Process development for the continuous production of heterologous proteins by the industrial yeast, Komagataella phaffii.

The current trend in industrial biotechnology is to move from batch or fed-batch fermentations to continuous operations. The success of this transition will require the development of genetically stable production strains, the use of strong constitutive promoters, and the development of new medium formulations that allow an appropriate balance between cell growth and product formation. We identified genes that showed high expression in Komagataella phaffii during different steady-state conditions and explored the utility of promoters of these genes (Chr1-4_0586 and FragB_0052) in optimizing the expression of two different r-proteins, human lysozyme (HuLy), and the anti-idiotypic antibody fragment, Fab-3H6, in comparison with the widely used glyceraldehyde-3-phosphate dehydrogenase promoter. Our results showed that the promoter strength was highly dependent on the cultivation conditions and thus constructs should be tested under a range of conditions to determine both the best performing clone and the ideal promoter for the expression of the protein of interest. An important benefit of continuous production is that it facilitates the use of the genome-scale metabolic models in the design of strains and cultivation media. In silico flux distributions showed that production of either protein increased the flux through aromatic amino acid biosynthesis. Tyrosine supplementation increased the productivity for both proteins, whereas tryptophan addition did not cause any significant change and, phenylalanine addition increased the expression of HuLy but decreased that of Fab-3H6. These results showed that a genome-scale metabolic model can be used to assess the metabolic burden imposed by the synthesis of a specific r-protein and then this information can be used to tailor a cultivation medium to increase production.

Transcriptomic responses of mixed cultures of ascomycete fungi to lignocellulose using dual RNA-seq reveal inter-species antagonism and limited beneficial effects on CAZyme expression.

Gaining new knowledge through fungal monoculture responses to lignocellulose is a widely used approach that can lead to better cocktails for lignocellulose saccharification (the enzymatic release of sugars which are subsequently used to make biofuels). However, responses in lignocellulose mixed cultures are rarely studied in the same detail even though in nature fungi often degrade lignocellulose as mixed communities. Using a dual RNA-seq approach, we describe the first study of the transcriptional responses of wild-type strains of Aspergillus niger, Trichoderma reesei and Penicillium chrysogenum in two and three mixed species shake-flask cultures with wheat straw. Based on quantification of species-specific rRNA, a set of conditions was identified where mixed cultures could be sampled so as to obtain sufficient RNA-seq reads for analysis from each species. The number of differentially-expressed genes varied from a couple of thousand to fewer than one hundred. The proportion of carbohydrate active enzyme (CAZy) encoding transcripts was lower in the majority of the mixed cultures compared to the respective straw monocultures. A small subset of P. chrysogenum CAZy genes showed five to ten-fold significantly increased transcript abundance in a two-species mixed culture with T. reesei. However, a substantial number of T. reesei CAZy transcripts showed reduced abundance in mixed cultures. The highly induced genes in mixed cultures indicated that fungal antagonism was a major part of the mixed cultures. In line with this, secondary metabolite producing gene clusters showed increased transcript abundance in mixed cultures and also mixed cultures with T. reesei led to a decrease in the mycelial biomass of A. niger. Significantly higher monomeric sugar release from straw was only measured using a minority of the mixed culture filtrates and there was no overall improvement. This study demonstrates fungal interaction with changes in transcripts, enzyme activities and biomass in the mixed cultures and whilst there were minor beneficial effects for CAZy transcripts and activities, the competitive interaction between T. reesei and the other fungi was the most prominent feature of this study.

Metabolic activity in dormant conidia of Aspergillus niger and developmental changes during conidial outgrowth.

The early stages of development of Aspergillus niger conidia during outgrowth were explored by combining genome-wide gene expression analysis (RNAseq), proteomics, Warburg manometry and uptake studies. Resting conidia suspended in water were demonstrated for the first time to be metabolically active as low levels of oxygen uptake and the generation of carbon dioxide were detected, suggesting that low-level respiratory metabolism occurs in conidia for maintenance. Upon triggering of spore germination, generation of CO2 increased dramatically. For a short period, which coincided with mobilisation of the intracellular polyol, trehalose, there was no increase in uptake of O2 indicating that trehalose was metabolised by fermentation. Data from genome-wide mRNA profiling showed the presence of transcripts associated with fermentative and respiratory metabolism in resting conidia. Following triggering of conidial outgrowth, there was a clear switch to respiration after 25min, confirmed by cyanide inhibition. No effect of SHAM, salicylhydroxamic acid, on respiration suggests electron flow via cytochrome c oxidase. Glucose entry into spores was not detectable before 1h after triggering germination. The impact of sorbic acid on germination was examined and we showed that it inhibits glucose uptake. O2 uptake was also inhibited, delaying the onset of respiration and extending the period of fermentation. In conclusion, we show that conidia suspended in water are not completely dormant and that conidial outgrowth involves fermentative metabolism that precedes respiration.

Uncovering the genome-wide transcriptional responses of the filamentous fungus Aspergillus niger to lignocellulose using RNA sequencing.

A key challenge in the production of second generation biofuels is the conversion of lignocellulosic substrates into fermentable sugars. Enzymes, particularly those from fungi, are a central part of this process, and many have been isolated and characterised. However, relatively little is known of how fungi respond to lignocellulose and produce the enzymes necessary for dis-assembly of plant biomass. We studied the physiological response of the fungus Aspergillus niger when exposed to wheat straw as a model lignocellulosic substrate. Using RNA sequencing we showed that, 24 hours after exposure to straw, gene expression of known and presumptive plant cell wall-degrading enzymes represents a huge investment for the cells (about 20% of the total mRNA). Our results also uncovered new esterases and surface interacting proteins that might form part of the fungal arsenal of enzymes for the degradation of plant biomass. Using transcription factor deletion mutants (xlnR and creA) to study the response to both lignocellulosic substrates and low carbon source concentrations, we showed that a subset of genes coding for degradative enzymes is induced by starvation. Our data support a model whereby this subset of enzymes plays a scouting role under starvation conditions, testing for available complex polysaccharides and liberating inducing sugars, that triggers the subsequent induction of the majority of hydrolases. We also showed that antisense transcripts are abundant and that their expression can be regulated by growth conditions.

Biochemical and molecular characterization of an atypical manganese peroxidase of the litter-decomposing fungus Agrocybe praecox.

Agrocybe praecox is a litter-decomposing Basidiomycota species of the order Agaricales, and is frequently found in forests and open woodlands. A. praecox grows in leaf-litter and the upper soil and is able to colonize bark mulch and wood chips. It produces extracellular manganese peroxidase (MnP) activities and mineralizes synthetic lignin. In this study, the A. praecox MnP1 isozyme was purified, cloned and enzymatically characterized. The enzyme catalysed the oxidation of Mn(2+) to Mn(3+), which is the specific reaction for manganese-dependent class II heme-peroxidases, in the presence of malonate as chelator with an activity maximum at pH 4.5; detectable activity was observed even at pH 7.0. The coding sequence of the mnp1 gene demonstrates a short-type of MnP protein with a slightly modified Mn(2+) binding site. Thus, A. praecox MnP1 may represent a novel group of atypical short-MnP enzymes. In lignocellulose-containing cultures composed of cereal bran or forest litter, transcription of mnp1 gene was followed by quantitative real-time RT-PCR. On spruce needle litter, mnp1 expression was more abundant than on leaf litter after three weeks cultivation. However, the expression was constitutive in wheat and rye bran cultures. Our data show that the atypical MnP of A. praecox is able to catalyse Mn(2+) oxidation, which suggests its involvement in lignocellulose decay by this litter-decomposer.

The role of carbon starvation in the induction of enzymes that degrade plant-derived carbohydrates in Aspergillus niger.

Fungi are an important source of enzymes for saccharification of plant polysaccharides and production of biofuels. Understanding of the regulation and induction of expression of genes encoding these enzymes is still incomplete. To explore the induction mechanism, we analysed the response of the industrially important fungus Aspergillus niger to wheat straw, with a focus on events occurring shortly after exposure to the substrate. RNA sequencing showed that the transcriptional response after 6h of exposure to wheat straw was very different from the response at 24h of exposure to the same substrate. For example, less than half of the genes encoding carbohydrate active enzymes that were induced after 24h of exposure to wheat straw, were also induced after 6h exposure. Importantly, over a third of the genes induced after 6h of exposure to wheat straw were also induced during 6h of carbon starvation, indicating that carbon starvation is probably an important factor in the early response to wheat straw. The up-regulation of the expression of a high number of genes encoding CAZymes that are active on plant-derived carbohydrates during early carbon starvation suggests that these enzymes could be involved in a scouting role during starvation, releasing inducing sugars from complex plant polysaccharides. We show, using proteomics, that carbon-starved cultures indeed release CAZymes with predicted activity on plant polysaccharides. Analysis of the enzymatic activity and the reaction products, indicates that these proteins are enzymes that can degrade various plant polysaccharides to generate both known, as well as potentially new, inducers of CAZymes.

Germination of Aspergillus niger conidia is triggered by nitrogen compounds related to L-amino acids.

Conidial germination is fundamentally important to the growth and dissemination of most fungi. It has been previously shown (K. Hayer, M. Stratford, and D. B. Archer, Appl. Environ. Microbiol. 79:6924-6931, 2013, http://dx.doi.org/10.1128/AEM.02061-13), using sugar analogs, that germination is a 2-stage process involving triggering of germination and then nutrient uptake for hyphal outgrowth. In the present study, we tested this 2-stage germination process using a series of nitrogen-containing compounds for the ability to trigger the breaking of dormancy of Aspergillus niger conidia and then to support the formation of hyphae by acting as nitrogen sources. Triggering and germination were also compared between A. niger and Aspergillus nidulans using 2-deoxy-D-glucose (trigger), D-galactose (nontrigger in A. niger but trigger in A. nidulans), and an N source (required in A. niger but not in A. nidulans). Although most of the nitrogen compounds studied served as nitrogen sources for growth, only some nitrogen compounds could trigger germination of A. niger conidia, and all were related to L-amino acids. Using L-amino acid analogs without either the amine or the carboxylic acid group revealed that both the amine and carboxylic acid groups were essential for an L-amino acid to serve as a trigger molecule. Generally, conidia were able to sense and recognize nitrogen compounds that fitted into a specific size range. There was no evidence of uptake of either triggering or nontriggering compounds over the first 90 min of A. niger conidial germination, suggesting that the germination trigger sensors are not located within the spore.

Development of an unmarked gene deletion system for the filamentous fungi Aspergillus niger and Talaromyces versatilis.

In this article, we present a method to delete genes in filamentous fungi that allows recycling of the selection marker and is efficient in a nonhomologous end-joining (NHEJ)-proficient strain. We exemplify the approach by deletion of the gene encoding the transcriptional regulator XlnR in the fungus Aspergillus niger. To show the efficiency and advantages of the method, we deleted 8 other genes and constructed a double mutant in this species. Moreover, we showed that the same principle also functions in a different genus of filamentous fungus (Talaromyces versatilis, basionym Penicillium funiculosum). This technique will increase the versatility of the toolboxes for genome manipulation of model and industrially relevant fungi.

Genome-wide transcriptional response of Trichoderma reesei to lignocellulose using RNA sequencing and comparison with Aspergillus niger.

BACKGROUND: A major part of second generation biofuel production is the enzymatic saccharification of lignocellulosic biomass into fermentable sugars. Many fungi produce enzymes that can saccarify lignocellulose and cocktails from several fungi, including well-studied species such as Trichoderma reesei and Aspergillus niger, are available commercially for this process. Such commercially-available enzyme cocktails are not necessarily representative of the array of enzymes used by the fungi themselves when faced with a complex lignocellulosic material. The global induction of genes in response to exposure of T. reesei to wheat straw was explored using RNA-seq and compared to published RNA-seq data and model of how A. niger senses and responds to wheat straw. RESULTS: In T. reesei, levels of transcript that encode known and predicted cell-wall degrading enzymes were very high after 24h exposure to straw (approximately 13% of the total mRNA) but were less than recorded in A. niger (approximately 19% of the total mRNA). Closer analysis revealed that enzymes from the same glycoside hydrolase families but different carbohydrate esterase and polysaccharide lyase families were up-regulated in both organisms. Accessory proteins which have been hypothesised to possibly have a role in enhancing carbohydrate deconstruction in A. niger were also uncovered in T. reesei and categories of enzymes induced were in general similar to those in A. niger. Similarly to A. niger, antisense transcripts are present in T. reesei and their expression is regulated by the growth condition. CONCLUSIONS: T. reesei uses a similar array of enzymes, for the deconstruction of a solid lignocellulosic substrate, to A. niger. This suggests a conserved strategy towards lignocellulose degradation in both saprobic fungi. This study provides a basis for further analysis and characterisation of genes shown to be highly induced in the presence of a lignocellulosic substrate. The data will help to elucidate the mechanism of solid substrate recognition and subsequent degradation by T. reesei and provide information which could prove useful for efficient production of second generation biofuels.

Trancriptional landscape of Aspergillus niger at breaking of conidial dormancy revealed by RNA-sequencing.

BACKGROUND: Genome-wide analysis was performed to assess the transcriptional landscape of germinating A. niger conidia using both next generation RNA-sequencing and GeneChips. The metabolism of storage compounds during conidial germination was also examined and compared to the transcript levels from associated genes. RESULTS: The transcriptome of dormant conidia was shown to be highly differentiated from that of germinating conidia and major changes in response to environmental shift occurred within the first hour of germination. The breaking of dormancy was associated with increased transcript levels of genes involved in the biosynthesis of proteins, RNA turnover and respiratory metabolism. Increased transcript levels of genes involved in metabolism of nitrate at the onset of germination implies its use as a source of nitrogen. The transcriptome of dormant conidia contained a significant component of antisense transcripts that changed during germination. CONCLUSION: Dormant conidia contained transcripts of genes involved in fermentation, gluconeogenesis and the glyoxylate cycle. The presence of such transcripts in dormant conidia may indicate the generation of energy from non-carbohydrate substrates during starvation-induced conidiation or for maintenance purposes during dormancy. The immediate onset of metabolism of internal storage compounds after the onset of germination, and the presence of transcripts of relevant genes, suggest that conidia are primed for the onset of germination. For some genes, antisense transcription is regulated in the transition from resting conidia to fully active germinants.

Structural features of sugars that trigger or support conidial germination in the filamentous fungus Aspergillus niger.

The asexual spores (conidia) of Aspergillus niger germinate to produce hyphae under appropriate conditions. Germination is initiated by conidial swelling and mobilization of internal carbon and energy stores, followed by polarization and emergence of a hyphal germ tube. The effects of different pyranose sugars, all analogues of d-glucose, on the germination of A. niger conidia were explored, and we define germination as the transition from a dormant conidium into a germling. Within germination, we distinguish two distinct stages, the initial swelling of the conidium and subsequent polarized growth. The stage of conidial swelling requires a germination trigger, which we define as a compound that is sensed by the conidium and which leads to catabolism of d-trehalose and isotropic growth. Sugars that triggered germination and outgrowth included d-glucose, d-mannose, and d-xylose. Sugars that triggered germination but did not support subsequent outgrowth included d-tagatose, d-lyxose, and 2-deoxy-d-glucose. Nontriggering sugars included d-galactose, l-glucose, and d-arabinose. Certain nontriggering sugars, including d-galactose, supported outgrowth if added in the presence of a complementary triggering sugar. This division of functions indicates that sugars are involved in two separate events in germination, triggering and subsequent outgrowth, and the structural features of sugars that support each, both, or none of these events are discussed. We also present data on the uptake of sugars during the germination process and discuss possible mechanisms of triggering in the absence of apparent sugar uptake during the initial swelling of conidia.

Investigating the physiological response of Pichia (Komagataella) pastoris GS115 to the heterologous expression of misfolded proteins using chemostat cultures.

Pichia pastoris is widely used as a host system for heterologous protein expression in both academia and industry. Production is typically accomplished by a fed-batch induction process that is known to have negative impacts on cell physiology that impose limits on both protein yields and quality. We have analysed recombinant protein production in chemostat cultures to understand the physiological responses associated with methanol-induced production of two human lysozyme variants with different degrees of misfolding by P. pastoris. Confounding variables associated with nutrient stress or growth-rate are minimised during steady-state growth in chemostats. Comparison of transcriptome-level data obtained during the non-inducing and inducing steady states identified changes in expression of only about 1 % of the genome during production of either an amyloidogenic human lysozyme variant prone to intracellular aggregation (I56T) or a misfolded but secretable variant (T70N), indicating near-complete acclimation to their production. A marked, but temporary, stress response involving both the unfolded protein response (UPR) and ER-associated degradation pathway was observed during the transient between steady states, particularly following induction of the T70N variant synthesis, and was accompanied by changes in expression of around 50 antisense transcripts. The results suggest that optimal heterologous protein production could best be achieved by a continuous process that minimises the number of methanol-induced transients experienced by the cultures. The processing of HAC1 mRNA required for the UPR was found to be constitutive in the culture conditions used, even in the absence of recombinant protein induction.

Heterologous expression and structural characterization of two low pH laccases from a biopulping white-rot fungus Physisporinus rivulosus.

The lignin-degrading, biopulping white-rot fungus Physisporinus rivulosus secretes several laccases of distinct features such as thermostability, extremely low pH optima and thermal activation for oxidation of phenolic substrates. Here we describe the cloning, heterologous expression and structural and enzymatic characterisation of two previously undescribed P. rivulosus laccases. The laccase cDNAs were expressed in the methylotrophic yeast Pichia pastoris either with the native or with Saccharomyces cerevisiae α-factor signal peptide. The specific activity of rLac1 and rLac2 was 5 and 0.3 µkat/µg, respectively. However, mutation of the last amino acid in the rLac2 increased the specific laccase activity by over 50-fold. The recombinant rLac1 and rLac2 enzymes demonstrated low pH optima with both 2,6-dimethoxyphenol (2,6-DMP) and 2,2'-azino-bis(3-ethylbenzathiazoline-6-sulfonate). Both recombinant laccases showed moderate thermotolerance and thermal activation at +60 °C was detected with rLac1. By homology modelling, it was deduced that Lac1 and Lac2 enzymes demonstrate structural similarity with the Trametes versicolor and Trametes trogii laccase crystal structures. Comparison of the protein architecture at the reducing substrate-binding pocket near the T1-Cu site indicated the presence of five amino acid substitutions in the structural models of Lac1 and Lac2. These data add up to our previous reports on laccase production by P. rivulosus during biopulping and growth on Norway spruce. Heterologous expression of the novel Lac1 and Lac2 isoenzymes in P. pastoris enables the detailed study of their properties and the evaluation of their potential as oxidative biocatalysts for conversion of wood lignin, lignin-like compounds and soil-polluting xenobiotics.

Evaluating the potential of natural product combinations with sorbic acid for improving preservative action against food-spoilage yeasts.

Fungal control methods commonly involve the use of antifungals or preservatives, which can raise concerns about broader effects of these stressors on non-target organisms, spread of resistance and regulatory hurdles. Consequently, control methods enabling lower usage of such stressors are highly sought, for example chemical combinations that synergistically inhibit target-organisms. Here, we investigated how well such a principle extends to improving efficacy of an existing but tightly controlled food preservative, sorbic acid. A screen of ∼200 natural products for synergistic fungal inhibition in combinations with sorbic acid, in either 2% or 0.1% (w/v) glucose to simulate high or reduced-sugar foods, did not reveal reproducible synergies in either of the spoilage yeast species Saccharomyces cerevisiae or Zygosaccharomyces bailii. Potentially promising screen candidates (e.g. lactone parthenolide, ethyl maltol) or a small additional panel of rationally-selected compounds (e.g. benzoic acid) all gave Fractional Inhibitory Concentration Indices (FICI) ≥ 0.5 in combinations with sorbic acid, corroborating absence of synergy in either glucose condition (although FICI values did differ between the glucose conditions). Synergies were not achieved either in a tripartite combination with screen candidates or in a soft-drink formulation as matrix. In previous work with other stressors synergy 'hits' have been comparatively frequent, suggesting that sorbic acid could be unusually resistant to forming synergies with other potential inhibitors and this may relate to the weak acid's known multifactorial inhibitory-actions on cells. The study highlights a challenge in developing appropriate natural product or other chemical combinations applicable to food and beverage preservation.

Adaptation to sorbic acid in low sugar promotes resistance of yeast to the preservative.

The weak acid sorbic acid is a common preservative used in soft drink beverages to control microbial spoilage. Consumers and industry are increasingly transitioning to low-sugar food formulations, but potential impacts of reduced sugar on sorbic acid efficacy are barely characterised. In this study, we report enhanced sorbic acid resistance of yeast in low-glucose conditions. We had anticipated that low glucose would induce respiratory metabolism, which was shown previously to be targeted by sorbic acid. However, a shift from respiratory to fermentative metabolism upon sorbic acid exposure of Saccharomyces cerevisiae was correlated with relative resistance to sorbic acid in low glucose. Fermentation-negative yeast species did not show the low-glucose resistance phenotype. Phenotypes observed for certain yeast deletion strains suggested roles for glucose signalling and repression pathways in the sorbic acid resistance at low glucose. This low-glucose induced sorbic acid resistance was reversed by supplementing yeast cultures with succinic acid, a metabolic intermediate of respiratory metabolism (and a food-safe additive) that promoted respiration. The results indicate that metabolic adaptation of yeast can promote sorbic acid resistance at low glucose, a consideration for the preservation of foodstuffs as both food producers and consumers move towards a reduced sugar landscape.

Genome-wide expression analysis upon constitutive activation of the HacA bZIP transcription factor in Aspergillus niger reveals a coordinated cellular response to counteract ER stress.

BACKGROUND: HacA/Xbp1 is a conserved bZIP transcription factor in eukaryotic cells which regulates gene expression in response to various forms of secretion stress and as part of secretory cell differentiation. In the present study, we replaced the endogenous hacA gene of an Aspergillus niger strain with a gene encoding a constitutively active form of the HacA transcription factor (HacACA). The impact of constitutive HacA activity during exponential growth was explored in bioreactor controlled cultures using transcriptomic analysis to identify affected genes and processes. RESULTS: Transcription profiles for the wild-type strain (HacAWT) and the HacACA strain were obtained using Affymetrix GeneChip analysis of three replicate batch cultures of each strain. In addition to the well known HacA targets such as the ER resident foldases and chaperones, GO enrichment analysis revealed up-regulation of genes involved in protein glycosylation, phospholipid biosynthesis, intracellular protein transport, exocytosis and protein complex assembly in the HacACA mutant. Biological processes over-represented in the down-regulated genes include those belonging to central metabolic pathways, translation and transcription. A remarkable transcriptional response in the HacACA strain was the down-regulation of the AmyR transcription factor and its target genes. CONCLUSIONS: The results indicate that the constitutive activation of the HacA leads to a coordinated regulation of the folding and secretion capacity of the cell, but with consequences on growth and fungal physiology to reduce secretion stress.

Presence and functionality of mating type genes in the supposedly asexual filamentous fungus Aspergillus oryzae.

The potential for sexual reproduction in Aspergillus oryzae was assessed by investigating the presence and functionality of MAT genes. Previous genome studies had identified a MAT1-1 gene in the reference strain RIB40. We now report the existence of a complementary MAT1-2 gene and the sequencing of an idiomorphic region from A. oryzae strain AO6. This allowed the development of a PCR diagnostic assay, which detected isolates of the MAT1-1 and MAT1-2 genotypes among 180 strains assayed, including industrial tane-koji isolates. Strains used for sake and miso production showed a near-1:1 ratio of the MAT1-1 and MAT1-2 mating types, whereas strains used for soy sauce production showed a significant bias toward the MAT1-2 mating type. MAT1-1 and MAT1-2 isogenic strains were then created by genetic manipulation of the resident idiomorph, and gene expression was compared by DNA microarray and quantitative real-time PCR (qRT-PCR) methodologies under conditions in which MAT genes were expressed. Thirty-three genes were found to be upregulated more than 10-fold in either the MAT1-1 host strain or the MAT1-2 gene replacement strain relative to each other, showing that both the MAT1-1 and MAT1-2 genes functionally regulate gene expression in A. oryzae in a mating type-dependent manner, the first such report for a supposedly asexual fungus. MAT1-1 expression specifically upregulated an α-pheromone precursor gene, but the functions of most of the genes affected were unknown. The results are consistent with a heterothallic breeding system in A. oryzae, and prospects for the discovery of a sexual cycle are discussed.

Sequencing of Aspergillus nidulans and comparative analysis with A. fumigatus and A. oryzae.

The aspergilli comprise a diverse group of filamentous fungi spanning over 200 million years of evolution. Here we report the genome sequence of the model organism Aspergillus nidulans, and a comparative study with Aspergillus fumigatus, a serious human pathogen, and Aspergillus oryzae, used in the production of sake, miso and soy sauce. Our analysis of genome structure provided a quantitative evaluation of forces driving long-term eukaryotic genome evolution. It also led to an experimentally validated model of mating-type locus evolution, suggesting the potential for sexual reproduction in A. fumigatus and A. oryzae. Our analysis of sequence conservation revealed over 5,000 non-coding regions actively conserved across all three species. Within these regions, we identified potential functional elements including a previously uncharacterized TPP riboswitch and motifs suggesting regulation in filamentous fungi by Puf family genes. We further obtained comparative and experimental evidence indicating widespread translational regulation by upstream open reading frames. These results enhance our understanding of these widely studied fungi as well as provide new insight into eukaryotic genome evolution and gene regulation.

Genomic sequence of the pathogenic and allergenic filamentous fungus Aspergillus fumigatus.

Aspergillus fumigatus is exceptional among microorganisms in being both a primary and opportunistic pathogen as well as a major allergen. Its conidia production is prolific, and so human respiratory tract exposure is almost constant. A. fumigatus is isolated from human habitats and vegetable compost heaps. In immunocompromised individuals, the incidence of invasive infection can be as high as 50% and the mortality rate is often about 50% (ref. 2). The interaction of A. fumigatus and other airborne fungi with the immune system is increasingly linked to severe asthma and sinusitis. Although the burden of invasive disease caused by A. fumigatus is substantial, the basic biology of the organism is mostly obscure. Here we show the complete 29.4-megabase genome sequence of the clinical isolate Af293, which consists of eight chromosomes containing 9,926 predicted genes. Microarray analysis revealed temperature-dependent expression of distinct sets of genes, as well as 700 A. fumigatus genes not present or significantly diverged in the closely related sexual species Neosartorya fischeri, many of which may have roles in the pathogenicity phenotype. The Af293 genome sequence provides an unparalleled resource for the future understanding of this remarkable fungus.

Genome sequencing and analysis of Aspergillus oryzae.

The genome of Aspergillus oryzae, a fungus important for the production of traditional fermented foods and beverages in Japan, has been sequenced. The ability to secrete large amounts of proteins and the development of a transformation system have facilitated the use of A. oryzae in modern biotechnology. Although both A. oryzae and Aspergillus flavus belong to the section Flavi of the subgenus Circumdati of Aspergillus, A. oryzae, unlike A. flavus, does not produce aflatoxin, and its long history of use in the food industry has proved its safety. Here we show that the 37-megabase (Mb) genome of A. oryzae contains 12,074 genes and is expanded by 7-9 Mb in comparison with the genomes of Aspergillus nidulans and Aspergillus fumigatus. Comparison of the three aspergilli species revealed the presence of syntenic blocks and A. oryzae-specific blocks (lacking synteny with A. nidulans and A. fumigatus) in a mosaic manner throughout the genome of A. oryzae. The blocks of A. oryzae-specific sequence are enriched for genes involved in metabolism, particularly those for the synthesis of secondary metabolites. Specific expansion of genes for secretory hydrolytic enzymes, amino acid metabolism and amino acid/sugar uptake transporters supports the idea that A. oryzae is an ideal microorganism for fermentation.