George Szakacs

Evidence for sexuality in the opportunistic fungal pathogen Aspergillus fumigatus

Aspergillus fumigatus is a medically important opportunistic pathogen and a major cause of respiratory allergy. The species has long been considered an asexual organism. However, genome analysis has revealed the presence of genes associated with sexual reproduction, including a MAT-2 high-mobility group mating-type gene and genes for pheromone production and detection (Galagan et al., personal communication; Nierman et al., personal communication). We now demonstrate that A. fumigatus has other key characteristics of a sexual species. We reveal the existence of isolates containing a complementary MAT-1 alpha box mating-type gene and show that the MAT locus has an idiomorph structure characteristic of heterothallic (obligate sexual outbreeding) fungi. Analysis of 290 worldwide clinical and environmental isolates with a multiplex-PCR assay revealed the presence of MAT1-1 and MAT1-2 genotypes in similar proportions (43% and 57%, respectively). Further population genetic analyses provided evidence of recombination across a global sampling and within North American and European subpopulations. We also show that mating-type, pheromone-precursor, and pheromone-receptor genes are expressed during mycelial growth. These results indicate that A. fumigatus has a recent evolutionary history of sexual recombination and might have the potential for sexual reproduction. The possible presence of a sexual cycle is highly significant for the population biology and disease management of the species.

Production, purification and properties of microbial phytases

Phytases (myo-inositol hexakisphosphate phosphohydrolase, EC 3.1.3.8) catalyse the release of phosphate from phytate (mycoinositol hexakiphosphate). Several cereal grains, legumes and oilseeds, etc., store phosphorus as phytate. Environmental pollution due to the high-phosphate manure, resulting in the accumulation of P at various locations has raised serious concerns. Phytases appear of significant value in effectively controlling P pollution. They can be produced from a host of sources including plants, animals and micro-organisms. Microbial sources, however, are promising for their commercial exploitations. Strains of Aspergillus sp., chiefly A. ficuum and A. niger have most commonly been employed for industrial purposes. Phytases are considered as a monomeric protein, generally possessing a molecular weight between 40 and 100 kDa. They show broad substrate specificity and have generally pH and temperature optima around 4.5-6.0 and 45-60 degrees C. The crystal structure of phytase has been determined at 2.5 A resolution. Immobilization of phytase has been found to enhance its thermostability. This article reviews recent trends on the production, purification and properties of microbial phytases.

Phylogeny and evolution of the genus Trichoderma: a multigene approach

The phylogeny of Trichoderma and the phylogenetic relationships of its species was investigated by maximum parsimony analysis and distance analysis of DNA sequences from multiple genetic loci. 18S rDNA sequence analysis suggests that the genus Trichoderma evolved at the same time as Hypomyces and Fusarium and thus about 110 Myr ago. 28S rDNA sequence analysis shows that the genus Trichoderma is part of a monophyletic branch within the Hypocreaceae , which also includes Arachnocrea and Aphysiostroma in basal positions. Gene trees inferred from a combined analysis of the nuclear ribosomal internal transcribed spacer (ITS1 and 2), the D1 and D2 region of the 28S rDNA, the small subunit of the mitochondrial rDNA (mitSSU), the fifth and part of the sixth exon of translation elongation factor 2 ( tef1 ), and a fragment of ech42 provide strong statistical support for a phylogeny consistent with the existence of four clades: clade A comprises species of Bissetts (1991) sect. Trichoderma but also T. hamatum, T. pubescens, T. asperellum , and T. strigosum ; clade C comprises all the species contained in section Longibrachiatum as revised by Samuels et al. (1998), and clade D contains only T. aureoviride which is genetically most distant to all other species. Clade B, on the other hand, contains a large and taxonomically heterogeneous mixture of species, among which several subclades could be identified: subclade B1 containing H. lactea, H. citrina, H. citrina var. americana, H. lutea , and an unnamed T. sp. 1; subclade B2 containing T. stromaticum , and an unnamed T. sp. PPRI3559; subclade B3 containing T. fertile, T. oblongisporum , and H. hunua ; subclade B5 containing T. polysporum, T. croceum, Hypocrea pilulifera , and T. minutisporum ; and a larger subclade (B4), in which three strain clusters could be distinguished: one comprising T. harzianum, T. inhamatum, H. vinosa , and T. aggressivum , another one containing T. fasciculatum, T. longipile , and T. strictipile ; and a third containing T. virens, T. flavofuscum , and T. crassum. The position of the remaining species of subclade B4 ( T. spirale, T. cfr aureoviride, H. tawa, and T. tomentosum ) was not resolved. A comparison of the topologies of the individual gene trees was concordant with the topology of the combined tree in most cases, but also revealed incongruent positions for a few species ( T. oblongisporum, T. longipile, T. fasciculatum ) which was most pronounced in the ITS1 and 2 tree. The results confirm the recent concept for sects Longibrachiatum and Trichoderma , indicate that the sects Hypocreanum and Pachybasium cannot be distinguished phylogenetically, and provide a first phylogenetic basis for dissection of the latter two sections.

Genetic and metabolic diversity of Trichoderma: a case study on South-East Asian isolates

We have used isolates of Trichoderma spp. collected in South-East Asia, including Taiwan and Western Indonesia, to assess the genetic and metabolic diversity of endemic species of Trichoderma. Ninety-six strains were isolated in total, and identified at the species level by analysis of morphological and biochemical characters (Biolog system), and by sequence analysis of their internal transcribed spacer regions 1 and 2 (ITS1 and 2) of the rDNA cluster, using ex-type strains and taxonomically established isolates of Trichoderma as reference. Seventy-eight isolates were positively identified as Trichoderma harzianum/Trichoderma inhamatum (37 strains) Trichoderma virens (16 strains), Trichoderma spirale (8 strains), Trichoderma koningii (3 strains), Trichoderma atroviride (3 strains), Trichoderma asperellum (4 strains), Hypocrea jecorina (anamorph: Trichoderma reesei; 2 strains), Trichoderma viride (2 strains), Trichoderma hamatum (1 strain), and Trichoderma ghanense (1 strain). Analysis of biochemical characters revealed that T. virens, T. spirale, T. asperellum, T. koningii, H. jecorina, and T. ghanense formed clearly defined clusters, thus exhibiting species-specific metabolic properties. In biochemical character analysis T. atroviride and T. viride formed partially overlapping clusters, indicating that these two species may share overlapping metabolic characteristics. This behavior was even more striking with T. harzianum/T. inhamatum where genotypes defined on the basis of ITS1 and 2 sequences overlapped significantly with adjacent genotypes in the biochemical character analysis, and four strains from the same location (Bali, Indonesia) even clustered with species from section Longibrachiatum. The data indicate that the T. harzianum/T. inhamatum group represents species with high metabolic diversity and partially unique metabolic characteristics. Nineteen strains yielded three different ITS1/2 sequence types which were not alignable with any known species. They were also uniquely characterized by morphological and biochemical characters and therefore represent three new taxa of Trichoderma.

Comparative enzymatic hydrolysis of pretreated spruce by supernatants, whole fermentation broths and washed mycelia of Trichoderma reesei and Trichoderma atroviride

Cellulase and beta-glucosidase production on steam pretreated spruce (SPS) with Trichoderma reesei Rut C30, Trichoderma atroviride TUB F-1505 and TUB F-1663 was investigated. The enzymes were compared in term of activity, temperature optima and hydrolytic capacity. The T. atroviride cellulases proved to have lower temperature optima for filter paper activity (FPA) assay (50 degrees C) and for hydrolysis of SPS (40 degrees C) than the Rut C30 enzymes (60 degrees C for FPA and 50 degrees C for hydrolysis). Due to high levels of extracellular beta-glucosidases, the T. atroviride enzyme supernatants hydrolyzed the washed SPS to glucose more efficiently than the enzymes produced by T. reesei. On the other hand, when the whole fermentation broths were used instead of the supernatants, thus the mycelium bound enzymes were also present, the hydrolytic capacity of T. reesei Rut C30 was enhanced by approximately 200%, while an improvement of only 15% was observed in case of the T. atroviride isolates.

Enzymatic hydrolysis of steam-pretreated lignocellulosic materials with Trichoderma atroviride enzymes produced in-house

BackgroundImprovement of the process of cellulase production and development of more efficient lignocellulose-degrading enzymes are necessary in order to reduce the cost of enzymes required in the biomass-to-bioethanol process.ResultsLignocellulolytic enzyme complexes were produced by the mutant Trichoderma atroviride TUB F-1663 on three different steam-pretreated lignocellulosic substrates, namely spruce, wheat straw and sugarcane bagasse. Filter paper activities of the enzymes produced on the three materials were very similar, while β-glucosidase and hemicellulase activities were more dependent on the nature of the substrate. Hydrolysis of the enzyme preparations investigated produced similar glucose yields. However, the enzymes produced in-house proved to degrade the xylan and the xylose oligomers less efficiently than a commercial mixture of cellulase and β-glucosidase. Furthermore, accumulation of xylose oligomers was observed when the TUB F-1663 supernatants were applied to xylan-containing substrates, probably due to the low β-xylosidase activity of the enzymes. The efficiency of the enzymes produced in-house was enhanced by supplementation with extra commercial β-glucosidase and β-xylosidase. When the hydrolytic capacities of various mixtures of a commercial cellulase and a T. atroviride supernatant produced in the lab were investigated at the same enzyme loading, the glucose yield appeared to be correlated with the β-glucosidase activity, while the xylose yield seemed to be correlated with the β-xylosidase level in the mixtures.ConclusionEnzyme supernatants produced by the mutant T. atroviride TUB F-1663 on various pretreated lignocellulosic substrates have good filter paper activity values combined with high levels of β-glucosidase activities, leading to cellulose conversion in the enzymatic hydrolysis that is as efficient as with a commercial cellulase mixture. On the other hand, in order to achieve good xylan conversion, the supernatants produced by the mutant have to be supplemented with additional β-xylosidase activity.

Alpha amylase from a fungal culture grown on oil cakes and its properties

Fermentacao no Estado Solido foi empregada na producao de alfa-amilase usando Aspergillus niger. Diferentes tipos de torta foram utilizadas, como torta de oleo de coco (COC), torta de de oleo de amendoim (GOC) torta de oleo de sesamo (SOC), torta de palma (PKC) e torta de oleo de oliva (OOC) foram selecionadas para serem usadas como substratos para producao de enzima e comparadas com o farelo de trigo (WB), GOC foi escolhido por ser o que produziu maiores concentracoes de enzima. A combinacao WB e GOC (1:1) resultou em maiores titulos da enzima quando em comparacao com os substratos individuais. A maxima concentracao de enzima (9196 U/ gms) foi obtida quando a FES foi conduzida utilizando WB + GOC, com umidade de 64% e suplementada com lactose e nitrato de amonia (1% cada) a 300C por 72 horas utilizando 2 mL de uma suspensao de esporo (6x107sporos/ml). A purificacao parcial da enzima usando fracoes de sulfato de amonio resultou num aumento de 2-4 vezes o aumento da atividade. A enzima apresentou um peso molecular de 68 Kda pelo SDS_PAGE. Exceto Mn, todos os outros ions metalicos como Ca, K, Na, Mg sao inibitorios na producao da enzima.

Low Genetic Variation and No Detectable Population Structure in Aspergillus fumigatus Compared to Closely Related Neosartorya Species

ABSTRACT Aspergillus fumigatus is an anamorphic euascomycete mold with a ubiquitous presence worldwide. Despite intensive work to understand its success as a pathogen infecting immunosuppressed patients, the population dynamics and recent evolutionary history of A. fumigatus remain understudied. We examined patterns of genetic variation at three intergenic loci for 70 natural isolates from Europe, North America, South America, Asia, Africa, and Australia. The same loci were used to analyze within-population genetic variation for 33 isolates obtained from five geographic locations. Neither data set detected evidence of population differentiation or found any association between the genetic and geographic distances among these isolates. No evidence for genetic differentiation within the two A. fumigatus mating types was detected. The genetic diversity of A. fumigatus, contrasted with that of its close teleomorphic relatives, Neosartorya fischeri and Neosartorya spinosa, is remarkably low.

Production of phytase by Mucor racemosus in solid-state fermentation.

Phytase production was studied by three Mucor and eight Rhizopus strains by solid‐state fermentation (SSF) on three commonly used natural feed ingredients (canola meal, coconut oil cake, wheat bran). Mucor racemosusNRRL 1994 (ATCC 46129) gave the highest yield (14.5 IU/g dry matter phytase activity) on coconut oil cake. Optimizing the supplementation of coconut oil cake with glucose, casein and (NH4)2SO4, phytase production in solid‐state fermentation was increased to 26 IU/g dry matter (DM). Optimization was carried out by Plackett‐Burman and central composite experimental designs. Using the optimized medium phytase, α‐amylase and lipase production of Mucor racemosusNRRL 1994 was compared in solid‐state fermentation and in shake flask (SF) fermentation. SSF yielded higher phytase activity than did SF based on mass of initial substrate. Because this particular isolate is a food‐grade fungus that has been used for sufu fermentation in China, the whole SSF material (crude enzyme, in situ enzyme) may be used directly in animal feed rations with enhanced cost efficiency.

Thermostable phytase production by Thermoascus aurantiacus in submerged fermentation

Phytases act on phytic acid, an antinutrient factor present in animal feeds, and release inorganic phosphate. We optimized the production parameters for phytase production using Thermoascus aurantiacus (TUB F 43), a thermophilic fungal culture, by submerged fermentation. A semisynthetic medium containing glucose, starch, peptone, and minerals supplemented with 3.75% (w/v) wheat bran particles was found to be the best production medium among the various combinations tried. Further supplementation of this medium with surfactants such as Tween-20 and Tween-80 considerably enhanced the enzyme yield. A maximum phytase activity (468.22 U/mL) was obtained using this production medium containing 2% (v/v) Tween-20 after 72 h of fermentation at 45°C in shake-flask cultures with a rotation of 150 rpm. Herein we present details of a few of the process parameter optimizations. The phytase enzyme was found to be thermostable, and the optimal temperature for phytase activity was found to be 55°C. However, 80% of the activity still remained when the temperature was shifted to 70°C.