Characterization of a bifunctional xylanase/endoglucanase from yak rumen microorganisms

A new gene, RuCelA, encoding a bifunctional xylanase/endoglucanase, was cloned from a metagenomic library of yak rumen microorganisms. RuCelA showed activity against xylan and carboxymethylcellulose (CMC), suggesting bifunctional xylanase/endoglucanase activity. The optimal conditions for xylanase and endoglucanase activities were 65°C, pH 7.0 and 50°C, pH 5.0, respectively. In addition, the presence of Co⁺ and Co²⁺ can greatly improve RuCelA's endoglucanase activity, while inhibits its xylanase activity. Further examination of substrate preference showed a higher activity against barley glucan and lichenin than against xylan and CMC. Using xylan and barley glucan as substrates, RuCelA displayed obvious synergistic effects with β-1,4-xylosidase and β-1,4-glucosidase. Generation of soluble oligosaccharides from lignocellulose is the key step in bioethanol production, and it is greatly notable that RuCelA can produce xylo-oligosaccharides and cello-oligosaccharides in the continuous saccharification of pretreated rice straw, which can be further degraded into fermentable sugars. Therefore, the bifunctional RuCelA distinguishes itself as an ideal candidate for industrial applications.     

Construction of a gorilla fosmid library and its PCR screening system

A gorilla fosmid library of 261,120 independent clones was constructed and characterized. The fosmid vector is similar to the cosmid in average insert size of ca. 40 kb but contains the F factor for replication, and it is more resistant to recombination. This clone library represents about 3.7 times coverage of the gorilla genome. A simple screening system by PCR was established, and we successfully found 9 clones that cover the entire Hox A gene cluster of the gorilla genome. This gorilla fosmid DNA library is a useful resource for comparative genomics of human and apes.     

Rapid screening of an ordered fosmid library to clone multiple polyketide synthase genes of the phytopathogenic fungus Cladosporium phlei

In previous studies, the biological characteristics of the fungus Cladosporium phlei and its genetic manipulation by transformation were assessed to improve production of the fungal pigment, phleichrome, which is a fungal perylenequinone that plays an important role in the production of a photodynamic therapeutic agent. However, the low production of this metabolite by the wild-type strain has limited its application. Thus, we attempted to clone and characterize the genes that encode polyketide synthases (PKS), which are responsible for the synthesis of fungal pigments such as perylenequinones including phleichrome, elsinochrome and cercosporin. Thus, we performed genomic DNA PCR using 11 different combinations of degenerate primers targeting conserved domains including β-ketoacyl synthase and acyltransferase domains. Sequence comparison of the PCR amplicons revealed a high homology to known PKSs, and four different PKS genes showing a high similarity to three representative types of PKS genes were amplified. To obtain full-length PKS genes, an ordered gene library of a phleichrome-producing C. phlei strain (ATCC 36193) was constructed in a fosmid vector and 4800 clones were analyzed using a simple pyramidal arrangement system. This hierarchical clustering method combines the efficiency of PCR with enhanced specificity. Among the three representative types of PKSs, two reducing, one partially reducing, and one non-reducing PKS were identified. These genes were subsequently cloned, sequenced, and characterized. Biological characterization of these genes to determine their roles in phleichrome production is underway, with the ultimate aim of engineering this pathway to overproduce the desired substance.     

Cloning and characterization of a cold-active xylanase enzyme from an environmental DNA library

There is a great interest in xylanases due to the wide variety of industrial applications for these enzymes. We cloned a xylanase gene (xyn8) from an environmental genomic DNA library. The encoded enzyme was predicted to be 399 amino acids with a molecular weight of 45.9 kD. The enzyme was categorized as a glycosyl hydrolase family 8 member based on sequence analysis of the putative catalytic domain. The purified enzyme was thermolabile, had an activity temperature optimum of 20°C on native xylan substrate, and retained significant activity at lower temperatures. At 4°C, the apparent K _m was 3.7 mg/ml, and the apparent k _cat was 123/s.Reference to a company and/or products is only for the purposes of information and does not imply approval or recommendation of the product to the exclusion of others which may also be suitable. All programs and services of the U.S. Department of Agriculture are offered on a nondiscriminatory basis without regard to race, color, national origin, religion, sex, age, marital status, or handicap.     

Molecular diversity of rumen methanogens from sheep in Western Australia

The molecular diversity of rumen methanogens in sheep in Australia was investigated by using individual 16S rRNA gene libraries prepared from the rumen contents obtained from six merino sheep grazing pasture (326 clones), six sheep fed an oaten hay-based diet (275 clones), and five sheep fed a lucerne hay-based diet (132 clones). A total of 733 clones were examined, and the analysis revealed 65 phylotypes whose sequences (1,260 bp) were similar to those of cultivated methanogens belonging to the order Methanobrevibacter: Pasture-grazed sheep had more methanogen diversity than sheep fed either the oaten hay or lucerne hay diet. Methanobrevibacter strains SM9, M6, and NT7 accounted for over 90% of the total number of clones identified. M6 was more prevalent in grazing sheep, and SM9, despite being found in 16 of the 17 sheep, was more prevalent in sheep fed the lucerne-based diet. Five new species were identified. Two of these species exhibited very little sequence similarity to any cultivated methanogens and were found eight times in two of the six sheep that were grazing pasture. These unique sequences appear to represent a novel group of rumen archaea that are atypical for the rumen environment.     

Enriching plant microbiota for a metagenomic library construction

Plant microbiota (the microorganisms that live in any associations with plant tissues) represents a rather unexplored area of metagenomic research compared with soils and oceans. Constructing a metagenomic library for plant microbiota is technically challenging. Using all the biomass without pre-enrichment could lead to vast proportions of the host plant DNA in the metagenomic library, doubtless obliterating the microbial contribution. Therefore, the first and essential step is to enrich for the constituent microorganisms from plant tissues. Here, a strong enrichment for plant microbiota was achieved by coupling SDS (sodium dodecyl sulfate) with NaCl, creating a predominantly microbial metagenomic library that contains 88% bacterial inserts. 16S rDNA sequence analysis revealed that the metagenomic DNA of enrichments originates from very diverse microorganisms. At least 74 distinct ribotypes (at a 97% threshold) from seven different bacterial phyla were identified and mainly distributed among Actinobacteria and Proteobacteria. Additionally, a simplified version of Amplified Ribosomal DNA Restriction Analysis (ARDRA) was developed for a quick and efficient assessment of the enriching procedures. This work opens further insight into the great biotechnical potential of plant microbiota, holding more potential for drug discovery through a metagenomic strategy, and paving the way for recovery and biochemical characterization of functional gene repertoire from plant microbiota.     

Screening and characterization of a novel esterase from a metagenomic library

Metagenomes from various environmental soils were screened using alpha-naphthyl acetate and Fast Blue RR for a novel ester-hydrolyzing enzyme on Escherichia coli. Stepwise fragmentations and subcloning of the initial insert DNA (30-40 kb) using restriction enzymes selected to exclude already known esterases with subsequent screenings resulted in a positive clone with a 2.5-kb DNA fragment. The cloned sequence included an open reading frame consisting of 1089 bp, designated as est25, encoding a protein of 363 amino acids with a molecular mass of about 38.3 kDa. Amino acid sequence analysis revealed only moderate identity (< or = 48%) to the known esterases/lipases in the databases containing the conserved sequence motifs of esterases/lipases, such as HGGG (residues 124-127), GxSxG (residues 199-203), and the putative catalytic triad composed of Ser201, Asp303, and His333. Est25 was functionally overexpressed in a soluble form in E. coli with optimal activity at pH 7.0 and 25 degrees C. The purified Est25 exhibited hydrolyzing activity toward p-nitrophenyl (NP)-fatty acyl esters with short-length acyl chains (< or = C6) with the highest activity toward p-NP-acetate (Km=1.0 mM and Vmax = 63.7 U/mg), but not with chain lengths > or = C8, demonstrating that Est25 is an esterase originated most likely from a mesophilic microorganism in soils. Est25 efficiently hydrolyzed (R,S)-ketoprofen ethyl ester with Km of 16.4 mM and Vmax of 59.1 U/mg with slight enantioselectivity toward (R)-ketoprofen ethyl ester. This study demonstrates that functional screening combined with the sequential uses of restriction enzymes to exclude already known enzymes is a useful approach for isolating novel enzymes from a metagenome.     

Identification and characterization of a cellulase-encoding gene from the buffalo rumen metagenomic library

Microorganisms residing in the rumens of cattle represent a rich source of lignocellulose-degrading enzymes, since their diet consists of plant-based materials that are high in cellulose and hemicellulose. In this study, a metagenomic library was constructed from buffalo rumen contents using pCC1FOS fosmid vector. Ninety-three clones from the pooled library of approximately 10,000 clones showed degrading activity against AZCL-HE-Cellulose, whereas four other clones showed activity against AZCL-Xylan. Contig analysis of pyrosequencing data derived from the selected strongly positive clones revealed 15 ORFs that were closely related to lignocellulose-degrading enzymes belonging to several glycosyl hydrolase families. Glycosyl hydrolase family 5 (GHF5) was the most abundant glycosyl hydrolase found, and a majority of the GHF5s in our metagenomes were closely related to several ruminal bacteria, especially ones from other buffalo rumen metagenomes. Characterization of BT-01, a selected clone with highest cellulase activity from the primary plate screening assay, revealed a cellulase encoding gene with optimal working conditions at pH 5.5 at 50 °C. Along with its stability over acidic pH, the capability efficiently to hydrolyze cellulose in feed for broiler chickens, as exhibited in an in vitro digestibility test, suggests that BT-01 has potential application as a feed supplement.     

Ureolytic bacteria in sheep rumen.

Estimates were made of the numbers of viable bacteria in the rumens of sheep receiving different rations. Representative colonies were isolated and tested for urease production. Some urease-positive isolates were characterized and identified. The ureolytic activities of the urease-producing isolates were determined and compared with the activity of rumen fluid. The rations fed to the sheep did not exert a significant influence on the relative numbers of the urease-producting organisms in the rumen. No obligately anaerobic ureolytic bacteria were found. All urease-positive isolates were facultatively anaerobic, Gram-positive, catalase-positive cocci. Out of ten isolates, nine were identified as Staphylococcus saprophyticus and one as Micrococcus varians. The total urease activity of the different isolates based on the lowest numbers in which they were present in the rumen, compared favourably with the urease activity of rumen fluid. The facultatively anaerobic Gram-positive cocci were probably responsible for a large proportion of the urease activity of the rumen fluid. Conditions prevailing in the rumen were found to be conducive to urease production by the isolates tested.     

Adrenergic reactions of sheep rumen in vitro

The alpha and beta-adrenergic responses of the isolated muscle of sheep rumen were analysed by pharmacodynamic methods after administration of alpha and beta-adrenergic agonists and alpha and beta-adrenergic antagonists. It was found that phenylephrine, and in a lower degree propranolol, stimulated contractions of isolated muscle of sheep rumen while adrenaline, noradrenaline, isoprenaline, phenoxybenzamine and regitine inhibited these contractions. Propranolol abolished the dilating (atonic) effect of catecholamines on the isolated muscles of sheep rumen and previous blockade of beta-adrenergic receptors with propranolol reversed the dilating effects of catecholamines. It is concluded that noradrenaline has an ambiceptor effect (similar to that of adrenaline) on the isolated muscle of the rumen.     

Adrenergic reactions of sheep rumen in vivo

Pharmacodynamical analysis of alpha-adrenergic and beta-adrenergic reactions of sheep rumen was performed in vivo after administration of agonists and antagonists of alpha-adrenergic and beta-adrenergic receptors. It was found that phenylephrine, and in a lower degree propranolol, stimulated the motor activity of sheep rumen, while adrenaline, noradrenaline, isoprenaline, phenoxybenzamine and regitine depressed this activity. Propranolol abolished atonia produced by catecholamines in sheep rumen, and previous blockade of beta-adrenergic receptors with propranolol reversed the relaxing action of catecholamines on the muscular elements in the rumen. The experiments in vivo confirmed the adrenergic effects on the motor activity of the rumen of sheep obtained in earlier investigations on isolated muscles of the rumen. It is suggested that noradrenaline exerts an ambireceptro effect (similar to that of adrenaline) on the motor activity in sheep rumen.