Xianghua Tang

Metagenomic Analysis of the Pygmy Loris Fecal Microbiome Reveals Unique Functional Capacity Related to Metabolism of Aromatic Compounds

The animal gastrointestinal tract contains a complex community of microbes, whose composition ultimately reflects the co-evolution of microorganisms with their animal host. An analysis of 78,619 pyrosequencing reads generated from pygmy loris fecal DNA extracts was performed to help better understand the microbial diversity and functional capacity of the pygmy loris gut microbiome. The taxonomic analysis of the metagenomic reads indicated that pygmy loris fecal microbiomes were dominated by Bacteroidetes and Proteobacteria phyla. The hierarchical clustering of several gastrointestinal metagenomes demonstrated the similarities of the microbial community structures of pygmy loris and mouse gut systems despite their differences in functional capacity. The comparative analysis of function classification revealed that the metagenome of the pygmy loris was characterized by an overrepresentation of those sequences involved in aromatic compound metabolism compared with humans and other animals. The key enzymes related to the benzoate degradation pathway were identified based on the Kyoto Encyclopedia of Genes and Genomes pathway assignment. These results would contribute to the limited body of primate metagenome studies and provide a framework for comparative metagenomic analysis between human and non-human primates, as well as a comparative understanding of the evolution of humans and their microbiome. However, future studies on the metagenome sequencing of pygmy loris and other prosimians regarding the effects of age, genetics, and environment on the composition and activity of the metagenomes are required.

Novel low-temperature-active, salt-tolerant and proteases-resistant endo-1,4-β-mannanase from a new Sphingomonas strain.

Sphingomonas sp. JB13, isolated from slag of a >20-year-old phosphate rock-stacking site, showed the highest 16S rDNA (1343bp) identity of 97.2% with Sphingomonas sp. ERB1-3 (FJ948169) and <97% identities with other identified Sphingomonas strains. A mannanase-coding gene (1191bp) was cloned and encodes a 396-residue polypeptide (ManAJB13) showing the highest amino acid sequence identities of 56.2% with the putative glycosyl hydrolase (GH) family 26 endo-1,4-β-mannanase from Rhodothermus marinus (YP_004824245), and 44.2% with the identified GH 26 endo-1,4-β-mannanase from Cellvibrio japonicus (2VX5_A). The recombinant ManAJB13 (rManAJB13) was expressed in Escherichia coli BL21 (DE3). Purified rManAJB13 displayed the typical characteristics of low-temperature-active enzymes: showing apparent optimal at 40°C, ~55% of the maximum activity at 20°C and ~20% at 10°C, and thermolability at 45°C (~15min half-life). The potential mechanism for low-temperature-activity of GH 26 endo-1,4-β-mannanases might be ascribed to the more hydrophobic residues (AILFWV) and less polar residues (NCQSTY) compared with typical thermophilic and mesophilic counterparts. The purified rManAJB13 exhibited >85% mannanase activity at the concentration of 0-4.0M NaCl. No loss of enzyme activity was observed after incubating the enzyme with 1M or 2M NaCl, or trypsin or proteinase K at 37°C and pH 6.5 for 1h. The K(m), V(max) and k(cat) values were 5.0mgml(-1), 277.8μmol min(-1)mg(-1), and 211.9s(-1), respectively, using locust bean gum as the substrate.

Properties of a Newly Identified Esterase from Bacillus sp. K91 and Its Novel Function in Diisobutyl Phthalate Degradation

The widely used plasticizer phthalate esters (PAEs) have become a public concern because of their effects on environmental contamination and toxicity on mammals. However, the biodegradation of PAEs, especially diisobutyl phthalate (DiBP), remains poorly understood. In particular, genes involved in the hydrolysis of these compounds were not conclusively identified. In this study, the CarEW gene, which encodes an enzyme that is capable of hydrolyzing ρ-nitrophenyl esters of fatty acids, was cloned from a thermophilic bacterium Bacillus sp. K91 and heterologously expressed in Escherichia coli BL21 using the pEASY-E2 expression system. The enzyme showed a monomeric structure with a molecular mass of approximately 53.76 kDa and pI of 4.88. The enzyme exhibited maximal activity at pH 7.5 and 45°C, with ρ-NP butyrate as the best substrate. The enzyme was fairly stable within the pH range from 7.0 to 8.5. High-pressure liquid chromatography (HPLC) and electrospray ionization mass spectrometry (ESI-MS) were employed to detect the catabolic pathway of DiBP. Two intermediate products were identified, and a potential biodegradation pathway was proposed. Altogether, our findings present a novel DiBP degradation enzyme and indicate that the purified enzyme may be a promising candidate for DiBP detoxification and for environmental protection.

Characterization of two glycoside hydrolase family 36 α-galactosidases: novel transglycosylation activity, lead-zinc tolerance, alkaline and multiple pH optima, and low-temperature activity.

Two α-galactosidases, AgaAJB07 from Mesorhizobium and AgaAHJG4 from Streptomyces, were expressed in Escherichia coli. Recombinant AgaAJB07 showed a 2.9-fold and 22.6-fold increase in kcat with a concomitant increase of 2.3-fold and 16.3-fold in Km in the presence of 0.5mM ZnSO4 and 30.0mM Pb(CH3COO)2, respectively. Recombinant AgaAHJG4 showed apparent optimal activity at pH 8.0 in McIlvaine or Tris-HCl buffer and 9.5 in glycine-NaOH or HCl-borax-NaOH buffer, retention of 23.6% and 43.2% activity when assayed at 10 and 20°C, respectively, and a half-life of approximately 2min at 50°C. The activation energies for p-nitrophenyl-α-d-galactopyranoside hydrolysis by AgaAJB07 and AgaAHJG4 were 71.9±0.8 and 48.2±2.0kJmol(-1), respectively. Both AgaAJB07 and AgaAHJG4 exhibited transglycosylation activity, but they required different acceptors and produced different compounds. Furthermore, potential factors for alkaline and multiple pH optima and low-temperature adaptations of AgaAHJG4 were presumed.

Metagenomic analysis of the Rhinopithecus bieti fecal microbiome reveals a broad diversity of bacterial and glycoside hydrolase profiles related to lignocellulose degradation

BackgroundThe animal gastrointestinal tract contains a complex community of microbes, whose composition ultimately reflects the co-evolution of microorganisms with their animal host and the diet adopted by the host. Although the importance of gut microbiota of humans has been well demonstrated, there is a paucity of research regarding non-human primates (NHPs), especially herbivorous NHPs.ResultsIn this study, an analysis of 97,942 pyrosequencing reads generated from Rhinopithecus bieti fecal DNA extracts was performed to help better understanding of the microbial diversity and functional capacity of the R. bieti gut microbiome. The taxonomic analysis of the metagenomic reads indicated that R. bieti fecal microbiomes were dominated by Firmicutes, Bacteroidetes, Proteobacteria and Actinobacteria phyla. The comparative analysis of taxonomic classification revealed that the metagenome of R. bieti was characterized by an overrepresentation of bacteria of phylum Fibrobacteres and Spirochaetes as compared with other animals. Primary functional categories were associated mainly with protein, carbohydrates, amino acids, DNA and RNA metabolism, cofactors, cell wall and capsule and membrane transport. Comparing glycoside hydrolase profiles of R. bieti with those of other animal revealed that the R. bieti microbiome was most closely related to cow rumen.ConclusionsMetagenomic and functional analysis demonstrated that R. bieti possesses a broad diversity of bacteria and numerous glycoside hydrolases responsible for lignocellulosic biomass degradation which might reflect the adaptations associated with a diet rich in fibrous matter. These results would contribute to the limited body of NHPs metagenome studies and provide a unique genetic resource of plant cell wall degrading microbial enzymes. However, future studies on the metagenome sequencing of R. bieti regarding the effects of age, genetics, diet and environment on the composition and activity of the metagenomes are required.

De novo sequencing and analysis of the termite mushroom (Termitomyces albuminosus) transcriptome to discover putative genes involved in bioactive component biosynthesis

A one-eighth 454 sequencing run produced 82,386 high-quality reads. De novo assembly generated 6494 unique sequences. Based on the bioinformatic analysis, we found many the known enzymes involved in the biosynthesis of triterpene saponin in Termitomyces albuminosus, including 6 cytochrome P450 and 22 glycosyltransferase unique genes.

Cold-active and NaCl-tolerant exo-inulinase from a cold-adapted Arthrobacter sp. MN8 and its potential for use in the production of fructose at low temperatures

An exo-inulinase gene was cloned from Arthrobacter sp. MN8, a cold-adapted bacterium isolated from lead-zinc-rich soil. The gene was expressed in Escherichia coli BL21(DE3). The resultant 505-residue polypeptide (InuAMN8) showed the highest identity (81.1%) with the putative levanase from Arthrobacter phenanthrenivorans Sphe3 (ADX73279) and shared 57.8% identity with the exo-inulinase from Bacillus sp. snu-7 (AAK00768). The purified recombinant InuAMN8 (rInuAMN8) showed an apparently optimal activity at 35°C, and 75.3%, 39.4%, and 15.8% of its maximum activity at 20°C, 10°C, and 0°C, respectively. After pre-incubation for 60 min at 50°C and 55°C, the rInuAMN8 exhibited 69.8% and 17.7% of its initial activity, respectively. The apparent Km values of rInuAMN8 towards inulin were 2.8, 1.5, 1.2, 5.3, and 8.2 mM at 0°C, 10°C, 20°C, 30°C, and 35°C, respectively. Inulin and Jerusalem artichoke tubers were effectively hydrolyzed to release fructose by rInuAMN8 at 0°C, 10°C, and 35°C. Compared with its hyperthermophilic and thermophilic counterparts, the exo-inulinase had less aromatic amino acid F and more hydrophobic amino acid A. In addition, the purified rInuAMN8 retained 127.9%-88.4% inulinase activity at 3.5%-15.0% (w/v) NaCl concentrations. Zn(2+) and Pb(2+) at 10 mM exhibited little or no effect on the enzyme activity. This paper is the first to report a cold-active and/or NaCl-tolerant exo-inulinase from the genus Arthrobacter. The exo-inulinase rInuAMN8 shows a potential for use in the production of fructose at low temperatures.

Molecular and Biochemical Characterization of a Novel Xylanase from Massilia sp. RBM26 Isolated from the Feces of Rhinopithecus bieti.

Xylanases sourced from different bacteria have significantly different enzymatic properties. Therefore, studying xylanases from different bacteria is important to their applications in different fields. A potential xylanase degradation gene in Massilia was recently discovered through genomic sequencing. However, its xylanase activity remains unexplored. This paper is the first to report a xylanase (XynRBM26) belonging to the glycosyl hydrolase family (GH10) from the genus Massilia. The gene encodes a 383-residue polypeptide (XynRBM26) with the highest identity of 62% with the endoxylanase from uncultured bacterium BLR13. The XynRBM26 expressed in Escherichia coli BL21 is a monomer with a molecular mass of 45.0 kDa. According to enzymatic characteristic analysis, pH 5.5 is the most appropriate for XynRBM26, which could maintain more than 90% activity between pH 5.0 and 8.0. Moreover, XynRBM26 is stable at 37°C and could maintain at least 96% activity after being placed at 37°C for 1 h. This paper is the first to report that GH10 xylanase in an animal gastrointestinal tract (GIT) has salt tolerance, which could maintain 86% activity in 5 M NaCl. Under the optimum conditions, Km, Vmax, and kcat of XynRBM26 to beechwood xylan are 9.49 mg/ml, 65.79 μmol/min/mg, and 47.34 /sec, respectively. Considering that XynRBM26 comes from an animal GIT, this xylanase has potential application in feedstuff. Moreover, XynRBM26 is applicable to high-salt food and seafood processing, as well as other high-salt environmental biotechnological fields, because of its high catalytic activity in high-concentration NaCl.

Study of fecal bacterial diversity in Yunnan snub-nosed monkey (Rhinopithecus bieti) using phylogenetic analysis of cloned 16S rRNA gene sequences

The bacterial diversity in fecal samples from Yunnan snub-nosed monkey ( Rhinopithecus bieti ) was investigated by constructed 16S rRNA gene clone library and restriction fragment length polymorphism analysis. As a result, a total of 156 representative clones for each profile, comprising nearly full length sequences (with a mean length of 1.5 kb) were sequenced and submitted to an on-line similarity search and neighbor-joining phylogenetic analysis. Using the criterion of 97%, these 16S rRNA gene sequences were binned in 129 OTUs. 11 sequences whose similarity is 97% were affiliated to the cultured bacteria and accounted for 7.05% of the total clones. For 23 sequences (14.74%), the similarity with the database was in the range of 89 - 97%. The remaining 122 sequences (78.21%) were uncultured and unidentified bacteria. Based on the phylogenetic analysis, the fecal bacteria of R. bieti distributed mainly in 6 bacteriophyta of Firmicutes, Proteobacteria, Bacteroidetes, Fibrobacteres, Spirochaetes and Actinobacteria , and belonged to 17 genera. Besides, there were a large number of uncultured and unidentified bacteria. These results illustrate the fecal bacteria diversity of R. bieti. Key Words : Rhinopithecus bieti, fecal bacterial diversity, 16S rRNA gene, phylogenetic analysis.

Characterization of a Glycoside Hydrolase Family 27 α-Galactosidase from Pontibacter Reveals Its Novel Salt-Protease Tolerance and Transglycosylation Activity.

α-Galactosidases are of great interest in various applications. A glycoside hydrolase family 27 α-galactosidase was cloned from Pontibacter sp. harbored in a saline soil and expressed in Escherichia coli. The purified recombinant enzyme (rAgaAHJ8) was little or not affected by 3.5-30.0% (w/v) NaCl, 10.0-100.0 mM Pb(CH3COO)2, 10.0-60.0 mM ZnSO4, or 8.3-100.0 mg mL(-1) trypsin and by most metal ions and chemical reagents at 1.0 and 10.0 mM concentrations. The degree of synergy on enzymatic degradation of locust bean gum and guar gum by an endomannanase and rAgaAHJ8 was 1.22-1.54. In the presence of trypsin, the amount of reducing sugars released from soybean milk treated by rAgaAHJ8 was approximately 3.8-fold compared with that treated by a commercial α-galactosidase. rAgaAHJ8 showed transglycosylation activity when using sucrose, raffinose, and 3-methyl-1-butanol as the acceptors. Furthermore, potential factors for salt adaptation of the enzyme were presumed.