The effects of fluctuating culture temperature on stress tolerance and antioxidase expression in Esteya vermicola.

The endoparasitic nematophagous fungus, Esteya vermicola, has shown great potential as a biological control agent against the pine wood nematode, Bursaphelenchus xylophilus. Fluctuating culture temperatures can affect fungal yields and fungal tolerance to desiccation, UV radiation, H2O2, and heat stress, as well as antioxidase expression. To explore these effects, E. vermicola cultured under five temperature ranges, 26°C, 15-26°C, 26-35°C, 20-30°C, and 15-35°C, were compared. The cultures grown at lower temperatures showed better growth, stronger tolerance to desiccation, UV, and H2O2 stresses, and increased catalase expression, However, these cultures also showed weaker heat stress tolerance and lower superoxide dismutase expression than the higher-temperature cultures. In particular, the E. vermicola cultured at 20-30°C, i.e., fluctuating in a narrow range around the optimal temperature, showed the best performance. Therefore, for production in practical applications, this narrowly fluctuating, moderate temperature appears to be optimal for yield and stress tolerance in E. vermicola.

Gene discovery for enzymes involved in limonene modification or utilization by the mountain pine beetle-associated pathogen Grosmannia clavigera.

To successfully colonize and eventually kill pine trees, Grosmannia clavigera (Gs cryptic species), the main fungal pathogen associated with the mountain pine beetle (Dendroctonus ponderosae), has developed multiple mechanisms to overcome host tree chemical defenses, of which terpenoids are a major component. In addition to a monoterpene efflux system mediated by a recently discovered ABC transporter, Gs has genes that are highly induced by monoterpenes and that encode enzymes that modify or utilize monoterpenes [especially (+)-limonene]. We showed that pine-inhabiting Ophiostomale fungi are tolerant to monoterpenes, but only a few, including Gs, are known to utilize monoterpenes as a carbon source. Gas chromatography-mass spectrometry (GC-MS) revealed that Gs can modify (+)-limonene through various oxygenation pathways, producing carvone, p-mentha-2,8-dienol, perillyl alcohol, and isopiperitenol. It can also degrade (+)-limonene through the C-1-oxygenated pathway, producing limonene-1,2-diol as the most abundant intermediate. Transcriptome sequencing (RNA-seq) data indicated that Gs may utilize limonene 1,2-diol through beta-oxidation and then valine and tricarboxylic acid (TCA) metabolic pathways. The data also suggested that at least two gene clusters, located in genome contigs 108 and 161, were highly induced by monoterpenes and may be involved in monoterpene degradation processes. Further, gene knockouts indicated that limonene degradation required two distinct Baeyer-Villiger monooxygenases (BVMOs), an epoxide hydrolase and an enoyl coenzyme A (enoyl-CoA) hydratase. Our work provides information on enzyme-mediated limonene utilization or modification and a more comprehensive understanding of the interaction between an economically important fungal pathogen and its host's defense chemicals.

Influence of temperature, pH and metal ions on guaiacol oxidation of purified laccase from Leptographium qinlingensis.

The bark beetle Dendroctonus armandi is able to kill living Pinus armandi and has caused serious damage to pine forest in Northern China. As the most important symbiotic fungus of D. armandi, Leptographium qinlingensis plays an important role in the invasion process of the bark beetle. The laccase secreted by it are involved in lignin degradation to provide utilizable nutrition for D. armandi, and catalyze some biochemical reactions, causing the damages of tree tissue. In present study, the extracellular laccase of L. qinlingensis was purified by using the ammonium sulfate precipitation and DEAE-cellulose (DE-52) column chromatography. Furthermore, the effects of temperature, pH value and metal ions on it were investigated and characterized. The purified enzyme exerted its optimal activity with guaiacol. The catalytic efficiencies K(m) and V(max) determined for substrate guaiacol were 15.4 µM and 372.9 IU mg⁻¹, respectively. The optimum pH and temperature for the purified enzyme was 4.4 and 45 °C, respectively, with the highest enzyme specific activity of 7,000 IU mg⁻¹. Moreover, the metal ions, Co²âº, Mn²âº, Ca²âº, Mg²âº, Fe²âº and Cd²âº, especially Hg²âº, showed significantly inhibition effects on its activity. To understand the characteristics of this laccase might provide an opportunity and theoretical basis to promote integrated pest management of D. armandi.

Microbial transformation of ginsenoside Rg3 to ginsenoside Rh2 by Esteya vermicola CNU 120806.

In the present investigation, we successfully employed a cell-free extract of Esteya vermicola CNU 120806 to convert ginsenoside Rg3 to Rh2. Three important factors including pH, temperature and substrate concentration were optimized for the preparation of Rh(2). The optimal condition was obtained as follows: 50°C, pH 5.0 and substrate concentration of 3 mg ml(-1). The yield of conversion was up to 90.7%. In order to identify the specificity of the ß-glucosidase activity of Esteya vermicola CNU 120806, ginsenoside Re (protopanaxatriol saponins) was treated under the same reaction system. Interestingly, no new metabolite was generated, which elucidated that the enzymatic process only occurred by hydrolysis of the terminal glucopyranosyl moieties at the C-3 carbon of ginsenoside Rg(3). The crude enzyme extract can be used for commercial ginsenoside Rh(2) preparation.

Highly selective microbial transformation of major ginsenoside Rb1 to gypenoside LXXV by Esteya vermicola CNU120806.

AIMS: This study examined the biotransformation pathway of ginsenoside Rb(1) by the fungus Esteya vermicola CNU 120806. METHODS AND RESULTS: Ginsenosides Rb(1) and Rd were extracted from the root of Panax ginseng. Liquid fermentation and purified enzyme hydrolysis were employed to investigate the biotransformation of ginsenoside Rb(1) . The metabolites were identified and confirmed using NMR analysis as gypenoside XVII and gypenoside LXXV. A mole yield of 95·4% gypenoside LXXV was obtained by enzymatic conversion (pH 5·0, temperature 50°C). Ginsenoside Rd was used to verify the transformation pathway under the same reaction condition. The product Compound K (mole yield 49·6%) proved a consecutive hydrolyses occurred at the C-3 position of ginsenoside Rb(1) . CONCLUSIONS: Strain CNU 120806 showed a high degree of specific ß-glucosidase activity to convert ginsenosides Rb(1) and Rd to gypenoside LXXV and Compound K, respectively. The maximal activity of the purified glucosidase for ginsenosides transformation occurred at 50°C and pH 5·0. Compared with its activity against pNPG (100%), the ß-glucosidase exhibited quite lower level of activity against other aryl-glycosides. Enzymatic hydrolysate, gypenoside LXXV and Compound K were produced by consecutive hydrolyses of the terminal and inner glucopyranosyl moieties at the C-3 carbon of ginsenoside Rb(1) and Rd, giving the pathway: ginsenoside Rb(1) → gypenoside XVII → gypenoside LXXV; ginsenoside Rd→F(2) →Compound K, but did not hydrolyse the 20-C, ß-(1-6)-glucoside of ginsenoside Rb(1) and Rd. SIGNIFICANCE AND IMPACT OF THE STUDY: The results showed an important practical application on the preparation of gypenoside LXXV. Additionally, this study for the first time provided a high efficient preparation method for gypenoside LXXV without further conversion, which also gives rise to a potential commercial enzyme application.

A group II intron encodes a functional LAGLIDADG homing endonuclease and self-splices under moderate temperature and ionic conditions.

A group II intron encoding a protein belonging to the LAGLIDADG family of homing endonucleases was identified in the mitochondrial rns gene of the filamentous fungus Leptographium truncatum, and the catalytic activities of both the intron and its encoded protein were characterized. A model of the RNA secondary structure indicates that the intron is a member of the IIB1 subclass and the open reading frame is inserted in ribozyme domain III. In vitro assays carried out with two versions of the intron, one in which the open reading frame was removed and the other in which it was present, demonstrate that both versions of the intron readily self-splice at 37 degrees C and at a concentration of MgCl(2) as low as 6 mM. The open reading frame encodes a functional LAGLIDADG homing endonuclease that cleaves 2 (top strand) and 6 (bottom strand) nucleotides (nt) upstream of the intron insertion site, generating 4 nt 3' OH overhangs. In vitro splicing assays carried out in the absence and presence of the intron-encoded protein indicate that the protein does not enhance intron splicing, and RNA-binding assays show that the protein does not appear to bind to the intron RNA precursor transcript. These findings raise intriguing questions concerning the functional and evolutionary relationships of the two components of this unique composite element.