, Setaria or Triticum were exclusively infected by isolates from P. oryzae, while some host plant such as Cenchrus, Echinochloa, Lolium, Pennisetum or Zingiber were infected by different Pyricularia species. This demonstrates that host range cannot be used as taxonomic criterion without extensive pathotyping. Our results also show that the typical pyriform, 2-septate conidium morphology of P. grisea/P. oryzae is restricted to Pyricularia and Neopyricularia, while most other genera have obclavate to more ellipsoid 2-septate conidia. Some related genera (Deightoniella, Macgarvieomyces) have evolved 1-septate conidia. Therefore, conidium morphology cannot be used as taxonomic criterion at generic level without phylogenetic data. We also identified 10 novel genera, and seven novel species. A re-evaluation of generic and species concepts within Pyriculariaceae is presented, and novelties are proposed based on morphological and phylogenetic data.

Growth and hydrolase profiles can be used as characteristics to distinguish Aspergillus niger and other black aspergilli.

Wild type Aspergillus niger isolates from different biotopes from all over the world were compared to each other and to the type strains of other black Aspergillus species with respect to growth and extracellular enzyme profiles. The origin of the A. niger isolate did not result in differences in growth profile with respect to monomeric or polymeric carbon sources. Differences were observed in the growth rate of the A. niger isolates, but these were observed on all carbon sources and not specific for a particular carbon source. In contrast, carbon source specific differences were observed between the different species. Aspergillus brasiliensis is the only species able to grow on D-galactose, and A. aculeatus had significantly better growth on Locus Bean gum than the other species. Only small differences were found in the extracellular enzyme profile of the A. niger isolates during growth on wheat bran, while large differences were observed in the profiles of the different black aspergilli. In addition, differences were observed in temperature profiles between the black Aspergillus species, but not between the A. niger isolates, demonstrating no isolate-specific adaptations to the environment.These data indicate that the local environment does not result in stable adaptations of A. niger with respect to growth profile or enzyme production, but that the potential is maintained irrespective of the environmental parameters. It also demonstrates that growth, extracellular protein and temperature profiles can be used for species identification within the group of black aspergilli.

Analysis of regulation of pentose utilisation in Aspergillus niger reveals evolutionary adaptations in Eurotiales.

Aspergilli are commonly found in soil and on decaying plant material. D-xylose and L-arabinose are highly abundant components of plant biomass. They are released from polysaccharides by fungi using a set of extracellular enzymes and subsequently converted intracellularly through the pentose catabolic pathway (PCP).In this study, the L-arabinose responsive transcriptional activator (AraR) is identified in Aspergillus niger and was shown to control the L-arabinose catabolic pathway as well as expression of genes encoding extracellular L-arabinose releasing enzymes. AraR interacts with the D-xylose-responsive transcriptional activator XlnR in the regulation of the pentose catabolic pathway, but not with respect to release of L-arabinose and D-xylose.AraR was only identified in the Eurotiales, more specifically in the family Trichocomaceae and appears to have originated from a gene duplication event (from XlnR) after this order or family split from the other filamentous ascomycetes. XlnR is present in all filamentous ascomycetes with the exception of members of the Onygenales. Since the Onygenales and Eurotiales are both part of the subclass Eurotiomycetidae, this indicates that strong adaptation of the regulation of pentose utilisation has occurred at this evolutionary node. In Eurotiales a unique two-component regulatory system for pentose release and metabolism has evolved, while the regulatory system was lost in the Onygenales. The observed evolutionary changes (in Eurotiomycetidae) mainly affect the regulatory system as in contrast, homologues for most genes of the L-arabinose/D-xylose catabolic pathway are present in all the filamentous fungi, irrespective of the presence of XlnR and/or AraR.

Binding of avian coronavirus spike proteins to host factors reflects virus tropism and pathogenicity.

The binding of viruses to host cells is the first step in determining tropism and pathogenicity. While avian infectious bronchitis coronavirus (IBV) infection and avian influenza A virus (IAV) infection both depend on α2,3-linked sialic acids, the host tropism of IBV is restricted compared to that of IAV. Here we investigated whether the interaction between the viral attachment proteins and the host could explain these differences by using recombinant spike domains (S1) of IBV strains with different pathogenicities, as well as the hemagglutinin (HA) protein of IAV H5N1. Protein histochemistry showed that S1 of IBV strain M41 and HA of IAV subtype H5N1 displayed sialic acid-dependent binding to chicken respiratory tract tissue. However, while HA bound with high avidity to a broad range of α2,3-linked sialylated glycans, M41 S1 recognized only one particular α2,3-linked disialoside in a glycan array. When comparing the binding of recombinant IBV S1 proteins derived from IBV strains with known differences in tissue tropism and pathogenicity, we observed that while M41 S1 displayed binding to cilia and goblet cells of the chicken respiratory tract, S1 derived from the vaccine strain H120 or the nonvirulent Beaudette strain had reduced or no binding to chicken tissues, respectively, in agreement with the reduced abilities of these viruses to replicate in vivo. While the S1 protein derived from the nephropathogenic IBV strain B1648 also hardly displayed binding to respiratory tract cells, distinct binding to kidney cells was observed, but only after the removal of sialic acid from S1. In conclusion, our data demonstrate that the attachment patterns of the IBV S proteins correlate with the tropisms and pathogenicities of the corresponding viruses.

Vaccination with a soluble recombinant hemagglutinin trimer protects pigs against a challenge with pandemic (H1N1) 2009 influenza virus.

In 2009 a new influenza A/H1N1 virus strain ("pandemic (H1N1) 2009", H1N1v) emerged that rapidly spread around the world. The virus is suspected to have originated in swine through reassortment and to have subsequently crossed the species-barrier towards humans. Several cases of reintroduction into pigs have since been reported, which could possibly create a reservoir for human exposure or ultimately become endemic in the pig population with similar clinical disease problems as current swine influenza strains. A soluble trimer of hemagglutinin (HA), derived from the H1N1v, was used as a vaccine in pigs to investigate the extent to which this vaccine would be able to protect pigs against infection with the H1N1v influenza strain, especially with respect to reducing virus replication and excretion. In a group of unvaccinated control pigs, no clinical symptoms were observed, but (histo)pathological changes consistent with an influenza infection were found on days 1 and 3 after inoculation. Live virus was isolated from the upper and lower respiratory tract, with titres up to 10(6) TCID(50) per gram of tissue. Furthermore, live virus was detected in brain samples. Control pigs were shedding live virus for up to 6 days after infection, with titres of up to 10(5) TCID(50) per nasal or oropharyngeal swab. The soluble H1N1v HA trimer diminished virus replication and excretion after a double vaccination and subsequent challenge. Live virus could not be detected in any of the samples taken from the vaccinated pigs. Vaccines based on soluble HA trimers provide an attractive alternative to the current inactivated vaccines.

Technical advance in fungal biotechnology: development of a miniaturized culture method and an automated high-throughput screening.

AIMS: The goal of the study was to develop a reliable, reproducible and rapid method of culture in order to screen a large number of fungal transformants. METHODS AND RESULTS: The method is based upon miniaturized cell cultures and automated expression screening in microwell plates. For the method development, 50 recombinant Aspergillus vadensis clones producing feruloyl esterase B (FaeB) from Aspergillus niger were screened in 6 days. Then a panel of clones showing various behaviours was checked in flasks in order to demonstrate the reproducibility of the method. Using this method, a transformant of A. vadensis producing 1.2 g l(-1) of FaeB was selected (12-fold more than the A. niger overproducing strain). CONCLUSIONS: This miniaturized culture method allows to obtain reliable and reproducible results. The procedure has the advantages of being efficient, time-saving and more efficient than conventional in-flask culture screening as it can screen 800 clones per day after a culture of 3 days. SIGNIFICANCE AND IMPACT OF THE STUDY: This method could be applied to any other fungal strain culture, enzyme activity or biodiversity screening.

Regulation of Aspergillus genes encoding plant cell wall polysaccharide-degrading enzymes; relevance for industrial production.

The genus Aspergillus is widely used for the production of plant cell wall polysaccharide-degrading enzymes. The range of enzymes purified from these fungi covers nearly every function required for the complete degradation of cellulose, xyloglucan, xylan, galacto(gluco)mannan and pectin. This paper describes the Aspergillus enzymes involved in the degradation of these polysaccharides and discusses the regulatory systems involved in the expression of the genes encoding these proteins. The latter is of major importance in the large-scale production of these enzymes for industrial applications.

Regulation of the alpha-glucuronidase-encoding gene ( aguA) from Aspergillus niger.

The alpha-glucuronidase gene aguA from Aspergillus niger was cloned and characterised. Analysis of the promoter region of aguA revealed the presence of four putative binding sites for the major carbon catabolite repressor protein CREA and one putative binding site for the transcriptional activator XLNR. In addition, a sequence motif was detected which differed only in the last nucleotide from the XLNR consensus site. A construct in which part of the aguA coding region was deleted still resulted in production of a stable mRNA upon transformation of A. niger. The putative XLNR binding sites and two of the putative CREA binding sites were mutated individually in this construct and the effects on expression were examined in A. niger transformants. Northern analysis of the transformants revealed that the consensus XLNR site is not actually functional in the aguA promoter, whereas the sequence that diverges from the consensus at a single position is functional. This indicates that XLNR is also able to bind to the sequence GGCTAG, and the XLNR binding site consensus should therefore be changed to GGCTAR. Both CREA sites are functional, indicating that CREA has a strong influence on aguA expression. A detailed expression analysis of aguA in four genetic backgrounds revealed a second regulatory system involved in activation of aguA gene expression. This system responds to the presence of glucuronic and galacturonic acids, and is not dependent on XLNR.

Aspergillus enzymes involved in degradation of plant cell wall polysaccharides.

Degradation of plant cell wall polysaccharides is of major importance in the food and feed, beverage, textile, and paper and pulp industries, as well as in several other industrial production processes. Enzymatic degradation of these polymers has received attention for many years and is becoming a more and more attractive alternative to chemical and mechanical processes. Over the past 15 years, much progress has been made in elucidating the structural characteristics of these polysaccharides and in characterizing the enzymes involved in their degradation and the genes of biotechnologically relevant microorganisms encoding these enzymes. The members of the fungal genus Aspergillus are commonly used for the production of polysaccharide-degrading enzymes. This genus produces a wide spectrum of cell wall-degrading enzymes, allowing not only complete degradation of the polysaccharides but also tailored modifications by using specific enzymes purified from these fungi. This review summarizes our current knowledge of the cell wall polysaccharide-degrading enzymes from aspergilli and the genes by which they are encoded.

The Aspergillus niger D-xylulose kinase gene is co-expressed with genes encoding arabinan degrading enzymes, and is essential for growth on D-xylose and L-arabinose.

The Aspergillus niger D-xylulose kinase encoding gene has been cloned by complementation of a strain deficient in D-xylulose kinase activity. Expression of xkiA was observed in the presence of L-arabinose, L-arabitol and D-xylose. Expression of xkiA is not mediated by XLNR, the xylose-dependent positively-acting xylanolytic regulator. Although the expression of xkiA is subject to carbon catabolite repression, the wide domain regulator CREA is not directly involved. The A. niger D-xylulose kinase was purified to homogeneity, and the molecular mass determined using electrospray ionization mass spectrometry agreed with the calculated molecular mass of 62816.6 Da. The activity of XKIA is highly specific for D-xylulose. Kinetic parameters were determined as Km(D-xylulose) = 0.76 mM and Km(ATP) = 0.061 mM. Increased transcript levels of the genes encoding arabinan and xylan degrading enzymes, observed in the xylulose kinase deficient strain, correlate with increased accumulation of L-arabitol and xylitol, respectively. This result supports the suggestion that L-arabitol may be the specific low molecular mass inducer of the genes involved in arabinan degradation. It also suggests a possible role for xylitol in the induction of xylanolytic genes. Conversely, overproduction of XKIA did not reduce the size of the intracellular arabitol and xylitol pools, and therefore had no effect on expression of genes encoding xylan and arabinan degrading enzymes nor on the activity of the enzymes of the catabolic pathway.