Soil Metagenomic Library Research Articles

Characterization of a novel salt- and solvent-tolerant esterase Dhs82 from soil metagenome capable of hydrolyzing estrogenic phthalate esters

Esterases that can function under extreme conditions are important for industrial processing and environmental remediation. Here, we report the identification of a salt- and solvent-tolerant esterase, Dhs82, from a soil metagenomic library. Dhs82 prefers short-chain p-nitrophenyl (p-NP) esters and exhibits enzymatic activity up to 1460 ± 61 U/mg towards p-NP butyrate. Meanwhile, Dhs82 can catalyze the hydrolysis of dialkyl phthalate esters, especially the widely-used diethyl phthalate (DEP), dipropyl phthalate (DPP) and di-n-butyl phthalate (DBP). Importantly, as an acidic protein with negative charges dominating its surface, Dhs82 is highly active and extraordinarily stable at high salinity. This property is quite rare among previously reported esterases/hydrolases capable of degrading phthalate esters (PAEs). In addition, Dhs82 activity can be significantly enhanced in the presence of solvents over a concentration range of 10–30 % (v/v). Notably, Dhs82 also showed high stability towards these solvents and solvent concentrations as high as 50–60 % (v/v) are required to inactivate Dhs82. Furthermore, molecular docking revealed the key residues, including the catalytic triad (Ser156, His281, and Asp251) and the surrounding Gly84 and Gly85, involved in the interaction of Dhs82 with DBP, depicting how Dhs82 degrades PAEs as a family IV esterase. Together, these diverse properties make Dhs82 a valuable candidate for both basic research and biotechnological applications.

Characterization of a novel amidohydrolase with promiscuous esterase activity from a soil metagenomic library and its application in degradation of amide herbicides.

Amide herbicides have been extensively used worldwide and have received substantial attention due to their adverse environmental effects. Here, a novel amidohydrolase gene was identified from a soil metagenomic library using diethyl terephthalate (DET) as a screening substrate. The recombinant enzyme, AmiH52, was heterologously expressed in Escherichia coli and later purified and characterized, with the highest activity occurring at 40 ℃ and pH 8.0. AmiH52 was demonstrated to have both esterase and amidohydrolase activities, which exhibited highly specific activity for p-nitrophenyl butyrate (2669 U/mg) and degrading activity against several amide herbicides. In particular, it displayed the strongest activity against propanil, with a high degradation rate of 84% at 8h. A GC-MS analysis revealed that propanil was transformed into 3,4-dichloroaniline (3,4-DCA) during this degradation. The molecular interactions and binding stability were then analyzed by molecular docking and molecular dynamics simulation, which revealed that several key amino acid residues, including Tyr164, Trp66, Ala59, Val283, Arg58, His33, His191, and His226, are involved in the specific interactions with propanil. This study provides a function-driven screening method for amide herbicide hydrolase from the metagenomic libraries and a promising propanil-degrading enzyme (AmiH52) for potential applications in environmental remediation.

Screening, cloning, immobilization and application prospects of a novel β-glucosidase from the soil metagenome

The soil environment for straw return is a rich and valuable library containing many microorganisms and proteins. In this study, we aimed to screen a high-quality β-glucosidase (BGL) from the soil metagenomic library and to overcome the limitation of the low extraction rate of resveratrol in Polygonum cuspidatum. This includes the construction of a soil metagenomic library, screening of BGL, bioinformatics analysis, cloning, expression, immobilization, enzymatic property analysis, and application for the transformation of polydatin. The results showed that the soil metagenomic library of straw return was successfully constructed, and a novel BGL was screened. The identified 1356 bp long BGL belonged to the glycoside hydrolase 1 (GH1) family and was named Bgl1356. After successful cloning and expression of Bgl1356, it was immobilized using chitosan. The optimum temperature of immobilized Bgl1356 was 50 °C, and the pH was 5. It exhibited good tolerance for various metal ions (CO2+, Ni2+, Cu2+, Mn2+, Na2+, Ca2+, and Ag+) and organic solvents (DMSO, Triton-X-10, and ethanol). Enzymatic kinetics assays showed that Bgl1356 had good affinity for the substrate, and the specific enzyme activity was 234.03 U/mg. The conversion rate of polydatin by immobilized Bgl1356 was 95.70 ± 1.08%, facilitating the production of high amounts of resveratrol. Thus, this paper reports a novel temperature-, organic solvent-, and metal ion-tolerant BGL that has good application prospects in the pharmaceutical industry.

Discovery and characterization of a novel PKD-Fn3 domains containing GH44 endoglucanase from a Tibetan metagenomic library.

To explore novel microbial endoglucanases with unique properties derived from extreme environments by using metagenomics approach. A Tibetan soil metagenomic library was applied for screening cellulase-active clones by function-based metagenomics. The candidate genes in the active clones were identified through bioinformatic analyses and heterologously expressed using an Escherichia coli system. The recombinant endoglucanases were purified and characterized using enzyme assays to determine their bioactivities, stabilities, substrate specificities, and other enzymatic properties. A novel endoglucanase gene Zfeg1907 was identified, which consisted of a glycoside hydrolase family 44 (GH44) catalytic domain along with a polycystic kidney disease (PKD) domain and a fibronectin type Ⅲ (Fn3) domain at the C terminal. Recombinant enzyme ZFEG1907 and its truncated mutant ZFEG1907t (ΔPKDΔFn3) were successfully expressed and purified. The two recombinants exhibited catalytic activities towards carboxymethyl cellulose, konjac glucomannan and lichenan. Both enzymes had an optimal temperature of 50°C and an optimal pH value of 5.0. The catalytic activities of both recombinant enzymes were promoted by adding Zn2+ and Ca2+ at the final concentration of 10mM. The Km value of ZFEG1907 were lower while the kcat/Km value of ZFEG1907 was higher than those of of ZFEG1907t when using carboxymethyl cellulose, konjac glucomannan and lichenan as substrates. Structure prediction of two recombinants revealed that PKD-Fn3 domains consisted of a flexible linker and formed a β-sandwich structure. A novel endoglucanase ZFEG1907 contained a GH44 catalytic domain and a PKD-Fn3 domain was characterized. The PKD-Fn3 domains were not indispensable for the activity but contributed to the enzyme binding of the polysaccharide substrates as a carbohydrate-binding module (CBM).

Discovery of two bifunctional/multifunctional cellulases by functional metagenomics

Cellulases are widely used in industry, and the usage in bioconversion of biofuels makes cellulases more valuable. In this study, two tandem genes that encoded cellulases ZF994–1 and ZF994–2, respectively, were identified on a cosmid from a soil metagenomic library. Phylogenetic analysis indicated that ZF994–1 and ZF994–2 belonged to glycoside hydrolase family 12 (GH12), and GH3, respectively. Based on the substrate specificity analysis, the recombinant ZF994–1 exhibited weak endoglucanase activity, moderate β-1,3-glucanase and β-1,4-mannanase activities, and strong β-glucosidase activity, while the recombinant ZF994–2 exhibited moderate endoglucanase activity and strong β-glucosidase activity. More than 45% β-glucosidase activity of the recombinant ZF994–1 retained in the buffer containing 3 M glucose, indicating the good tolerance against glucose. The recombinant ZF994–2 showed high activity in the presence of metal ions and organic reagents, exhibiting potential industrial applications.

Mining metagenomes reveals diverse antibiotic biosynthetic genes in uncultured microbial communities

Pathogens resistant to antimicrobials form a significant threat to public health worldwide. Tackling multidrug-resistant pathogens via screening metagenomic libraries has become a common approach for the discovery of new antibiotics from uncultured microorganisms. This study focuses on capturing nonribosomal peptide synthase (NRPS) gene clusters implicated in the synthesis of many natural compounds of industrial relevance. A NRPS PCR assay was used to screen 2976 Escherichia coli clones in a soil metagenomic library to target NRPS genes. DNA extracts from 4 clones were sequenced and subjected to bioinformatic analysis to identify NRPS domains, their phylogeny, and substrate specificity.Successfully, 17 NRPS-positive hits with a biosynthetic potential were identified. DNA sequencing and BLAST analysis confirmed that NRPS protein sequences shared similarities with members of the genus Delftia in the Proteobacteria taxonomic position. Multiple alignment and phylogenetic analysis demonstrated that clones no. 15cd35 and 15cd37 shared low bootstrap values (54%) and were distantly far from close phylogenetic neighbors. Additionally, NRPS domain substrate specificity has no hits with the known ones; hence, they are more likely to use different substrates to produce new diverse antimicrobials. Further analysis confirmed that the NRPS hits resemble several transposon elements from other bacterial taxa, confirming its diversity. We confirmed that the analyses of the soil metagenomic library revealed a diverse set of NRPS related to the genus Delftia. An in-depth understanding of those positive NRPS hits is a crucial step for genetic manipulation of NRPS, shedding light on alternative novel antimicrobial compounds that can be used in drug discovery and hence supports the pharmaceutical sector.

Engineering Modular and Highly Sensitive Cell-Based Biosensors for Aromatic Contaminant Monitoring and High-Throughput Enzyme Screening.

Although a variety of whole-cell-based biosensors have been developed for different applications in recent years, most cannot meet practical requirements due to insufficient sensing performance. Here, we constructed two sets of modular genetic circuits by serial and parallel modes capable of significantly amplifying the input/output signal in Escherichia coli. The biosensors are engineered using σ54-dependent phenol-responsive regulator DmpR as a sensor and enhanced green fluorescent protein as a reporter. Cells harboring serial and parallel genetic circuits displayed nearly 9- and 16-fold higher sensitivity than the general circuit. The genetic circuits enabled rapid detection of six phenolic contaminants in 12 h and showed the low limit of detection of 2.5 and 2.2 ppb for benzopyrene (BaP) and tetracycline (Tet), with a broad detection range of 0.01-1 and 0.005-5 μM, respectively. Furthermore, the positive rate was as high as 73% when the biosensor was applied to screen intracellular enzymes with ester-hydrolysis activity from soil metagenomic libraries using phenyl acetate as a phenolic substrate. Several novel enzymes were isolated, identified, and biochemically characterized, including serine peptidases and alkaline phosphatase family protein/metalloenzyme. Consequently, this study provides a new signal amplification method for cell-based biosensors that can be widely applied to environmental contaminant assessment and screening of intracellular enzymes.

A low-cost, high-throughput microfluidic nano-culture platform for functional metagenomics.

Functional metagenomics is an attractive culture-independent approach for functional screening of diverse microbiomes to identify known and novel genes. Since functional screening can involve sifting through tens of thousands of metagenomic library clones, an easy high-throughput screening approach is desirable. Here, we demonstrate a proof-of-concept application of a low-cost, high-throughput droplet based microfluidic assay to the selection of antibiotic resistance genes from a soil metagenomic library. Metagenomic library members encapsulated in nanoliter volume water-in-oil droplets were printed on glass slides robotically, and cell growth in individual drops in the presence of ampicillin was imaged and quantified to identify ampicillin-resistant clones. From the hits, true positives were confirmed by sequencing and functional validation. The ease of liquid handling, ease of set-up, low cost, and robust workflow makes the droplet-based nano-culture platform a promising candidate for screening and selection assays for functional metagenomic libraries.

Discovery and Biosynthesis of the Amodesmycins, Aromatic Polyketide-Siderophore Hybrid Conjugates.

A type II polyketide synthase biosynthetic gene cluster (amd) containing three P450 genes was identified from a soil metagenomic library, and novel benz[h]isoquinoline-desferrioxamine B conjugated compound amodesmycins were isolated from Streptomyces albus J1074 harboring the amd gene cluster. Genetic evidence showed that the benz[h]isoquinoline part and desferrioxamine B part in amodesmycins were derived from the amd gene cluster and S. albus J1074, respectively, while P450 enzymes played critical roles in the conjunction of these two parts.

Regulated Expression of an Environmental DNA-Derived Type II Polyketide Gene Cluster in Streptomyces Hosts Identified a New Tetracenomycin Derivative TCM Y.

As bacterial natural products have been proved to be the most important source of many therapeutic medicines, the need to discover novel natural products becomes extremely urgent. Despite the fact that the majority of bacterial species are yet to be cultured in a laboratory setting, and that most of the bacterial natural product biosynthetic genes are silent, "metagenomics technology" offers a solution to help clone natural product biosynthetic genes from environmental samples, and genetic engineering enables the silent biosynthetic genes to be activated. In this work, a type II polyketide biosynthetic gene cluster was identified from a soil metagenomic library and was activated by over-expression of a SARP regulator gene in the gene cluster in Streptomyces hosts. A new tetracenomycin type compound tetracenomycin Y was identified from the fermentation broth. This study shows that metagenomics and genetic engineering could be combined to provide access to new natural metabolites.

Crystal structure of glycoside hydrolase family 31 α-xylosidase from a soil metagenome.

MeXyl31, a member of glycoside hydrolase family 31 (GH31), is the α-xylosidase isolated from a soil metagenomic library. The enzyme degrades α-xylosyl substrate such as isoprimeverose, α-d-xylopyranosyl-(1→6)-glucopyranose. The crystal structure of MeXyl31 was determined at 1.80 Å resolution. MeXyl31 forms the tetrameric state. The complexed structure with a xylose in the-1 subsite (α-xylose binding site) shows that the enzyme strictly recognizes α-xylose. Structural comparison between MeXyl31 and its homologue, Aspergillus niger α-xylosidase in GH31, gave insights into the positive subsite of MeXyl31. First, in the tetrameric enzyme, two monomers (a catalytic monomer and the adjacent monomer), are involved in substrate recognition. Second, the adjacent monomer composes a part of positive subsites in MeXyl31. Docking simulation and site-directed mutagenesis suggested that the Arg100 from the adjacent monomer is partially involved in the recognizing of a glucopyranose of isoprimeverose.

Transmission of Escherichia coli from Manure to Root Zones of Field-Grown Lettuce and Leek Plants.

Pathogenic Escherichia coli strains are responsible for food-borne disease outbreaks upon consumption of fresh vegetables and fruits. The aim of this study was to establish the transmission route of E. coli strain 0611, as proxy for human pathogenic E. coli, via manure, soil and plant root zones to the above-soil plant compartments. The ecological behavior of the introduced strain was established by making use of a combination of cultivation-based and molecular targeted and untargeted approaches. Strain 0611 CFUs and specific molecular targets were detected in the root zones of lettuce and leek plants, even up to 272 days after planting in the case of leek plants. However, no strain 0611 colonies were detected in leek leaves, and only in one occasion a single colony was found in lettuce leaves. Therefore, it was concluded that transmission of E. coli via manure is not the principal contamination route to the edible parts of both plant species grown under field conditions in this study. Strain 0611 was shown to accumulate in root zones of both species and metagenomic reads of this strain were retrieved from the lettuce rhizosphere soil metagenome library at a level of Log 4.11 CFU per g dry soil.

Discovery and characterization of a novel protease from the Antarctic soil

The extensive use of proteases in many areas, especially in the textile, food, and detergent industries, has led increases in the discovery and characterization of novel proteolytic enzymes. Microorganism from the Antarctic may contain novel proteases because of the extreme environment; however, to our knowledge, few relevant studies have been reported to date. Here, an Antarctic soil metagenomic fosmid library was successfully constructed, and a 36 kb positive fosmid clone was screened using lactose-free skim milk agar. Sequence alignment and phylogenetic analysis indicated that the positive fosmid clone encoded a new protein belonging to the metalloprotease subfamily M43B (36.6 % identity), which was named as Pr18H-2. Enzymatic analysis revealed that Pr18H-2 possess several features: (1). it demonstrated high thermal stability with the optimal temperature was 60 °C; (2). it was active in the broad pH range of 5.0–10.0, with the optimal pH of 8.0 which allowed it to be an alkaline protease; (3). it was able to degrade a wide range of protein substrates and exhibit the substrate preferences for small substrates, such as casein hydrolysate and fibrin; (4). it showed that the enzymatic activity was increased by approximately four times in the presence of 5 mM Co2+.

Identification and characterization of a novel endo-β-1,4-glucanase from a soil metagenomic library

A cosmid clone cZFYN1413 with CMCase activity was identified from a soil metagenomic library. The sequence analysis of a subclone of cZFYN1413 revealed an endo-β-1,4-glucanase gene ZFYN1413 belonging to glycoside hydrolase family 6 and a transmembrane region in the N-terminal of ZFYN1413. Expression of ZFYN1413 in Escherichia coli BL21 (DE3) resulted in ZFYN1413-87, which was a truncated protein cleaved in transmembrane region of ZFYN1413. ZFYN1413-87 was expressed and its enzyme properties were studied. ZFYN1413-87 possessed strong endo-β-1,4-glucanase activity, and 52% of the activity could be retained after the protein was treated in buffer of pH 3.0 for 2 h. The study provided a special example of endo-β-1,4-glucanase in GH6 family.

Metagenome-Guided Analogue Synthesis Yields Improved Gram-Negative-Active Albicidin- and Cystobactamid-Type Antibiotics.

Natural products are a major source of new antibiotics. Here we utilize biosynthetic instructions contained within metagenome-derived congener biosynthetic gene clusters (BGCs) to guide the synthesis of improved antibiotic analogues. Albicidin and cystobactamid are the first members of a new class of broad-spectrum ρ-aminobenzoic acid (PABA)-based antibiotics. Our search for PABA-specific adenylation domain sequences in soil metagenomes revealed that BGCs in this family are common in nature. Twelve BGCs that were bio-informatically predicted to encode six new congeners were recovered from soil metagenomic libraries. Synthesis of these six predicted structures led to the identification of potent antibiotics with changes in their spectrum of activity and the ability to circumvent resistance conferred by endopeptidase cleavage enzymes.

Metagenome‐Guided Analogue Synthesis Yields Improved Gram‐Negative‐Active Albicidin‐ and Cystobactamid‐Type Antibiotics

AbstractNatural products are a major source of new antibiotics. Here we utilize biosynthetic instructions contained within metagenome‐derived congener biosynthetic gene clusters (BGCs) to guide the synthesis of improved antibiotic analogues. Albicidin and cystobactamid are the first members of a new class of broad‐spectrum ρ‐aminobenzoic acid (PABA)‐based antibiotics. Our search for PABA‐specific adenylation domain sequences in soil metagenomes revealed that BGCs in this family are common in nature. Twelve BGCs that were bio‐informatically predicted to encode six new congeners were recovered from soil metagenomic libraries. Synthesis of these six predicted structures led to the identification of potent antibiotics with changes in their spectrum of activity and the ability to circumvent resistance conferred by endopeptidase cleavage enzymes.

A chlorogenic acid esterase from a metagenomic library with unique substrate specificity and its application in caffeic and ferulic acid production from agricultural byproducts

Soil microbes are an abundant source of enzymes with unique properties that may be useful for industrial applications. As most wild-type strains show low chlorogenic acid esterase expression and activity, and most microbes cannot be cultured in the laboratory, a metagenomic approach provides methods of identifying new enzymes. In this study, a gene encoding a chlorogenic acid esterase, named Tan410, was isolated from a soil metagenomic library and overexpressed in Escherichia coli BL21 (DE3). The recombinant enzyme, with a predicted molecular weight of 54.88 kDa, was purified to homogeneity. The K m and V max values for Tan410 were 1.26 mM and 0.33 mM min–1, respectively, with chlorogenic acid as the substrate. Its optimum temperature and pH for reaction were 30 °C and 7.5, respectively. The enzyme exhibited moderate thermostability and broad pH stability (3.0–10.0). Tan410 was also able to hydrolyse ethyl ferulate, methyl caffeate, propyl gallate, ethyl gallate, methyl vanillate, methyl benzoate, ethyl benzoate, methyl 2,5-dihydroxybenzoate, and methyl 3,5-dihydroxybenzoate, and it released caffeic and ferulic acids from agricultural byproducts (destarched wheat bran and coffee pulp). Tan140 has potential for industrial application in biomass valorization.

Identification and Characterization of a Novel Carboxylesterase Belonging to Family VIII with Promiscuous Acyltransferase Activity Toward Cyanidin-3-O-Glucoside from a Soil Metagenomic Library.

An alkaline esterase, designated as EstXT1, was identified through functional screening from a metagenomic library. Sequence analysis revealed that EstXT1 belonged to the family VIII carboxylesterases and contained a characteristic conserved S-x-x-K motif and a deduced catalytic triad Ser56-Lys59-Tyr165. EstXT1 exhibited the strongest activity toward methyl ferulate at pH 8.0 and temperature 55°C and retained over 80% of its original activity after incubation in the pH range of 7.0-10.6 buffers. Biochemical characterization of the recombinant enzyme showed that it was activated by Zn2+ and Co2+ metal ion, while inhibited by Cu2+ and CTAB. EstXT1 exhibited significant promiscuous acyltransferase activity preferred to the acylation of benzyl alcohol acceptor using short-chain pNP-esters (C2-C8) as acyl-donors. A structure-function analysis indicated that a WAG motif is essential to acyltransferase activity. This is the first report example that WAG motif plays a pivotal role in acyltransferase activity in family VIII carboxylesterases beside WGG motif. Further experiment indicated that EstXT1 successfully acylated cyanidin-3-O-glucoside in aqueous solution. The results from the current investigation provided new insights for the family VIII carboxylesterase and lay a foundation for the potential applications of EstXT1 in food and biotechnology fields.

Characterization of a novel isoflavone glycoside-hydrolyzing β-glucosidase from mangrove soil metagenomic library

For the beneficial pharmacological properties of isoflavonoids and their related glycoconjugates, there is increasingly interest in their enzymatic conversion. In this study, a novel β-glucosidase gene isolated from metagenomic library of mangrove sediment was cloned and overexpressed in Escherichia coli BL21(DE3). The purified recombination β-glucosidase, designated as r-Bgl66, showed high catalytic activity for soy isoflavone glycosides. It converted soy isoflavone flour extract with the productivities of 0.87 mM/h for daidzein, 0.59 mM/h for genistein and 0.42 mM/h for glycitein. The kcat/Km values for daidzin, genistin and glycitin were 208.73, 222.37 and 288.07 mM−1 s−1, respectively. In addition, r-Bgl66 also exhibited the characteristic of glucose-tolerance, and the inhibition constant Ki was 471.4 mM. These properties make it a good candidate in the enzymatic hydrolysis of soy isoflavone glycosides. This study also highlights the utility of metagenomic approach in discovering novel β-glucosidase for soy isoflavone glycosides hydrolysis.

Identification and characterization of a novel carboxylesterase EstQ7 from a soil metagenomic library

A novel lipolytic gene, estq7, was identified from a fosmid metagenomic library. The recombinant enzyme EstQ7 consists of 370 amino acids with an anticipated molecular mass of 42kDa. Multiple sequence alignments showed that EstQ7 contained a pentapeptide motif GHSMG, and a putative catalytic triad Ser174-Asp306-His344. Interestingly, EstQ7 was found to have very little similarity to the characterized lipolytic enzymes. Phylogenetic analysis revealed that EstQ7 may be a member of a novel family of lipolytic enzymes. Biochemical characterization of the recombinant enzyme revealed that it constitutes a slightly alkalophilic, moderate thermophilic and highly active carboxylesterase against short-chain fatty acid esters with optimum temperature 50 ℃ and pH 8.2. The Km and kcat values toward p-nitrophenyl acetate were determined to be 0.17mM and 1910s-1, respectively. Moreover, EstQ7 was demonstrated to have acyltransferase activity by GC-MS analysis. Structural modeling of the three-dimensional structure of this new enzyme showed that it exhibits a typical α/β hydrolase fold, and the catalytic triad residues are spatially close. Molecular docking revealed the interactions between the enzyme and the ligand. The high levels of lipolytic activity of EstQ7, combined with its moderate thermophilic property and acyltransferase activity, render this novel enzyme a promising candidate biocatalyst for food, pharmaceutical and biotechnological applications.