Cellulases are crucial biocatalysts with extensive industrial applications, yet their study has been constrained by cultivation limitations of native microorganisms. Here, we report the discovery and characterization of a novel multifunctional cellulase (ZF580) from the extreme environment of Mount Everest using metagenomic approaches. Functional screening revealed ZF580's unique capacity to hydrolyze diverse substrates, including 4-nitrophenyl-β-D-glucopyranoside (pNPG), chitin, microcrystalline cellulose, and carboxymethyl cellulose sodium (CMC-Na). Phylogenetically, ZF580 forms an independent clade distinct from characterized β-glucosidases and known glycoside hydrolase (GH) families, suggesting its classification as a progenitor of a novel GH lineage. Structural modeling revealed a distinctive (β/α)8 TIM-barrel fold, diverging from canonical GH family architectures. Crucially, truncation analysis and site-directed mutagenesis identified the previously uncharacterized Domain of Unknown Function 5916 (DUF5916) as a catalytic functional region, with residue E373 serving as its essential proton donor. This study provides the first experimental evidence of DUF5916's enzymatic activity, redefining it as a novel catalytic domain. Overall, these findings suggest that ZF580 is a cellulolytic enzyme with β-glucosidase activity and that DUF5916 forms its catalytic core, offering insights that may be valuable for future studies on enzyme function and engineering.

Cellulases are crucial for converting biomass into renewable energy. Despite extensive research, there remains a significant industrial demand for novel cellulases, particularly those with multi-substrates catalytic activity. This study aimed to identify and characterize a novel cellulase from a high-altitude soil metagenome library using functional screening method. A novel 1218-bp GH6 family hydrolase gene, designated zfy1641, was identified from a Mount Everest soil library. Bioinformatics analysis indicated that it encoded a 405-amino-acid protein (43.7 kDa) and was classified into glycoside hydrolase family 6 (GH6). The target glycoside hydrolase gene was cloned and heterologously expressed, then the recombinant protein was purified, and its biochemical properties and kinetic parameters were characterized. The purified recombinant enzyme exhibited broad substrate specificity, demonstrating significant activity against carboxymethyl cellulose (CMC-Na; 69.87 ± 0.13 U/mg), locust bean gum (125.56 ± 0.18 U/mg) and chitin (77.06 ± 0.08 U/mg). ZFY1641 represented a novel member of the GH6 family, that exhibited detectable reducing sugar release from chitin-a function not previously documented for this family. Moreover, ZFY1641 demonstrated optimal activity at 50°C and pH 5.0, and exhibited moderate thermal stability, tolerance to selected metal ions, and halophilicity under the conditions tested. These characteristics suggest potential utility of ZFY1641 in industrial processes, though further validation is required. This work expanded the substrate diversity of GH6 family enzymes and provided a foundation for the development of new enzymatic preparations with a novel multi-functional GH6 family enzyme.

A type II polyketide biosynthetic gene cluster (agc) was identified from a soil metagenomic library. The gene cluster harbors several distinct oxidoreductase genes, suggesting that the heterologous expression of the agc gene cluster could yield novel polycyclic aromatic polyketides featuring unique redox-driven modifications. Eight angucycline derivatives were isolated from Streptomyces albus J1074 harboring the agc gene cluster, including two new S-bridged acetylcysteine-angucycline compounds, angucystemycins (1-2), a new angucycline congener, emycin H (3), along with five known analogues, rubiginone B2 (4), emycin C (5), rubiginone B1 (6), ochromycinone (7), and emycin A (8). Their structures were elucidated based on detailed High-Resolution Electrospray Ionization Mass Spectrometry and 1D and 2D NMR spectroscopy. The proposed biosynthetic pathway of angucystemycins indicated that the angucycline core and the acetylcysteine moiety are derived from the agc biosynthetic gene cluster and S. albus J1074, respectively. In addition, emycin H (3) and emycin C (5) exhibited inhibitory activity against Bacillus subtilis 168 and B. pumilus CMCC 63202. Structural analysis suggested that the saturated bond between C-5 and C-6 contributes to the activity, whereas the introduction of a C-8 O-methyl group diminishes the antimicrobial activity of the compounds of this structural class, implying a potential structure-activity relationship.

A novel actinomycete strain, Streptomyces sp. NRRL S-1813 was employed to study its secondary metabolites under different mediums to activate its cryptic gene clusters and produce antimicrobial secondary metabolites. During fermentation optimization, and purification, oxazolomycin A and oxazolomycin A2 were isolated from one strain simultaneously. Their structure was elucidated using a series of characterization techniques, including full wavelength scanning, mass spectrometry (MS), and nuclear magnetic resonance (NMR) spectroscopy. Oxazolomycin A2 was found not to be a typical enzymatic product of fermentation process. Instead, a spontaneous, non-enzymatic ring cleavage reaction was identified as mechanism for conversion of oxazolomycin A to oxazolomycin A2. Basing on these results, if the target product is oxazolomycin A2, the best fermentation condition of Streptomyces sp. NRRL S-1813 should be the Medium B under the alkalescence condition. For the biosynthesis of oxazolomycin A, the medium pH and reaction time were both important. A slightly acidic environment suppresses the side reactions such as hydrolysis of product, while reasonable reaction time minimizes accumulation of byproducts.

Metagenomics is increasingly recognized as a vital technique for exploring uncultured microorganisms, with one key application being the discovery of novel enzymes for industrial use. This study identified an endoglucanase gene from soil metagenome, termed ZFEG1801, which was expressed in E. coli BL21, purified, and characterized for its biochemical properties. The 72.8 kDa recombinant protein exhibited hydrolytic activity against sodium carboxymethyl cellulose (CMC) and konjac glucomannan (KG), with activities of 12.1 U/mg and 42.1 U/mg, respectively. The enzyme displayed optimal activity at pH 5 for CMC and pH 6 for KG, with broad pH stability ranging from 5 to 9. The optimal temperature was 40 °C, and it remained thermally stable between 20 and 40 °C, retaining over 60

Environmental microorganisms express enzymes with unique hydrolytic activity, stability, and kinetic parameters, which are of great interest for biotechnological applications. In this study, two novel endoglucanases, ZFEG1605 and ZFEG1663, were cloned from Mt. Everest soil metagenomic library, heterologously expressed in E. coli BL21(DE3), and characterized. Both enzymes exhibited high activity on konjac glucommanan (KG) and sodium carboxymethylcellulose (CMC), while ZFEG1605 also exhibited activity towards guar gum (GG). The optimal pH for both enzymes was slightly shifted toward acidic range (pH 5/6). The optimal reaction temperatures for ZFEG1605 and ZFEG1663 were 50 and 40 °C, respectively. ZFEG1605 was more thermostable than ZFEG1663 as it remained stable up to 50 °C, compared to 40 °C for ZFEG1663. Both enzymes showed broad pH stability, although they retained more mannanase activity than CMCase activity within the same pH range. The endoglucanases exhibited remarkable salt tolerance, retaining over 70 % of their enzymatic activity in the presence of 2.5 M NaCl. The purified enzymes hydrolyzed alkali-pretreated rice straw to release reducing sugars, demonstrating their potential usage for biomass saccharification.

[Objectives]This paper aimed to study the resistance mechanism of metagenome derived tetracycline destructase Tet（64）and analyze the gene environment of tet（64） in order to lay a foundation for monitoring the transmission of tetracycline-destroying enzyme genes and the development of antibiotics and adjuvants. [Methods]The degradation activity of Tet（64）on tetracycline was confirmed by biochemical reaction in vitro,and then the degradation products were analyzed by liquid chromatograph-mass spectrometer（LC-MS）to elucidate the resistance mechanism of the tetracycline destructase. The key amino acid residues of Tet（64）binding to tetracycline and the possibility of horizontal transfer of tet（64） were discussed by bioinformatics including multiple sequence alignment, phylogenetic tree construction, and molecular docking methods. [Results]Sequence analysis of MQ776 clone revealed that two IS1 insertions were located upstream of tet（64）,implying the potential transfer risk of the tetracycline destructase gene. Tet（64）was a flavin-dependent monooxygenase, and shared identities of 18.93% and approximate 70% with the first discovered tetracycline destructase Tet（X）and soil-derived tetracycline destructases Tet（47）-Tet（56）,respectively. The predicted 3D structure modeled by SWISS-MODEL revealed that Tet（64）had a similar structure with the soil-derived tetracycline destructase Tet（50）,which contained a tetracycline-binding domain, a FAD-binding domain, and two α-helices. Tet（64）degraded tetracycline into two products with m/z of 461.0 in vitro,which was same as that of Tet（X）. Thus, it was implied that Tet（64）hydroxylated the C11a site of tetracycline to destroy the β-diketone moiety（C11 and C12）,resulting in the failure of chelating magnesium ions and the loss of antibacterial activity. [Conclusions]The degradation activity of the tetracycline destructase Tet（64）on tetracycline was confirmed, and the resistance mechanism of Tet（64）was preliminarily analyzed.

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 (Delta PKD Delta Fn3) 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 degrees 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 K-m value of ZFEG1907 was lower, while the k(cat)/K-m 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 beta-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). Significance and impact of study: The current work broadened the distribution of PKD-Fn3 domains in glycoside hydrolase family members and provided a possible method for protein engineering of endoglucanases.

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 beta-1,3-glucanase and beta-1,4-mannanase activities, and strong beta-glucosidase activity, while the recombinant ZF994-2 exhibited moderate endoglucanase activity and strong beta-glucosidase activity. More than 45% beta-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.

Drug-resistant bacterial infections exhibit a major threat to public health. Thus, exploring a novel antibacterial with efficient inhibition is urgently needed. Herein, this paper describes three types of MSNs (MSNs-FC2-R1, MSNs-FC2-R0.75, MSNs-FC2-R0.5) with controllable pore size (4–6 nm) and particle size (30–90 nm) that were successfully prepared. The MSNs were loaded with tetracycline hydrochloride (TCH) for effective inhibition of Escherichia coli (ATCC25922) and TCH-resistant Escherichia coli (MQ776). Results showed that the loading capacity of TCH in three types of MSNs was as high as over 500 mg/g, and the cumulative release was less than 33% in 60 h. The inhibitory rate of MSNs-FC2-R0.5 loaded with TCH against E. coli and drug-resistant E. coli reached 99.9% and 92.9% at the concentration of MIC, respectively, compared with the other two types of MSNs or free TCH. Modified MSNs in our study showed a great application for long-term bacterial growth inhibition.

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.

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.

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.

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.

The performance of deep reinforcement learning methods prone to degenerate when applied to environments with non-stationary dynamics. In this paper, we utilize the latent context recurrent encoders motivated by recent Meta-RL materials, and propose the Latent Context-based Soft Actor Critic (LC-SAC) method to address aforementioned issues. By minimizing the contrastive prediction loss function, the learned context variables capture the information of the environment dynamics and the recent behavior of the agent. Then combined with the soft policy iteration paradigm, the LC-SAC method alternates between soft policy evaluation and soft policy improvement until it converges to the optimal policy. Experimental results show that the performance of LC-SAC is significantly better than the SAC algorithm on the MetaWorld ML1 tasks whose dynamics changes drasticly among different episodes, and is comparable to SAC on the continuous control benchmark task MuJoCo whose dynamics changes slowly or doesn't change between different episodes. In addition, we also conduct relevant experiments to determine the impact of different hyperparameter settings on the performance of the LC-SAC algorithm and give the reasonable suggestions of hyperparameter setting.

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.

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- β -1,4-glucanase and endo-1,4- β -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.

[Background] Only a small proportion of microbes can be cultured in laboratories,thereby hampering the discovery of novel β-glucosidases from microbes.Novel microbial β-glucosidases can be discovered using culture-independent metagenomic approach.[Objective] Discovery of new β-glucosidase from soil microbes by metagenomic technology.[Methods] Screening a metagenomic library on esculin-containing Luria-Bertani agar plates and the putative β-glucosidase gene derived from the active clone was heterologously expressed for characterization.[Results] We found a β-glucosidase active clone by screening 620 000 clones in the library and an ORF (YNBG3) whose product is homologous with the third family of β-glucosidase was identified from the active clone.YNBG3 was heterologously expressed and characterized.Its optimum pH is 5.2,and optimal temperature is 53 ℃.YNBG3 showed good tolerance to organic solvents including dimethyl sulphoxide,acetone and ethanol,and the activity of YNBG3 was enhanced in presence of EDTA and urea.[Conclusion] A new 3-glucosidase,with thermostability and stability in the presence of organic solvents and urea,was discovery in this study.

绝大部分微生物的不可培养性使微生物的开发利用受到了限制,而宏基因组学策略为研究土壤中的不可培养微生物提供了途径.天然卤化物具有抗菌活性和抗肿瘤活性等生物活性,卤化酶在催化化合物的卤化过程中,对化合物活性产生重要影响,而以卤化酶基因为探针,可以发现与之偶联的天然生物合成基因簇,为卤化物的发现提供基础.利用卤化酶基因序列的保守区域设计简并引物,筛选土壤宏基因组文库获得卤化酶阳性克隆,并对获得的卤化酶基因间的进化及其与天然产物合成的关系进行分析.结果显示:通过同源序列筛选获得了65个卤化酶阳性克隆,序列同源分析表明约85％的阳性克隆中的卤化酶基因序列与已知卤化酶的相似性低,所获卤化酶基因具有较好的新颖性和多样性.而对所获克隆中生物合成相关基因的分析表明其中一个克隆中同时存在聚酮合酶基因与卤化酶基因,其可能与卤代I型聚酮合成相关.本研究基于序列筛选的方法,从宏基因组文库中发现了新的卤化酶基因和聚酮合酶基因,为进一步发现新颖的天然卤化物生物合成基因簇及天然卤化物奠定了基础.

A competitive immunosensor was established using an electrochemical amperometric strategy for sensitive detection of tetrabromobisphenol A bis(2-hydroxyethyl) ether (TBBPA-DHEE), an important derivative of tetrabromobisphenol A (TBBPA). In this system, the amplified electrochemical signal towards the reduction of hydrogen peroxide (H2O2) was recorded by amperometric method. Meanwhile, the synthetized catalase functionalized AuNPs-loaded self-assembled polymer nanospheres showed an excellent electrocatalytic ability to catalyse H2O2, which was beneficial for strengthening the electrochemical signals. Under the optimized conditions, this method displayed: (i) low detection limits (0.12 ng/mL, 7 times lower than the traditional ELISA with the same antibody); (ii) satisfactory accuracy (recoveries, 78-124%; RSD, 2.1-8.3%) and good agreement with the corresponding ELISA; (iii) low sample consumption (6 mu L) and low cost. The proposed approach was applied for investigation of TBBPA-DHEE from environmental waters, and our results indicated that this immunosensor has great potential to detect the trace pollutants in aquatic environments. (C) 2018 Elsevier B.V. All rights reserved.