Aspergillus sydowii: Genome Analysis and Characterization of Two Heterologous Expressed, Non-redundant Xylanases.

A prerequisite for the transition toward a biobased economy is the identification and development of efficient enzymes for the usage of renewable resources as raw material. Therefore, different xylanolytic enzymes are important for efficient enzymatic hydrolysis of xylan-heteropolymers. A powerful tool to overcome the limited enzymatic toolbox lies in exhausting the potential of unexplored habitats. By screening a Vietnamese fungal culture collection of 295 undiscovered fungal isolates, 12 highly active xylan degraders were identified. One of the best xylanase producing strains proved to be an Aspergillus sydowii strain from shrimp shell (Fsh102), showing a specific activity of 0.6 U/mg. Illumina dye sequencing was used to identify our Fsh102 strain and determine differences to the A. sydowii CBS 593.65 reference strain. With activity based in-gel zymography and subsequent mass spectrometric identification, three potential proteins responsible for xylan degradation were identified. Two of these proteins were cloned from the cDNA and, furthermore, expressed heterologously in Escherichia coli and characterized. Both xylanases, were entirely different from each other, including glycoside hydrolases (GH) families, folds, substrate specificity, and inhibition patterns. The specific enzyme activity applying 0.1% birch xylan of both purified enzymes were determined with 181.1 ± 37.8 or 121.5 ± 10.9 U/mg for xylanase I and xylanase II, respectively. Xylanase I belongs to the GH11 family, while xylanase II is member of the GH10 family. Both enzymes showed typical endo-xylanase activity, the main products of xylanase I are xylobiose, xylotriose, and xylohexose, while xylobiose, xylotriose, and xylopentose are formed by xylanase II. Additionally, xylanase II showed remarkable activity toward xylotriose. Xylanase I is stable when stored up to 30°C and pH value of 9, while xylanase II started to lose significant activity stored at pH 9 after exceeding 3 days of storage. Xylanase II displayed about 40% activity when stored at 50°C for 24 h. The enzymes are tolerant toward mesophilic temperatures, while acting in a broad pH range. With site directed mutagenesis, the active site residues in both enzymes were confirmed. The presented activity and stability justify the classification of both xylanases as highly interesting for further development.

A unique fungal strain collection from Vietnam characterized for high performance degraders of bioecological important biopolymers and lipids.

Fungal strains are abundantly used throughout all areas of biotechnology and many of them are adapted to degrade complex biopolymers like chitin or lignocellulose. We therefore assembled a collection of 295 fungi from nine different habitats in Vietnam, known for its rich biodiversity, and investigated their cellulase, chitinase, xylanase and lipase activity. The collection consists of 70 isolates from wood, 55 from soil, 44 from rice straw, 3 found on fruits, 24 from oil environments (butchery), 12 from hot springs, 47 from insects as well as 27 from shrimp shells and 13 strains from crab shells. These strains were cultivated and selected by growth differences to enrich phenotypes, resulting in 211 visually different fungi. DNA isolation of 183 isolates and phylogenetic analysis was performed and 164 species were identified. All were subjected to enzyme activity assays, yielding high activities for every investigated enzyme set. In general, enzyme activity corresponded with the environment of which the strain was isolated from. Therefore, highest cellulase activity strains were isolated from wood substrates, rice straw and soil and similar substrate effects were observed for chitinase and lipase activity. Xylanase activity was similarly distributed as cellulase activity, but substantial activity was also found from fungi isolated from insects and shrimp shells. Seven strains displayed significant activities against three of the four tested substrates, while three degraded all four investigated carbon sources. The collection will be an important source for further studies. Therefore representative strains were made available to the scientific community and deposited in the public collection of the Leibniz-Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig.

The ATF/CREB transcription factor Atf1 is essential for full virulence, deoxynivalenol production, and stress tolerance in the cereal pathogen Fusarium graminearum.

Fusarium graminearum is a necrotrophic plant pathogen of cereals that produces mycotoxins such as deoxynivalenol (DON) and zearalenone (ZEA) in grains. The stress-activated mitogen-activated protein kinase FgOS-2 is a central regulator in F. graminearum and controls, among others, virulence and DON and ZEA production. Here, we characterized the ATF/CREB-activating transcription factor FgAtf1, a regulator that functions downstream of FgOS-2. We created deletion and overexpression mutants of Fgatf1, the latter being also in an FgOS-2 deletion mutant. FgAtf1 localizes to the nucleus and appears to interact with FgOS-2 under osmotic stress conditions. Deletion mutants in Fgatf1 (ΔFgatf1) are more sensitive to osmotic stress and less sensitive to oxidative stress compared with the wild type. Furthermore, sexual reproduction is delayed. ΔFgatf1 strains produced higher amounts of DON under in vitro induction conditions than that of the wild type. However, during wheat infection, DON production by ΔFgatf1 is strongly reduced. The ΔFgatf1 strains displayed strongly reduced virulence to wheat and maize. Interestingly, constitutive expression of Fgatf1 in the wild type led to hypervirulence on wheat, maize, and Brachypodium distachyon. Moreover, constitutive expression of Fgatf1 in the ΔFgOS-2 mutant background almost complements ΔFgOS-2-phenotypes. These data suggest that FgAtf1 may be the most important transcription factor regulated by FgOS-2.