Phylogenetic and functional analysis of gut microbiota of a fungus-growing higher termite: Bacteroidetes from higher termites are a rich source of ß-glucosidase genes.

Fungus-growing termites, their symbiotic fungi, and microbiota inhibiting their intestinal tract comprise a highly efficient cellulose-hydrolyzing system; however, little is known about the role of gut microbiota in this system. Twelve fosmid clones with ß-glucosidase activity were previously obtained by functionally screening a metagenomic library of a fungus-growing termite, Macrotermes annandalei. Ten contigs containing putative ß-glucosidase genes (bgl1-10) were assembled by sequencing data of these fosmid clones. All these contigs were binned to Bacteroidetes, and all these ß-glucosidase genes were phylogenetically closed to those from Bacteroides or Dysgonomonas. Six out of 10 ß-glucosidase genes had predicted signal peptides, indicating a transmembrane capability of these enzymes to mediate cellulose hydrolysis within the gut of the termites. To confirm the activities of these ß-glucosidase genes, three genes (bgl5, bgl7, and bgl9) were successfully expressed and purified. The optimal temperature and pH of these enzymes largely resembled the environment of the host's gut. The gut microbiota composition of the fungus-growing termite was also determined by 454 pyrosequencing, showing that Bacteroidetes was the most dominant phylum. The diversity and the enzyme properties of ß-glucosidases revealed in this study suggested that Bacteroidetes as the major member in fungus-growing termites contributed to cello-oligomer degradation in cellulose-hydrolyzing process and represented a rich source for ß-glucosidase genes.

N-glycosylation affects the proper folding, enzymatic characteristics and production of a fungal ß-glucosidase.

Heterologous expression of ß-glucosidase is one of the approaches to enhance the efficiency of fungal cellulase preparations. It has been reported that N-glycosylation affects the structure framework, function and stability of proteins. In this study, a ß-glucosidase from Aspergillus terreus (GenBank: XP_001216552, BglS) was heterologously expressed in Pichia pastoris and Trichoderma reesei. The four asparagine residues were all linked with high-mannose-type oligosaccharides in P. pastoris, whereas only N224 carried high-mannosetype glycan in T. reesei (the other three sites carried one N-acetylglucosamine). The long N-glycan chains on PpBglS weakened its substrate affinity, activity and thermostability. The moderate post-translational and post-secretory glycan modification in T. reesei makes it a suitable expression system for BglS. The N224 glycan played a critical role in BglS folding. The elucidation of the correlation between the different N-glycosylation patterns of BglS and their corresponding enzymatic characteristics is an important step towards improving the activity, thermostability and even production of heterologous ß-glucosidase by glycan engineering.

Biochemical characterization and crystal structure of a GH10 xylanase from termite gut bacteria reveal a novel structural feature and significance of its bacterial Ig-like domain.

Bacterial Ig-like (Big) domains are commonly distributed in glycoside hydrolases (GH), but their structure and function remains undefined. Xylanase is a GH, and catalyzes the hydrolysis of the internal ß-xylosidic linkages of xylan. In this study, we report the molecular cloning, biochemical and biophysical characterization, and crystal structure of a termite gut bacterial xylanase, Xyl-ORF19, which was derived from gut bacteria of a wood-feeding termite (Globitermes brachycerastes). The protein architecture of Xyl-ORF19 reveals that it has two domains, a C-terminal GH10 catalytic domain and an N-terminal Big_2 non-catalytic domain. The catalytic domain folds in an (α/ß)8 barrel as most GH10 xylanases do, but it has two extra ß-strands. The non-catalytic domain is structurally similar to an immunoglobulin-like domain of intimins. The recombinant enzyme without the non-catalytic domain has fairly low catalytic activity, and is different from the full-length enzyme in kinetic parameters, pH and temperature profiles, which suggests the non-catalytic domain could affect the enzyme biochemical and biophysical properties as well as the role for enzyme localization. This study provides a molecular basis for future efforts in xylanase bioengineering.

Discovery of (hemi-) cellulase genes in a metagenomic library from a biogas digester using 454 pyrosequencing.

In this study, 341, 246, and 386 positive clones with endo-ß-1,4-glucanase, ß-glucosidase, and endo-ß-1,4-xylanase activities, respectively, were identified by screening from a metagenomic fosmid library constructed from a biogas digester. Subsequently, pools of 4, 10, and 16 positive clones were subjected to 454 pyrosequencing in different subruns. In total, 21 unique glycosyl hydrolase (GH) genes were predicted by bioinformatic analysis, which showed similarities to their nearest neighbors from 39 % to 72 %. In addition to bioinformatics prediction, nine GH genes were expressed and purified to identify their activity with four kinds of substrates. The activities of the most expressed proteins were consistent with their annotation based on bioinformatics prediction; however, three GH genes belonging to the GH5 family showed different activities from their annotation. An efficient acidic cellulase En1 had an optimal condition at 55 °C, pH 5.5, with a specific activity toward carboxymethylcellulose at 118 U/mg and K m at 12.8 g/L. This study demonstrated that there are diverse GHs in the biogas digester system with potential industrial application in lignocellulose hydrolysis, and their activities should be investigated with different substrates before their application. Additionally, pool sequencing of positive fosmid clones might be a cost-effective approach to obtain functional genes from metagenomic libraries.

Expression and characterization of a novel metagenome-derived cellulase Exo2b and its application to improve cellulase activity in Trichoderma reesei.

A metagenomic fosmid library containing 1 × 10(5) clones was constructed from a biogas digester fed with pig ordure and rice straw. In total, 121 clones with activity of 4-methylumbelliferyl-cellobiosidase were screened from the metagenomic library. A novel GH5 cellulase gene exo2b was identified from a sequenced clone EXO02C10 and expressed in Escherichia coli BL21. The corresponding recombinant Exo2b protein showed high specific activity toward both carboxymethylcellulose (CMC; 260 U/mg protein) and ß-D-glucan from barley (849 U/mg), with an optimal pH and temperature of 7.5 and 58 °C, respectively. Exo2b showed stable activity at a wide pH range from 5.5 to 9.0 and was highly thermostable at 60 °C in the presence of 60 mM cysteine. Residual activity was maintained at nearly 100% when Exo2b was incubated at 60 °C for 15 h. A thin-layer chromatography analysis of the hydrolysis products confirmed that Exo2b was an endo-ß-1,4-glucanase and it could also produce oligosaccharide smaller than cellotetraose. The fragment encoding the Exo2b catalytic domain was then fused with the cbh1 gene from Trichoderma reesei, and the fused gene was successfully expressed in T. reesei Rut-C30. Compared to that of the parent strain, the filter paper activity and CMCase activity of the secreted proteins of a selected transformant A1 increased by 24% and 18%, respectively. Besides, the glucose concentration from the hydrolysis of pretreated corn stover by the A1 secreted proteins increased by 19.8%. The present study demonstrated the potential application of metagenome originated cellulase genes to modify cellulase producing fungi.

A flexible Mn0.5Ti2(PO4)3/C nanofiber film with superior cycling stability for potassium-ion batteries.

First-principles calculations indicate that Mn0.5Ti2(PO4)3 is more suited to potassium-ion storage because of its lower energy gap than that of KTi2(PO4)3. A flexible Mn0.5Ti2(PO4)3/C nanofiber film (F-MTP/C NFF) has been first synthesized via electrospinning and applied to potassium-ion batteries as a self-standing anode. An integral carbon conductive network, unique one-dimensional nanostructure, and exceptional mechanical flexibility give F-MTP/C NFF outstanding potassium-ion storage performance. Impressively, the self-standing F-MTP/C NFF electrode delivers a high specific capacity of 218 mA h g-1 at 0.02 A g-1 and shows ultra-long cycling stability of 2000 cycles at 1 A g-1. This work may give a new insight into developing NASICON-type Ti-based materials as flexible electrodes for potassium-ion batteries.

Engineering a high-performance, metagenomic-derived novel xylanase with improved soluble protein yield and thermostability.

The novel termite gut metagenomic-derived GH11 xylanase gene xyl7 was expressed in Escherichia coli BL21, and the purified XYL7 enzyme exhibited high specific activity (6340U/mg) and broad pH active range of 5.5-10.0. Directed evolution was employed to enhance the thermostability of XYL7; two mutants (XYL7-TC and XYL7-TS) showed a 250-fold increase in half-life at 55°C, with a 10°C increase in optimal temperature compared to that of wild-type XYL7. A truncated enzyme (XYL7-Tr3) acquired by protein engineering showed similar catalytic properties as the wild-type, with a tenfold increase in soluble protein yield by the mutant. The reducing sugar produced by XYL7-TC was about fourfold greater than that produced by their parents when incubated with xylan at 60°C for 4h. The engineered novel xylanase exhibited superior enzymatic performance and showed promise as an excellent candidate for industrial application due to its high specific activity, stability and soluble protein yield.

Characterization of a novel thermostable ß-glucosidase from a metagenomic library of termite gut.

A novel ß-glucosidase-encoding gene, bgl-gs1, which was identified from a positive fosmid clone in a metagenomic library of the gut of Globitermes brachycerastes, [corrected] encodes a 455 amino acid polypeptide that contains a catalytic domain belonging to glycoside hydrolase family 1 (GH1). It was expressed in Escherichia coli BL21 (DE3) and the expression product showed a molecular mass of ∼51.7 kDa by SDS-PAGE. The optimal temperature and pH for the activity of the purified recombinant enzyme Bgl-gs1 with p-nitrophenyl-ß-D-glucoside (pNPG) were 90°C and 6.0, respectively. The specific activities of Bgl-gs1 on pNPG and salicin were 110 and 14U/mg of protein, respectively, and its K(m) values were 0.18 and 2.59 mM, respectively. The residual activity of Bgl-gs1 was maintained above 70% after the recombinant enzyme was incubated at 75°C and pH 6.0 for 2h, and its half-life at 90°C was approximately 1h in the presence of 4mM pNPG. Bgl-gs1 showed synergistic effect with either a crude enzyme mixture of the fungal strain Trichoderma reesei Rut-C30 or a fusion protein (TcE1) created from the cellobiohydrolase cbh1 gene of T. reesei and endoglucanase from Acidothermus cellulolyticus; 87 and 137% increases in hydrolytic efficiency were noted on microcrystalline cellulose, respectively. These results suggest that the thermostable ß-glucosidase Bgl-gs1 is a likely candidate for industrial applications.

Bacillus qingdaonensis sp. nov., a moderately haloalkaliphilic bacterium isolated from a crude sea-salt sample collected near Qingdao in eastern China.

A moderately haloalkaliphilic, Gram-positive bacterium, designated as strain CM1(T), was isolated from a crude sea-salt sample collected near Qingdao in eastern China. Strain CM1(T) was found to grow optimally at 37 degrees C and pH 9.0. It was shown to be aerobic, rod-shaped and capable of growth at salinities of 2.5-20 % (w/v) NaCl (optimum, 12 %). The genomic DNA G+C content was about 48 mol%. The major cellular fatty acids were anteiso-C(15 : 0), anteiso-C(17 : 0) and iso-C(16 : 0) and the major isoprenoid quinones were MK-7(H(2)) and MK-6(H(2)). Phylogenetic analyses based on 16S rRNA gene sequences revealed that CM1(T) is a member of the genus Bacillus and has less than 95.2 % gene sequence similarity to the most closely related strain, Bacillus salarius BH169(T). Its DNA-DNA reassociation value with respect to B. salarius BH169(T) was 35.4 %. On the basis of phenotypic and molecular properties, strain CM1(T) represents a novel Bacillus species, for which the name Bacillus qingdaonensis sp. nov. is proposed. The type strain is CM1(T) (=CGMCC 1.6134(T)=JCM 14087(T)).

Halococcus qingdaonensis sp. nov., a halophilic archaeon isolated from a crude sea-salt sample.

A Gram-negative, extremely halophilic, coccoid archaeal strain, CM5(T), was isolated from a crude sea-salt sample collected near Qingdao, China. The organism grew optimally at 35-40 degrees C and pH 6.0 in the presence of 20 % (w/v) NaCl. Its colonies were red in colour and it could use glucose as a sole carbon source for growth. The 16S rRNA gene sequence of CM5(T) was most closely related to those of Halococcus species. Its pattern of antibiotic susceptibility was similar to those of other described Halococcus species. Biochemical tests revealed no sign of H(2)S production or gelatin liquefaction. The main polar lipids of strain CM5(T) were phosphatidylglycerol, phosphatidylglycerol methylphosphate and sulfated diglycosyl diether. No phosphatidylglycerol sulfate was present. The DNA G+C content of strain CM5(T) was 61.2 mol% and it gave DNA-DNA reassociation values of 33.7, 57.1 and 29.6 %, respectively, with Halococcus salifodinae DSM 8989(T), Halococcus dombrowskii DSM 14522(T) and Halococcus morrhuae ATCC 17082(T). Based on its morphological and chemotaxonomic properties and phylogenetic analysis of 16S rRNA gene sequence data, we propose that CM5(T) should be classified within a novel species, Halococcus qingdaonensis sp. nov., with strain CM5(T) (=CGMCC 1.4243(T)=JCM 13587(T)) as the type strain.