De novo transcriptome sequencing and comprehensive analysis of the heat stress response genes in the basidiomycetes fungus Ganoderma lucidum.

Ganoderma lucidum is a valuable basidiomycete with numerous pharmacological compounds, which is widely consumed throughout China. We previously found that the polysaccharide content of Ganoderma lucidum fruiting bodies could be significantly improved by 45.63% with treatment of 42 °C heat stress (HS) for 2 h. To further investigate genes involved in HS response and explore the mechanisms of HS regulating the carbohydrate metabolism in Ganoderma lucidum, high-throughput RNA-Seq was conducted to analyse the difference between control and heat-treated mycelia at transcriptome level. We sequenced six cDNA libraries with three from control group (mycelia cultivated at 28 °C) and three from heat-treated group (mycelia subjected to 42 °C for 2 h). A total of 99,899 transcripts were generated using Trinity method and 59,136 unigenes were annotated by seven public databases. Among them, 2790 genes were identified to be differential expressed genes (DEGs) under HS condition, which included 1991 up-regulated and 799 down-regulated. 176 DEGs were then manually classified into five main responsive-related categories according to their putative functions and possible metabolic pathways. These groups include stress resistance-related factors; protein assembly, transportation and degradation; signal transduction; carbohydrate metabolism and energy provision-related process; other related functions, suggesting that a series of metabolic pathways in Ganoderma lucidum are activated by HS and the response mechanism involves a complex molecular network which needs further study. Remarkably, 48 DEGs were found to regulate carbohydrate metabolism, both in carbohydrate hydrolysis for energy provision and polysaccharide synthesis. In summary, this comprehensive transcriptome analysis will provide enlarged resource for further investigation into the molecular mechanisms of basidiomycete under HS condition.

Effect of heat stress on production and in-vitro antioxidant activity of polysaccharides in Ganoderma lucidum.

Ganoderma lucidum is a traditional Chinese medicine, and its polysaccharides possess diverse and significant pharmacological activities. This study aimed to investigate the polysaccharide production, molecular characteristics and in-vitro antioxidant activity of G. lucidum fruiting body after the mushroom was harvested and treated with heat stress (HS). HS enhanced the production of polysaccharides after harvest and treatment of 42 °C HS for 2 h, and that resulted in the highest polysaccharide yield of 10.50%, which was 45.63% higher than that of the control, while 37, 45 °C HS had no significant effect on the production. In terms of molecular characteristics, 42 °C HS significantly changed monosaccharide ratio of polysaccharides, but no apparent molecular weight and functional group changes were found in polysaccharides after HS treatment. The results of in-vitro antioxidant activity assay revealed that 42 °C HS significantly improved the antioxidant activities of polysaccharides at the concentration of 2 mg/mL. In conclusion, this study provides a promising strategy to improve the production of G. lucidum fruiting body polysaccharides.

Intraspecific Variation and Phylogenetic Relationships Are Revealed by ITS1 Secondary Structure Analysis and Single-Nucleotide Polymorphism in Ganoderma lucidum.

Ganoderma lucidum is a typical polypore fungus used for traditional Chinese medical purposes. The taxonomic delimitation of Ganoderma lucidum is still debated. In this study, we sequenced seven internal transcribed spacer (ITS) sequences of Ganoderma lucidum strains and annotated the ITS1 and ITS2 regions. Phylogenetic analysis of ITS1 differentiated the strains into three geographic groups. Groups 1-3 were originated from Europe, tropical Asia, and eastern Asia, respectively. While ITS2 could only differentiate the strains into two groups in which Group 2 originated from tropical Asia gathered with Groups 1 and 3 originated from Europe and eastern Asia. By determining the secondary structures of the ITS1 sequences, these three groups exhibited similar structures with a conserved central core and differed helices. While compared to Group 2, Groups 1 and 3 of ITS2 sequences shared similar structures with the difference in helix 4. Large-scale evaluation of ITS1 and ITS2 both exhibited that the majority of subgroups in the same group shared the similar structures. Further Weblogo analysis of ITS1 sequences revealed two main variable regions located in helix 2 in which C/T or A/G substitutions frequently occurred and ITS1 exhibited more nucleotide variances compared to ITS2. ITS1 multi-alignment of seven spawn strains and culture tests indicated that a single-nucleotide polymorphism (SNP) site at position 180 correlated with strain antagonism. The HZ, TK and 203 fusion strains of Ganoderma lucidum had a T at position 180, whereas other strains exhibiting antagonism, including DB, RB, JQ, and YS, had a C. Taken together, compared to ITS2 region, ITS1 region could differentiated Ganoderma lucidum into three geographic originations based on phylogenetic analysis and secondary structure prediction. Besides, a SNP in ITS 1 could delineate Ganoderma lucidum strains at the intraspecific level. These findings will be implemented to improve species quality control in the Ganoderma industry.

Direct cloning, expression of a thermostable xylanase gene from the metagenomic DNA of cow dung compost and enzymatic production of xylooligosaccharides from corncob.

OBJECTIVES: To acquire a thermostable xylanase, that is suitable for xylooligosaccharide production from pretreated corncobs, the metagenomic method was used to obtain the gene from an uncultured environmental microorganism. RESULTS: A thermostable xylanase-encoding gene (xyn10CD18) was cloned directly from the metagenomic DNA of cow dung compost. When xyn10CD18 was expressed in Bacillus megaterium MS941, extracellular xylansae activity at 106 IU/ml was achieved. The purified recombinant Xyn10CD18 was optimally active at pH 7 and 75 °C as measured over 10 min. It retained over 55% of its initial activity at 70 °C and pH 7 after 24 h. Its action on birchwood xylan for 18 h liberated xylooligosaccharides with 2°-4° of polymerization, with xylobiose and xylotetraose as the main products. When pretreated corncobs were hydrolyzed by Xyn10CD18 for 18 h, the xylooligosaccharides (DP 2-4) products increased to 80% and the xylose was just increased by 3%. CONCLUSION: Xyn10CD18 is a thermostable endoxylanase and is a promising candidate for biomass conversion and xylooligosaccharide production.

Purification and characterization of a thermostable xylanase from Paenibacillus sp. NF1 and its application in xylooligosaccharides production.

High levels of extracellular xylanase activity (211.79 IU/mg) produced by Paenibacillus sp. NF1 were detected when it was submerged-cultured. After three consecutive purification steps using Octyl-Sepharose, Sephadex G75, and Q-Sepharose columns, a thermostable xylanase (XynNF) was purified to homogeneity and showed a molecular mass of 37 kDa according to SDS-PAGE. The specific activity of the purified XynNF was up to 3,081.05 IU/mg with a 14.55-fold purification. The activity of XynNF was stimulated by Ca(2+), Ba(2+), DTT, and ß-mercaptoethanol, but was inhibited by Fe(3+), Zn(2+), Fe(2+), Cu(2+), SDS, and EDTA. The purified XynNF displayed a greater affinity for oat spelt xylan with the maximal enzymatic activity at 60°C and pH 6.0. XynNF, which was shown to be cellulose-free, with high stability at high temperature (70°C-80°C) and low pH range (pH 4.0-7.0), is potentially valuable for various industrial applications. The end products of high efficient oat spelt xylan hydrolysis by XynNF (an endoxylanase) containing 95.8% xylooligosaccharides of 2-4 degree of polymerization (DP2-4) with the enrichment of xylobiose (61.5%) indicated that XynNF is a promising candidate for xylooligosaccharides production.

Improvement of alkali stability and thermostability of Paenibacillus campinasensis Family-11 xylanase by directed evolution and site-directed mutagenesis.

The extreme process condition of high temperature and high alkali limits the applications of most of natural xylanases in pulp and paper industry. Recently, various methods of protein engineering have been used to improve the thermal and alkalic tolerance of xylanases. In this work, directed evolution and site-directed mutagenesis were performed to obtain a mutant xylanase improved both on alkali stability and thermostability from the native Paenibacillus campinasensis Family-11 xylanase (XynG1-1). Mutant XynG1-1B43 (V90R/P172H) with two units increased in the optimum pH (pH 7.0-pH 9.0) and significant improvement on alkali stability was selected from the second round of epPCR library. And the further thermoduric mutant XynG1-1B43cc16 (V90R/P172H/T84C-T182C/D16Y) with 10 °C increased in the optimum temperature (60-70 °C) was then obtained by introducing a disulfide bridge (T84C-T182C) and a single amino acid substitution (D16Y) to XynG1-1B43 using site-directed mutagenesis. XynG1-1B43cc16 also showed higher thermostability and catalytic efficiency (k cat /K m ) than that of wild-type (XynG1-1) and XynG1-1B43. The attractive improved properties make XynG1-1B43cc16 more suitable for bioleaching of cotton stalk pulp under the extreme process condition of high temperature (70 °C) and high alkali (pH 9.0).

Flow-through pretreatment with strongly acidic electrolyzed water for hemicellulose removal and enzymatic hydrolysis of corn stover.

The application of strongly acidic electrolyzed water (SAEW) in a flow-through system for the treatment of recalcitrant corn stover resulted in enhanced hemicellulose degradation at 160°C and 180°C compared to hydrochloric acid treatment under the same pH. Pretreatment conditions were optimized by varying the four main factors: pH, flow rate, temperature, and reaction time. About 96% of the hemicellulose was removed when corn stover was treated at 180°C for 20min, with SAEW at pH 2.0 and a flow rate of 10mL/min. The recovery of total monomeric and oligomeric xylose in the liquid fraction reached 93%. Using 30 filter paper units of cellulase per gram of cellulose, a digestibility of 100% was achieved under these conditions. Therefore, flow-through SAEW pretreatment is an efficient way to remove hemicellulose and improve enzymatic digestibility of corn stover.

Corn stover pretreatment by inorganic salts and its effects on hemicellulose and cellulose degradation.

Inorganic salts, NaCl, KCl, CaCl(2), MgCl(2), FeCl(2), FeSO(4), FeCl(3), and Fe(2)(SO(4))(3), were studied as catalysts for the degradation of hemicellulose in corn stover. FeCl(3) significantly increased the hemicellulose degradation in aqueous solutions heated between 140 and 200 degrees C with high xylose recovery and low cellulose removal, amounting to approximately 90% and <10%, respectively. Hemicellulose removal increased 11-fold when the corn stover was pretreated with 0.1 M FeCl(3) compared to pretreatment with hot water under otherwise the same conditions, which was also 6-fold greater than pretreatment with dilute sulfuric acid at the same pH. Optimum pretreatment conditions were found where the corn stover was pretreated with 0.1 M FeCl(3) at 140 degrees C for 20 min. Under such conditions, 91% of hemicellulose was removed, and the recovery of monomeric and oligomeric xylose in liquid fraction achieved 89%, meanwhile, only 9% of cellulose was removed.

Enhanced enzymatic hydrolysis and structural features of corn stover by FeCl3 pretreatment.

Corn stover was pretreated with FeCl(3) to remove almost all of the hemicellulose present and then hydrolyzed with cellulase and beta-glucosidase to produce glucose. Enzymatic hydrolysis of corn stover that had been pretreated with FeCl(3) at 160 degrees C for 20 min resulted in an optimum yield of 98.0%. This yield was significantly higher than that of untreated corn stover (22.8%). FeCl(3) pretreatment apparently damaged the surface of corn stover and significantly increased the enzymatic digestibility, as evidenced by SEM and XRD analysis data. FTIR analysis indicated that FeCl(3) pretreatment could disrupt almost all the ether linkages and some ester linkages between lignin and carbohydrates but had no effect on delignification. The FeCl(3) pretreatment technique, as a novel pretreatment method, enhances enzymatic hydrolysis of lignocellulosic biomass by destructing chemical composition and altering structural features.

The novel antimicrobial peptides from skin of Chinese broad-folded frog, Hylarana latouchii (Anura:Ranidae).

Broad-folded frogs (Hylarana latouchii), one member of 12 species of the genus Hylarana in the Chinese frog fauna, are widely distributed in the South of China. In this study, we purified and characterized three antimicrobial peptides from the skin secretion of H. latouchii. Five different cDNA fragments encoding the precursors of these antimicrobial peptides were cloned, and five mature antimicrobial peptides belonging to two different families were deduced from the five cDNAs. Structural characterization of the mature peptides had identified them as members of the brevinin-1 and temporin families. They were named brevinin-1LTa (FFGTALKIAANVLPTAICKILKKC), brevinin-1LTb (FFGTALKIAANILPTAICKILKKC), temporin-LTa (FFPLVLGALGSILPKIF-NH(2)), temporin-LTb (FIITGLVRGLTKLF-NH(2)) and temorin-LTc (SLSRFLSFLKIVYPPAF-NH(2)). Brevinin-1LTa, temporin-LTa, temporin-LTb and temporin-LTc with different antimicrobial activities induced significant morphological alterations of the tested microbial surfaces as shown by scanning electron microscopy, which indicated strong membrane disruption.

[Mutation effect of ultra high pressure on microbe].

(Ultra) high pressure had many influences on microbe. It could regulate the expression of gene and protein, influence DNA's structure and function as well as change cell morphology and cell component. These effects not only make (ultra) high pressure to be applied into food sterilization, conserving and some processing, but also indicate it would play an important role in mutagenic breeding of microbe. Pressure can change the structure and function of microbe, yet it is possible that (ultra) high pressure could induce mutation of microbe. Now the feasibility of (ultra) high pressure's mutation effect was discussed according to the effects of it on microbe, some examples and author's studying.