Yunjuan Yang

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.

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.

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.

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.

Identification and Characterization of A New 7-Aminocephalosporanic Acid Deacetylase from Thermophilic Bacterium Alicyclobacillus tengchongensis

UNLABELLED Deacetylation of 7-aminocephalosporanic acid (7-ACA) at position C-3 provides valuable starting material for producing semisynthetic β-lactam antibiotics. However, few enzymes have been characterized in this process before now. Comparative analysis of the genome of the thermophilic bacterium Alicyclobacillus tengchongensis revealed a hypothetical protein (EstD1) with typical esterase features. The EstD1 protein was functionally cloned, expressed, and purified from Escherichia coli BL21(DE3). It indeed displayed esterase activity, with optimal activity at around 65°C and pH 8.5, with a preference for esters with short-chain acyl esters (C2 to C4). Sequence alignment revealed that EstD1 is an SGNH hydrolase with the putative catalytic triad Ser15, Asp191, and His194, which belongs to carbohydrate esterase family 12. EstD1 can hydrolyze acetate at the C-3 position of 7-aminocephalosporanic acid (7-ACA) to form deacetyl-7-ACA, which is an important starting material for producing semisynthetic β-lactam antibiotics. EstD1 retained more than 50% of its initial activity when incubated at pH values ranging from 4 to 11 at 65°C for 1 h. To the best of our knowledge, this enzyme is a new SGNH hydrolase identified from thermophiles that is able to hydrolyze 7-ACA. IMPORTANCE Deacetyl cephalosporins are highly valuable building blocks for the industrial production of various kinds of semisynthetic β-lactam antibiotics. These compounds are derived mainly from 7-ACA, which is obtained by chemical or enzymatic processes from cephalosporin C. Enzymatic transformation of 7-ACA is the main method because of the adverse effects chemical deacylation brought to the environment. SGNH hydrolases are widely distributed in plants. However, the tools for identifying and characterizing SGNH hydrolases from bacteria, especially from thermophiles, are rather limited. Here, our work demonstrates that EstD1 belongs to the SGNH family and can hydrolyze acetate at the C-3 position of 7-ACA. Moreover, this study can enrich our understanding of the functions of these enzymes from this family.

The 340-cavity in neuraminidase provides new opportunities for influenza drug development: A molecular dynamics simulation study

Influenza neuraminidase (NA) is a pivotal target for viral infection control. However, the accumulating of mutations compromise the efficacy of NA inhibitors. Thus, it is critical to design new drugs targeted to different motifs of NA. Recently, a new motif called 340-cavity was discovered in NA subtypes close to the calcium binding site. The presence of calcium is known to influence NA activity and thermostability. Therefore, the 340-cavity is a putative ligand-binding site for affecting the normal function of NA. In this study, we performed molecular dynamics simulations of different NA subtypes to explore the mechanism of 340-loop formation. Ligand-binding site prediction and fragment library screening were also carried out to provide evidence for the 340-cavity as a druggable pocket. We found that residues G342 and P/R344 in the 340-loop determine the size of the 340-cavity, and the calcium ion plays an important role in maintaining the conformation of the 340-loop through contacts with G345 and Q347. In addition, the 340-cavity is predicted to be a ligand-binding site by metaPocket, and a sequence analysis method is proposed to predict the existence of the 340-cavity. Our study shows that the 340-cavity is not an occasional or atypical domain in NA subtypes, and it has potential to function as a new hotspot for influenza drug binding.

Identification and Characterization of a New Alkaline SGNH Hydrolase from a Thermophilic Bacterium Bacillus sp. K91

est19 is a gene from Bacillus sp. K91 that encodes a new esterase. A comparison of the amino acid sequence showed that Est19 has typical Ser-Gly-Asn-His (SGNH) family motifs and could be grouped into the SGNH hydrolase family. The Est19 protein was functionally cloned, and expressed and purified from Escherichia coli BL21(DE3). The enzyme activity was optimal at 60°C and pH 9.0, and displayed esterase activity towards esters with short-chain acyl esters (C₂-C₆). A structural model of Est19 was constructed using phospholipase A1 from Streptomyces albidoflavus NA297 as a template. The structure showed an α/β-hydrolase fold and indicated the presence of the typical catalytic triad Ser49-Asp227-His230, which were further investigated by site-directed mutagenesis. To the best of our knowledge, Est19 is a new member of the SGNH hydrolase family identified from thermophiles, which may be applicable in the industrial production of semisynthetic β-lactam antibiotics after modification.

Characterization of a family 3 polysaccharide lyase with broad temperature adaptability, thermo-alkali stability, and ethanol tolerance

The 774-bp pectate lyase gene plyAI4 from Bacillus sp. I4 was cloned and expressed in E. coli. The gene encodes a 257-residue polypeptide (PlyAI4, 28.3 kDa) with the highest identities of 97.3% with a putative pectate lyase from Bacillus subtilis BSn5 (ADV94306) and 60.3% with an identified pectate lyase of the polysaccharide lyase family (PL) 3 from Paenibacillus amylolyticus 27C64 (ADB78774). The purified recombinant PlyAI4 (rPlyAI4) exhibited apparently optimal activity at pH 10.5 ∼ 11.0 and 50°C. Compared with the majority of reported alkaline pectate lyases, rPlyAI4 exhibited more residual enzyme activity at 20°C (∼45%) or at 70°C (∼50%) and better thermostability at 70°C (∼60 min half-life at 70°C). In the presence of 20% (v/v) ethanol, pectate lyase activity was enhanced by 0.2 fold. After incubation in 40% (v/v) ethanol at 37°C and pH 8.5 for 1 h, the purified rPelAI4 retained more than 75% of the initial activity. Sequence analysis proposed a new signature block, A-D-G-[V/I]-H, for PL 3 pectate lyases. These properties may prove to be important with regards to PlyAI4 for basic research and industrial application.

Genetic diversity of catechol 1,2‐dioxygenase in the fecal microbial metagenome

Catechol 1,2‐dioxygenase is the key enzyme that catalyzes the cleavage of the aromatic ring of catechol. We explored the genetic diversity of catechol 1,2‐dioxygenase in the fecal microbial metagenome by PCR with degenerate primers. A total of 35 gene fragments of C12O were retrieved from microbial DNA in the feces of pygmy loris. Based on phylogenetic analysis, most sequences were closely related to C12O sequences from Acinetobacter. A full‐length C12O gene was directly cloned, heterologously expressed in Escherichia coli, and biochemically characterized. Purified catPL12 had optimum pH and temperature pH 8.0 and 25 °C and retained 31 and 50% of its maximum activity when assayed at 0 and 35 °C, respectively. The enzyme was stable at 25 and 37 °C, retaining 100% activity after pre‐incubation for 1 h. The kinetic parameters of catPL12 were determined. The enzyme had apparent Km of 67 µM, Vmax of 7.3 U/mg, and kcat of 4.2 s−1 for catechol, and the cleavage activities for 3‐methylcatechol, 4‐methylcatechol, and 4‐chlorocatechol were much less than for catechol, and no activity with hydroquinone or protocatechuate was detected. This study is the first to report the molecular and biochemical characterizations of a cold‐adapted catechol 1,2‐dioxygenase from a fecal microbial metagenome.