Cloning and enzymatic characterization of a xylanase gene from a soil-derived metagenomic library with an efficient approach

Screening interesting biocatalysts directly from soil samples is a more convenient and applicable approach than conventional cultivation-dependent ones. In our present work, a soil-derived metagenomic library containing 24,000 transformants was constructed with an efficient strategy for cloning xylanase genes. A gene encoding the enzyme (XynH) able to hydrolyze xylan was obtained. Similarity analysis revealed that this enzyme is a new member in the family 10 of xylanases. The molecular mass of XynH purified from Escherichia coli was estimated to be 39 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis. It was found to display the maximal activity at lower temperature, under weakly alkaline conditions, different from most of xylanases. The K _m and V_max values of XynH with birchwood xylan as substrate are 7.5 mg/ml and 190 μmol min⁻¹ mg⁻¹, respectively. It is greatly interesting to note that the activity of XynH was not reduced significantly by Mn²⁺, Zn²⁺, Co²⁺, Ag⁺, and Cu²⁺, even at the concentration of 5 mM, which strongly inhibits most of the other xylanases studied previously. Yong Hu and Guimin Zhang contributed equally to this work.     

Polyketide synthase pathways identified from a metagenomic library are derived from soil Acidobacteria

Polyketides are structurally diverse secondary metabolites, many of which have antibiotic or anticancer activity. Type I modular polyketide synthase (PKS) genes are typically large and encode repeating enzymatic domains that elongate and modify the nascent polyketide chain. A fosmid metagenomic library constructed from an agricultural soil was arrayed and the macroarray was screened for the presence of conserved ketosynthase [β-ketoacyl synthase (KS)] domains, enzymatic domains present in PKSs. Thirty-four clones containing KS domains were identified by Southern hybridization. Many of the KS domains contained within metagenomic clones shared significant similarity to PKS or nonribosomal peptide synthesis genes from members of the Cyanobacteria or the Proteobacteria phyla. However, analysis of complete clone insert sequences indicated that the blast analysis for KS domains did not reflect the true phylogenetic origin of many of these metagenomic clones that had a %G+C content and significant sequence similarity to genes from members of the phylum Acidobacteria. This conclusion of an Acidobacteria origin for several clones was further supported by evidence that cultured soil Acidobacteria from different subdivisions have genetic loci closely related to PKS domains contained within metagenomic clones, suggesting that Acidobacteria may be a source of novel polyketides. This study also demonstrates the utility of combining data from culture-dependent and -independent investigations in expanding our collective knowledge of microbial genomic diversity.     

Expression and purification of organic solvent stable lipase from soil metagenomic library

Gene for organic solvent stable lipase was overexpressed from soil metagenomic library. The clone with maximum activity was selected, and enzyme was purified by gel-permeation chromatography with a molecular mass of approx. 40 kDa. The deduced aminoacid sequence indicated that the protein belongs to the lipase family I.3 and containing a C-terminal secretion signal for ABC dependent transport together with possible motifs for Ca(2+) binding sites. The enzyme expressed maximum activity at 30 °C and pH 7.0 and found to be stable in pH and temperature ranging from 6.0-9.0 and 20-60 °C, respectively. Furthermore, the enzyme was found highly resistant to many organic solvents, especially isopropanol, DMSO, methanol, xylene and hexane. The enzyme showed enhanced activity in the presence of divalent cations (Mg(2+), Mn(2+), Ca(2+), Hg(2+), Cu(2+)), whereas the presence of trivalent cation (Fe(3+)) inhibited the activity.     

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.     

Characterization of a novel amylolytic enzyme encoded by a gene from a soil-derived metagenomic library

It has been estimated that less than 1% of the microorganisms in nature can be cultivated by conventional techniques. Thus, the classical approach of isolating enzymes from pure cultures allows the analysis of only a subset of the total naturally occurring microbiota in environmental samples enriched in microorganisms. To isolate useful microbial enzymes from uncultured soil microorganisms, a metagenome was isolated from soil samples, and a metagenomic library was constructed by using the pUC19 vector. The library was screened for amylase activity, and one clone from among approximately 30,000 recombinant Escherichia coli clones showed amylase activity. Sequencing of the clone revealed a novel amylolytic enzyme expressed from a novel gene. The putative amylase gene (amyM) was overexpressed and purified for characterization. Optimal conditions for the enzyme activity of the AmyM protein were 42 degrees C and pH 9.0; Ca2+ stabilized the activity. The amylase hydrolyzed soluble starch and cyclodextrins to produce high levels of maltose and hydrolyzed pullulan to panose. The enzyme showed a high transglycosylation activity, making alpha-(1, 4) linkages exclusively. The hydrolysis and transglycosylation properties of AmyM suggest that it has novel characteristics and can be regarded as an intermediate type of maltogenic amylase, alpha-amylase, and 4-alpha-glucanotransferase.     

Biochemical screening of stable dinucleosomes using DNA fragments from a dinucleosome DNA library

The dinucleosome is an informative unit for analysis of the higher-order chromatin structure. DNA fragments forming stable dinucleosomes were screened from a dinucleosome DNA library after the reconstitution of nucleosomes in vitro and digestion with micrococcal nuclease. Reconstituted dinucleosomes showed a diversity of sensitivity to micrococcal nuclease, suggesting that the biochemical stability of a dinucleosome depends, in part, on the DNA fragments. The DNA fragments after the screening were classified into three groups represented by clones bf10, af14 and af32 according to the sensitivity to micrococcal nuclease. Mapping of the nucleosome boundaries by Southern blotting of the DNA after restriction digestion and by primer extension analysis showed that each nucleosome position of clone af32 was fixed. Analysis of reconstituted dinucleosomes using mutant DNA fragments of clone af32 revealed a unique property characteristic of a key nucleosome, given that the replacement of a DNA fragment corresponding to the right nucleosome position resulted in marked sensitivity to micrococcal nuclease, whereas the replacement of the other nucleosome fragment had almost no effect on sensitivity as compared to the original af32 construct. The mutant construct in which the right nucleosome was removed showed multiple nucleosome phases, suggesting that the right nucleosome stabilized first each mononucleosome and then the dinucleosome. An oligonucleotide bending assay revealed that the DNA fragment in the right nucleosome included curved DNA, suggesting that the positioning activity of the nucleosome was attributed to its DNA structure. These results suggest that information for forming stable dinucleosome is embedded in the genomic DNA and that a further characterization of the key nucleosome is useful for understanding the building up of the chromatin structure.     

Evaluation of functional gene enrichment in a soil metagenomic clone library

We evaluated the use of mixed oligonucleotide probes hybridized to metagenomic clones spotted on high density membranes. The pooled probes included oligonucleotides designed for genes associated with denitrification, antibiotic resistance, and dehalogenation among others. Pyrosequence comparison between the clones and the original DNA demonstrated the utility of clone screening with pooled probes.     

Cloning and heterologous expression of the vibrioferrin biosynthetic gene cluster from a marine metagenomic library

A biosynthetic gene cluster of siderophore consisting of five open reading frames (ORFs) was cloned by functional screening of a metagenomic library constructed from tidal-flat sediment. Expression of the cloned biosynthetic genes in Escherichia coli led to the production of vibrioferrin, a siderophore originally reported for the marine bacterium Vibrio parahaemolyticus. To the best of our knowledge, this is the first example of heterologous production of a siderophore by biosynthetic genes cloned from a metagenomic library. The cloned cluster was one of the largest of the clusters obtained by functional screening. In this study, we demonstrated and extended the possibility of function-based metagenomic research.     

Identification and characterization of a novel methionine γ-lyase gene from deep-sea sediment metagenomic library

World Journal of Microbiology and Biotechnology - A putative methionine γ-lyase (MGL) gene was identified from a deep-sea sediment metagenomic library. The gene (mgl_deepsea), consisting of...     

Isolation and characterization of a thermostable esterase from a metagenomic library

A novel esterase gene was isolated by functional screening of a metagenomic library prepared from an activated sludge sample. The gene (est-XG2) consists of 1,506 bp with GC content of 74.8 %, and encodes a protein of 501 amino acids with a molecular mass of 53 kDa. Sequence alignment revealed that Est-XG2 shows a maximum amino acid identity (47 %) with the carboxylesterase from Thermaerobacter marianensis DSM 12885 (YP_004101478). The catalytic triad of Est-XG2 was predicted to be Ser₁₉₂-Glu₃₁₃-His₄₁₂ with Ser₁₉₂ in a conserved pentapeptide (GXSXG), and further confirmed by site-directed mutagenesis. Phylogenetic analysis suggested Est-XG2 belongs to the bacterial lipase/esterase family VII. The recombinant Est-XG2, expressed and purified from Escherichia coli, preferred to hydrolyze short and medium length p-nitrophenyl esters with the best substrate being p-nitrophenyl acetate (K _m and k _cat of 0.33 mM and 36.21 s^?1, respectively). The purified enzyme also had the ability to cleave sterically hindered esters of tertiary alcohols. Biochemical characterization of Est-XG2 revealed that it is a thermophilic esterase that exhibits optimum activity at pH 8.5 and 70 °C. Est-XG2 had moderate tolerance to organic solvents and surfactants. The unique properties of Est-XG2, high thermostability and stability in the presence of organic solvents, may render it a potential candidate for industrial applications.     

Characterization of a new and thermostable esterase from a metagenomic library

A new gene encoding an esterase (designated as EstEP16) was identified from a metagenomic library prepared from a sediment sample collected from a deep-sea hydrothermal field in east Pacific. The open reading frame of this gene encoded 249 amino acid residues. It was cloned, overexpressed in Escherichia coli, and the recombinant protein was purified to homogeneity. The monomeric EstEP16 presented a molecular mass of 51.7 kDa. Enzyme assays using p-nitrophenyl esters with different acyl chain lengths as the substrates confirmed its esterase activity, yielding highest specific activity with p-nitrophenyl acetate. When p-nitrophenyl butyrate was used as a substrate, recombinant EstEP16 exhibited highest activity at pH 8.0 and 60 °C. The recombinant enzyme retained about 80% residual activity after incubation at 90 °C for 6 h, which indicated that EstEP16 was thermostable. Homology modeling of EstEP16 was developed with the monoacylglycerol lipase from Bacillus sp. H-257 as a template. The structure showed an α/β-hydrolase fold and indicated the presence of a typical catalytic triad. The activity of EstEP16 was inhibited by addition of phenylmethylsulfonyl fluoride, indicating that it contains serine residue, which plays a key role in the catalytic mechanism.     

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.     

云南土壤宏基因组文库中替加环素耐药基因的筛选与分析

[背景]随着抗生素的广泛应用以及滥用,出现了多重耐药的"超级细菌",并且在临床上已出现对治疗"超级细菌"感染的最后一线抗生素——替加环素耐药的细菌.[目的]对土壤环境中存在的替加环素耐药基因进行筛选调查,探讨替加环素耐药机制,为临床抗生素治疗提供借鉴.[方法]利用功能宏基因组学技术对土壤宏基因组文库进行替加环素耐药阳性...     

Characterization of alginate lyase gene using a metagenomic library constructed from the gut microflora of abalone

A metagenomic fosmid library was constructed using a genomic DNA mixture extracted from the gut microflora of abalone. The library gave an alginate lyase positive clone (AlyDW) harboring a 31.7-kbp insert. The AlyDW insert consisted of 22 open reading frames (ORFs). The deduced amino acid sequences of ORFs 11–13 were similar to those of known alginate lyase genes, which are found adjacent in the genome of Klebsiella pneumoniae subsp. aerogenes, Vibrio splendidus, and Vibrio sp. belonging to the phylum Gammaproteobacteria. Among the three recombinant proteins expressed from the three ORFs, alginate lyase activity was only observed in the recombinant protein (AlyDW11) coded by ORF 11. The expressed protein (AlyDW11) had the highest alginate lyase activity at pH 7.0 and 45°C in the presence of 1 mM AgNO₃. The alginate lyase activity of ORF 11 was confirmed to be endolytic by thin-layer chromatography. AlyDW11 preferred poly(β-d-mannuronate) as a substrate over poly(α-l-guluronate). AlyDW11 contained three highly conserved regions, RSEL, QIH, and YFKAGVYNQ, which may act to stabilize the three-dimensional conformation and function of the alginate lyase.     

源于红树林土壤宏基因组文库的新型磷脂酶A1基因的筛选、克隆表达及酶学性质

【目的】利用宏基因组学的方法从红树林土壤中筛选新型酯水解酶类。【方法】构建红树林土壤宏基因组文库,采用以三丁酸甘油酯为底物的功能筛选方法,对筛选出的阳性克隆进行系统发育树分析,实现新型磷脂酶A1基因的原核表达,研究重组酶的酶学性质。【结果】筛选到一个新的磷脂酶A1编码基因phop1413 (GenBank登录号KF767097),测序表明其全长1 413 bp,可编码470个氨基酸残基,表达蛋白约51.7 kD,表达量高达220 mg/L,NCBI中Blast比对及系统进化树分析显示该蛋白属于磷脂酶类的fAMILY Ⅵ家族;酶学性质分析表明,该重组酶的最适反应底物为对硝基苯酚己酸酯,比酶活为124 U/mg;最适反应温度为54 °C,最适pH 7.8;50 °C热处理1.5 h剩余相对酶活为44%,表现出很好的热稳定性。【结论】通过构建宏基因组文库利用功能筛选方法获得一个新型磷脂酶A1基因;研究中获得的新型磷脂酶A1性质较好,可用于植物油酶法脱胶。     

Screening and characterization of a cellulase gene from the gut microflora of abalone using metagenomic library

A metagenomic fosmid library was constructed using genomic DNA isolated from abalone intestine. Screening of a library of 3,840 clones revealed a 36 kb insert of a cellulase positive clone (pAMHElO). A shotgun clone library was constructed using the positive clone (pAMHElO) and further screening of 3,840 shotgun clones with an approximately 5 kb insert size using a Congo red overlay revealed only one cellulase positive clone (pAMHL9). The pAMHL9 consisted of a 5,293-bp DNA sequence and three open reading frames (ORFs). Among the three ORFs, cellulase activity was only shown in the recombinant protein (CelAMll) coded by ORF3, which showed 100% identity with outer membrane protein A from Vibrio alginolyticus 12G01, but no significant sequence homology to known cellulases. The expressed protein (CelAMll) has a molecular weight of approximately 37 kDa and the highest CMC hydrolysis activity was observed at pH 7.0 and 37°C. The carboxymethyl cellulase activity was determined by zymogram active staining and different degraded product profiles for CelAMll were obtained when cellotetraose and cellopentaose were used as the substrates, while no substrate hydrolysis was observed on oligosaccharides such as cellobiose and cellotriose.     

Wide variation in antibiotic resistance proteins identified by functional metagenomic screening of a soil DNA library

Most genes for antibiotic resistance present in soil microbes remain unexplored because most environmental microbes cannot be cultured. Only recently has the identification of these genes become feasible through the use of culture-independent methods. We screened a soil metagenomic DNA library in an Escherichia coli host for genes that can confer resistance to kanamycin, gentamicin, rifampin, trimethoprim, chloramphenicol, or tetracycline. The screen revealed 41 genes that encode novel protein variants of eight protein families, including aminoglycoside acetyltransferases, rifampin ADP-ribosyltransferases, dihydrofolate reductases, and transporters. Several proteins of the same protein family deviate considerably from each other yet confer comparable resistance. For example, five dihydrofolate reductases sharing at most 44% amino acid sequence identity in pairwise comparisons were equivalent in conferring trimethoprim resistance. We identified variants of aminoglycoside acetyltransferases and transporters that differ in the specificity of the drugs for which they confer resistance. We also found wide variation in protein structure. Two forms of rifampin ADP-ribosyltransferases, one twice the size of the other, were similarly effective at conferring rifampin resistance, although the short form was expressed at a much lower level. Functional metagenomic screening provides insight into the large variability in antibiotic resistance protein sequences, revealing divergent variants that preserve protein function.     

Construction of a large phage-displayed human antibody domain library with a scaffold based on a newly identified highly soluble, stable heavy chain variable domain

Currently, almost all U.S. Food and Drug Administration-approved therapeutic antibodies and the vast majority of those in clinical trials are full-size antibodies mostly in an immunoglobulin G1 format of about 150 kDa in size. Two fundamental problems for such large molecules are their poor penetration into tissues (e.g., solid tumors) and poor or absent binding to regions on the surface of some molecules [e.g., on the human immunodeficiency virus envelope glycoprotein (Env)] that are accessible by molecules of smaller size. We have identified a phage-displayed heavy chain-only antibody by panning of a large (size, ∼ 1.5 × 10¹⁰) human naive Fab (antigen-binding fragment) library against an Env and found that the heavy chain variable domain (V_H) of this antibody, designated as m0, was independently folded, stable, highly soluble, monomeric, and expressed at high levels in bacteria. m0 was used as a scaffold to construct a large (size, ∼ 2.5 × 10¹⁰), highly diversified phage-displayed human V_H library by grafting naturally occurring complementarity-determining regions (CDRs) 2 and 3 of heavy chains from five human antibody Fab libraries and by randomly mutating four putative solvent-accessible residues in CDR1 to A, D, S, or Y. The sequence diversity of all CDRs was determined from 143 randomly selected clones. Most of these V_Hs were with different CDR2 origins (six of seven groups of V_H germlines) or CDR3 lengths (ranging from 7 to 24 residues) and could be purified directly from the soluble fraction of the Escherichia coli periplasm. The quality of the library was also validated by successful selection of high-affinity V_Hs against viral and cancer-related antigens; all selected V_Hs were monomeric, easily expressed, and purified with high solubility and yield. This library could be a valuable source of antibodies targeting size-restricted epitopes and antigens in obstructed locations where efficient penetration could be critical for successful treatment.     

Codon-optimized expression and characterization of a pH stable fungal xylanase in Pichia pastoris

Novel xylanase (EC 3.2.1.8) is in great demand due to its industrial significance. In this study, we have developed and characterized a novel xylanase-producing yeast strain. This mature xylanase gene xyn11A consists of 870 base pairs and belongs to GH11 family. The gene sequence was optimized and synthesized, and was then cloned into yeast vector pGAPZαA under the control of the constitutive GAP promoter. SDS-PAGE analysis indicates that Xyn11A is extracellularly expressed as a glycosylated protein in P. pastoris. Xyn11A is optimally active at 70 °C and pH 7.4. This xylanase retained more than 90% of its activity after incubation at 50 °C and 60 °C for up to 1 h. Xyn11A is also stable over a wide range of pH (2.0–11.0). Most metal ions tested such as copper (Cu²⁺) and lead (Pb²⁺) have little inhibitory effects on Xyn11A. It is also resistant to pepsin and proteinase K digestion, retaining 80% and 90% of its activity after digestion at 37 °C for 1 h, respectively. Those superior properties make Xyn11A a robust xylanase with great potential for industrial use. To the best of our knowledge, this is the first report of xylanase from the fungus Corynascus thermophilus.     

云南腾冲热泉土壤微生物基因组文库的构建与分析

采用冻融、蛋白酶K、SDS-高盐-加热处理法联合的方法,直接从云南腾冲地区的一个弱碱性高温热泉沉积样品中提取和分离环境混合基因组DNA,产量为每克样品1~2μg DNA,用Promega试剂盒纯化后进行PstⅠ部分酶切处理,电泳回收3~8kb的片段后,构建了pSK( )为载体的基因组文库,共获得25000个阳性克隆,平均插入片段长度为4.6kb。通过随机DNA序列测定和基因注释,发现外源插入片段含有未见报道的序列。