The "depict" strategy for discovering new compounds in complex matrices: Lycibarbarspermidines as a case.

The comprehensive detection and identification of active ingredients in complex matrices is a crucial challenge. Liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) is the most prominent analytical platform for the exploration of novel active compounds from complex matrices. However, the LC-HRMS-based analysis workflow suffers from several bottleneck issues, such as trace content of target compounds, limited acquisition for fragment information, and uncertainty in interpreting relevant MS2 spectra. Lycibarbarspermidines are vital antioxidant active ingredients in Lycii Fructus, while the reported structures are merely focused on dicaffeoylspermidines due to their low content. To comprehensively detect the new structures of lycibarbarspermidine derivatives, a "depict" strategy was developed in this study. First, potential new lycibarbarspermidine derivatives were designed according to the biosynthetic pathway, and a comprehensive database was established, which enlarged the coverage of lycibarbarspermidine derivatives. Second, the polarity-oriented sample preparation of potential new compounds increased the concentration of the target compounds. Third, the construction of the molecular network based on the fragmentation pathway of lycibarbarspermidine derivatives broadened the comprehensiveness of identification. Finally, the weak response signals were captured by data-dependent scanning (DDA) followed by parallel reaction monitoring (PRM), and the efficiency of acquiring MS2 fragment ions of target compounds was significantly improved. Based on the integrated strategy above, 210 lycibarbarspermidine derivatives were detected and identified from Lycii Fructus, and in particular, 170 potential new compounds were structurally characterized. The integrated strategy improved the sensitivity of detection and the coverage of low-response components, and it is expected to be a promising pipeline for discovering new compounds.

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

AIMS: To explore novel microbial endoglucanases with unique properties derived from extreme environments by using metagenomics approach. METHODS AND RESULTS: 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 toward carboxymethyl cellulose, konjac glucomannan (KGM), 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 10 mM. The Km value of ZFEG1907 was lower, while the kcat/Km value of ZFEG1907 was higher than those of of ZFEG1907t when using carboxymethyl cellulose, KGM, and lichenan as substrates. Structure prediction of two recombinants revealed that PKD-Fn3 domains consisted of a flexible linker and formed a ß-sandwich structure. CONCLUSIONS: 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 ß-1,3-glucanase and ß-1,4-mannanase activities, and strong ß-glucosidase activity, while the recombinant ZF994-2 exhibited moderate endoglucanase activity and strong ß-glucosidase activity. More than 45% ß-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.

O-methyltransferases selectively modify anthraquinone natural products.

In this issue of Structure, Huber et al. identify five O-methyltransferases, and three of them catalyze the sequential methylation of the Gram-negative bacterium-derived aromatic polyketide anthraquinone AQ-256. They present co-crystal structures with bound AQ-256 and its methylated derivatives, which explains the specificities of these O-methyltransferases.

A funnel-type stepwise filtering strategy for identification of potential Q-markers of traditional Chinese medicine formulas.

Quality marker (Q-marker) serves as an important driver for the standardization of quality control in traditional Chinese medicine (TCM) formulas. However, it is still challenging to discover comprehensive and representative Q-markers. This study aimed to identify Q-markers of Hugan tablet (HGT), a famous TCM formula with ideal clinical effects in liver diseases. Here, we proposed a funnel-type stepwise filtering strategy that integrated secondary metabolites characterization, characteristic chromatogram, quantitative analysis, literature mining, biotransformation rules and network analysis. Firstly, the strategy of "secondary metabolites-botanical drugs-TCM formula" was applied to comprehensively identify the secondary metabolites of HGT. Then, the secondary metabolites with specificity and measurability in each botanical drug were identified by HPLC characteristic chromatogram, biosynthesis pathway and quantitative analysis. Based on literature mining, the effectiveness of botanical metabolites that met the above conditions was evaluated. Furthermore, the metabolism of the above metabolites in vivo was studied to reveal their biotransformation forms, which were used for network analysis. At last, according to biotransformation rules of the prototype drugs in vivo, the secondary metabolites were traced and preliminarily chosen as Q-markers. As a result, 128 plant secondary metabolites were identified in HGT, and 11 specific plant secondary metabolites were screened out. Then, the content of specific plant secondary metabolites in 15 batches of HGT was determined, which confirmed their measurability. And the results of literature mining showed that eight secondary metabolites had therapeutic effects in treating liver disease at the in vivo level, and three secondary metabolites inhibited liver disease-related indicators at the in vitro level. After that, 26 compounds absorbed into the blood (11 specific plant metabolites and their 15 metabolites in vivo) were detected in rats. Moreover, 14 compounds, including prototype components and their metabolites, were selected as Q-marker candidates by the "TCM formula-botanical drugs-compounds-targets-pathways" network. Finally, 9 plant secondary metabolites were defined as comprehensive and representative Q-markers. Our study not only provides a scientific basis for the improvement and secondary development of the quality standard of HGT, but also proposes a reference method for discovering and identifying Q-markers of TCM preparations.

Ultrasensitive SERS substrate for label-free therapeutic drug monitoring of chlorpromazine hydrochloride and aminophylline in human serum.

Surface-enhanced Raman spectroscopy (SERS) has been widely used in the field of therapeutic drug monitoring (TDM) because of its powerful fingerprinting capability. In this paper, we used an in situ synthesis method to anchor Ag nanoparticles (AgNPs) on the surface of MIL-101(Cr) to obtain MIL-101(Cr)@Ag. Owing to the large specific surface area and ultra-high porosity of MIL-101(Cr)@Ag, we developed a method for the determination of chlorpromazine hydrochloride (CPZ) and aminophylline (AMP) in human serum by using it as a solid-phase extraction sorbent and SERS substrate. The label-free TDM-SERS method was able to evaluate the levels of CPZ and AMP in serum samples with detection limits as low as 8.91 × 10-2 µg/mL and 3.4 × 10-2 µg/mL, respectively. In addition, influencing factors including sample solution pH, AgNO3 concentration, drug adsorption time, and the amount of sample solution were optimized. This protocol provides a new method with good selectivity, stability, reproducibility, homogeneity, and sensitivity for the determination of small-molecule drug content in serum samples. This label-free TDM-SERS method will help to achieve rapid individualized dosing regimens in clinical practice and has potential applications in the field of TDM.

Magnetic/Zeolitic Imidazolate Framework-67 Nanocomposite for Magnetic Solid-Phase Extraction of Five Flavonoid Components from Chinese Herb Dicranopteris pedata.

A magnetically functionalized Fe3O4@ZIF-67 metal-organic framework (MOF) was prepared by electrostatic self-assembly using magnetic Fe3O4 nanoparticles as the core and ZIF-67 as the shell. The composite was characterized by electron microscopy, X-ray diffraction, Fourier- transform infrared spectroscopy, and Brunauer-Emmett-Teller measurements. Magnetic solid-phase extraction (MSPE) was performed on five flavonoids from Dicranopteris pedata using Fe3O4@ZIF-67 as an adsorbent. The developed MSPE method was combined with high-performance liquid chromatography-ultraviolet detection to preconcentrate and separate five flavonoids (rutin, quercitrin, kaempferol-3-O-α-L-rhamnoside, quercetin, and kaempferol) from Dicranopteris pedata. The factors affecting the extraction, such as the amount of Fe3O4@ZIF-67 adsorbent, salt ion concentration in the sample solution, vortex time, type and amount of desorbing solvent, concentration of formic acid to acidify the desorbing solvent, and acetonitrile ratio, were optimized. The developed method showed good linearity over the concentration range of 1.09-70.0 µg∙mL-1 for the five flavonoids, with R2 values between 0.9901 and 0.9945. The limits of detection and average recoveries for the five flavonoids were in the ranges of 39.5-56.2 ng∙mL-1 and 92.2-100.7%, respectively. The method presented herein is simple, efficient, and sensitive; it can be used for enrichment analysis of the five flavonoids in Dicranopteris pedata.

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.

Biosynthesis of coelulatin for the methylation of anthraquinone featuring HemN-like radical S-adenosyl-L-methionine enzyme.

Bacterial aromatic polyketides are usually biosynthesized by the type II polyketide synthase (PKS-II) system. Advances in deoxyribonucleic acid (DNA) sequencing, informatics, and biotechnologies have broadened opportunities for the discovery of aromatic polyketides. Meanwhile, metagenomics is a biotechnology that has been considered as a promising approach for the discovery of novel natural products from uncultured bacteria. Here, we cloned a type II polyketide biosynthetic gene cluster (BGC) from the soil metagenome, and the heterologous expression of this gene cluster in Streptomyces coelicolor M1146 resulted in the production of three anthraquinones, two of which (coelulatins 2 and 3) had special hydroxymethyl and methyloxymethyl modifications at C2 of the polyketide scaffold. Gene deletion and in vitro biochemical characterization indicated that the HemN-like radical S-adenosyl-L-methionine (SAM) enzyme CoeI exhibits methylation and is involved in C2 modification.

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.

Novel amikacin resistance genes identified from human gut microbiota by functional metagenomics.

AIMS: The aim of this study was to evaluate the diversity and potential for horizontal transfer of amikacin resistance genes from the human gut. METHODS AND RESULTS: A library of human faecal microbiota was constructed and subjected to functional screening for amikacin resistance. In total, five amikacin resistance genes that conferred relatively high amikacin resistance, with minimum inhibitory concentrations (MICs) ranging from 64 to >512, were identified from the library, including a novel aminoglycoside acetyltransferase gene and a 16S rRNA methyltransferase (MTase) gene, labelled aac (6')-Iao and rmtI, respectively. AAC(6')-Iao showed the highest identity of 48% to AAC(6')-Ian from a clinical isolate Serratia marcescens, whereas RmtI shared the closest amino acid identity of 32% with ArmA from Klebsiella pneumonia. The MICs of these five subclones to six commonly used aminoglycosides were determined. Susceptibility analysis indicated that RmtI was associated with high resistance phenotype to 4,6-disubstituted 2-DOS aminoglycosides, whereas AAC(6')-Iao conferred resistance to amikacin and kanamycin. In addition, kinetic parameters of AAC(6')-Iao were determined, suggesting a strong catalytic effect on amikacin and kanamycin. CONCLUSIONS: Antibiotic resistance genes with low identity to known sequences can be uncovered by functional metagenomics. In addition, the diversity and prevalence of amikacin resistance genes merit further investigation in extended habitats, especially the 16S rRNA MTase gene that might have been underestimated in previous cognition. SIGNIFICANCE AND IMPACT OF STUDY: Two novel amikacin resistance genes were identified in this study, including a 16S rRNA methyltransferase gene rmtI and an aminoglycoside acetyltransferase gene aac(6')-Iao. This work would contribute to the in-depth study of the diversity and horizontal transfer potential of amikacin resistance genes in the microbiome of the human gut.

Identification and characterization of a novel endo-ß-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-ß-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-ß-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-ß-1,4-glucanase in GH6 family.

Identification and heterologous expression of an isoquinolinequinone biosynthetic gene cluster from Streptomyces albus J1074.

Nitrogen heterocycle small molecules display various pharmaceutically important bioactivities and have great potential in drug development and application. Microbes are an important source for discovering nitrogen heterocycle natural products, and the elucidation of their biosynthetic pathways in microbes facilitates genetic manipulation of new nitrogen heterocycle products. In this study, we isolated three isoquinolinequinones from a Streptomyces albus J1074 conjugant and identified their biosynthetic gene cluster in the S. albus J1074 genome. The function of the biosynthetic gene cluster was confirmed by heterologous expression of the gene cluster in S. coelicolor M1146. This study uncovered a new biosynthetic machinery to produce nitrogen heterocycle natural products in microbes.

Salumycin, a New Pyrazolequinone from a Streptomyces albus J1074 Mutant Strain.

Heterocyclic natural products with various bioactivities play significant roles in pharmaceuticals. Here, we isolated a heterocyclic compound salumycin (1) from a Streptomyces albus J1074 mutant strain. The structure of (1) was elucidated via single-crystal X-ray diffraction, mass spectrometry (MS), fourier transform infrared spectrometer (FTIR), and nuclear magnetic resonance (NMR) data analysis. Salumycin (1) contained a novel pyrazolequinone ring, which had never been previously reported in a natural product. Salumycin (1) exhibited moderate 2,2'-diphenyl-1-picrylhydrazyl (DPPH)-radical scavenging activity (EC50 = 46.3 ± 2.2 µM) compared with tert-butylhydroquinone (EC50 = 4.7 ± 0.3 µM). This study provides a new example of discovering novel natural products from bacteria.

Identification and characterization of a novel bifunctional cellulase/hemicellulase from a soil metagenomic library.

Microbes, especially the uncultured microbes, have been considered as an important resource for discovery of novel cellulases. In this study, a novel bifunctional cellulase/hemicellulase (ZFYN184) was identified by functional screening of a soil metagenomic library. Sequence analysis indicated that ZFYN184 shared at best 39% identity with glycoside hydrolase family 44 (GH44) proteins and contained a glutamic acid residue at 235 acting as the catalytic proton donor in hydrolysis of polysaccharides. The recombinant ZFYN184 was expressed in Escherichia coli BL21 (DE3), and the biochemical profiles of the enzyme, including optimum pH and temperature, pH and thermal stabilities, tolerance to various additives, and substrate specificity, were determined. ZFYN184 possessed strong endo-ß-1,4-glucanase and endo-1,4-ß-mannanase activities, as well as weak xylanase activity, while all these hydrolytic activities were derived from a single catalytic domain in this GH44 enzyme. KEY POINTS: • Discovery a novel bifunctional glycosyl hydrolase from uncultured microorganism. • ZFYN184 contains a single catalytic domain belonged to GH44.

A low degenerate primer pool improved the efficiency of high-efficiency thermal asymmetric interlaced PCR to amplify T-DNA flanking sequences in Arabidopsis thaliana.

We employed hi-TAIL-PCR to identify T-DNA loci in our Arabidopsis activation tagging library and only a total of 28 (39%) insertion sites from 72 samples were characterized when the recommended primer pools, C1 and C2 were used. By comparison, we found C1 harboring relatively low degeneracy was more efficient to amplify the flanking sequences of T-DNA insertion than C2. We replaced the degenerate sequences in long arbitrary degenerate (LAD) primers with a piece of 16-bp degenerate sequence originally used in TAIL-PCR, which had the relatively low degeneracy. Our results showed that the new LAD primer pool N increased the valid amplifications and a total of 37 (51%) T-DNA loci were identified, indicating a more effective amplification of T-DNA flanking sequences in A. thaliana.

[Discovery and analysis of Staphylococcus aureus biofilm inhibitors using functional metagenomics approach].

Although most microbes are not readily cultured in the lab, microbial DNA can be extracted directly from an environmental sample and be functionally expressed in a suitable host for natural products discovery, and this approach has been termed "metagenomics". An E'mei Mountain soil metagenomic library was constructed using an Escherichia coli-Streptomyces shuttle vector for functional based screening of anti-bacterial clones in Streptomyces albus host. Two active clones were obtained and their fermentation broths were studied for the inhibitory effect on Staphylococcus aureus biofilm. Their fermentation products have a good inhibitory effect on the formation of S. aureus biofilm, and the inhibitory effect could exceed 90% when the concentration of sample was 2 MIC (Minimum Inhibitory Concentration). In addition, two samples had significantly effect on S. aureus biofilm dispersal, and the clearance rate of EM110 was higher than EM123. In conclusion, substances with strong bioactivities on biofilm formation and dispersal of S. aureus could be discovered by using metagenomics technology.

Tetracycline Resistance Genes Identified from Distinct Soil Environments in China by Functional Metagenomics.

Soil microbiota represents one of the ancient evolutionary origins of antibiotic resistance and has been increasingly recognized as a potentially vast unstudied reservoir of resistance genes with possibilities to exchange with pathogens. Tetracycline resistance is one of the most abundant antibiotic resistances that may transfer among clinical and commensal microorganisms. To investigate tetracycline resistance genes from soil bacteria in different habitats, we performed functional analysis of three metagenomic libraries derived from soil samples collected from Yunnan, Sichuan, and Tibet, respectively, in China. We found efflux transporter genes form all the libraries, including 21 major facilitator superfamily efflux pump genes and one multidrug and toxic compound extrusion (MATE) transporter gene. Interestingly, we also identified two tetracycline destructase genes, belonging to a newly described family of tetracycline-inactivating enzymes that scarcely observed in clinical pathogens, from the Tibet library. The inactivation activity of the putative enzyme was confirmed in vitro by biochemical analysis. Our results indicated that efflux pumps distributed predominantly across habitats. Meanwhile, the mechanism of enzymatic inactivation for tetracycline resistance should not be neglected and merits further investigation.

Heterologous Production of the Marine Myxobacterial Antibiotic Haliangicin and Its Unnatural Analogues Generated by Engineering of the Biochemical Pathway.

Despite their fastidious nature, marine myxobacteria have considerable genetic potential to produce novel secondary metabolites. The marine myxobacterium Haliangium ochraceum SMP-2 produces the antifungal polyketide haliangicin (1), but its productivity is unsatisfactory. The biosynthetic gene cluster hli (47.8 kbp) associated with 1 was identified and heterologously expressed in Myxococcus xanthus to permit the production of 1 with high efficiency (tenfold greater amount and threefold faster in growth speed compared with the original producer), as well as the generation of bioactive unnatural analogues of 1 through gene manipulation. A unique acyl-CoA dehydrogenase was found to catalyse an unusual γ,δ-dehydrogenation of the diketide starter unit, leading to the formation of the terminal alkene moiety of 1. Biological evaluation of the analogues obtained through this study revealed that their bioactivities (anti-oomycete and cytotoxic activities) can be modified by manipulating the vinyl epoxide at the terminus opposite the ß-methoxyacrylate pharmacophore.

Global biogeographic sampling of bacterial secondary metabolism.

Recent bacterial (meta)genome sequencing efforts suggest the existence of an enormous untapped reservoir of natural-product-encoding biosynthetic gene clusters in the environment. Here we use the pyro-sequencing of PCR amplicons derived from both nonribosomal peptide adenylation domains and polyketide ketosynthase domains to compare biosynthetic diversity in soil microbiomes from around the globe. We see large differences in domain populations from all except the most proximal and biome-similar samples, suggesting that most microbiomes will encode largely distinct collections of bacterial secondary metabolites. Our data indicate a correlation between two factors, geographic distance and biome-type, and the biosynthetic diversity found in soil environments. By assigning reads to known gene clusters we identify hotspots of biomedically relevant biosynthetic diversity. These observations not only provide new insights into the natural world, they also provide a road map for guiding future natural products discovery efforts.