Genetic diversity detection and gene discovery of novel glycoside hydrolase family 48 from soil environmental genomic DNA

Sequence diversity within a family of functional enzymes provides a platform for novel gene development and protein engineering to improve the properties of these enzymes for further applications. Glycoside hydrolase family 48 (GH48) is an important group of microbial cellulases. However, the genetic diversity and gene discovery of GH48 enzyme in natural environments are rarely reported. In this study, the genetic diversity of GH48 from Changbai Mountain soil was evaluated by building a clone library via a culture-independent molecular method for the first time. Results showed that the genetic diversity of GH48 in Changbai Mountain soil was different from that in thermophilic compost and marine sediment libraries, and more than 80% of the sequences exhibited the highest identity with cellulase genes from Chloroflexi. Novel GH48 genes were also cloned, and the recombinants Cel48_hm01 and Cel48_hm02 were prokaryotically expressed, purified, and characterized. Characterization results suggested that they were probably endocellulases that adopted a catalytic mechanism similar to the GH48 cellulase from Clostridium. This study revealed the genetic distribution of glycoside hydrolases in soil environment, described Changbai Mountain soil as a valuable source for glycoside hydrolase gene screening, and presented supplementary property data on novel GH48 from natural soil environments.     

Carboxylic ester hydrolases from hyperthermophiles

Carboxylic ester hydrolyzing enzymes constitute a large group of enzymes that are able to catalyze the hydrolysis, synthesis or transesterification of an ester bond. They can be found in all three domains of life, including the group of hyperthermophilic bacteria and archaea. Esterases from the latter group often exhibit a high intrinsic stability, which makes them of interest them for various biotechnological applications. In this review, we aim to give an overview of all characterized carboxylic ester hydrolases from hyperthermophilic microorganisms and provide details on their substrate specificity, kinetics, optimal catalytic conditions, and stability. Approaches for the discovery of new carboxylic ester hydrolases are described. Special attention is given to the currently characterized hyperthermophilic enzymes with respect to their biochemical properties, 3D structure, and classification.     

Divergence and convergence in enzyme evolution: parallel evolution of paraoxonases from quorum-quenching lactonases

We discuss the basic features of divergent versus convergent evolution and of the common scenario of parallel evolution. The example of quorum-quenching lactonases is subsequently described. Three different quorum-quenching lactonase families are known, and they belong to three different superfamilies. Their key active-site architectures have converged and are strikingly similar. Curiously, a promiscuous organophosphate hydrolase activity is observed in all three families. We describe the structural and mechanistic features that underline this converged promiscuity and how this promiscuity drove the parallel divergence of organophosphate hydrolases within these lactonase families by either natural or laboratory evolution.     

Novel α-L-Fucosidases from a Soil Metagenome for Production of Fucosylated Human Milk Oligosaccharides

This paper describes the discovery of novel α-L-fucosidases and evaluation of their potential to catalyse the transglycosylation reaction leading to production of fucosylated human milk oligosaccharides. Seven novel α-L-fucosidase-encoding genes were identified by functional screening of a soil-derived metagenome library and expressed in E. coli as recombinant 6xHis-tagged proteins. All seven fucosidases belong to glycosyl hydrolase family 29 (GH 29). Six of the seven α-L-fucosidases were substrate-inhibited, moderately thermostable and most hydrolytically active in the pH range 6–7, when tested with para-nitrophenyl-α-L-fucopyranoside (pNP-Fuc) as the substrate. In contrast, one fucosidase (Mfuc6) exhibited a high pH optimum and an unusual sigmoidal kinetics towards pNP-Fuc substrate. When tested for trans-fucosylation activity using pNP-Fuc as donor, most of the enzymes were able to transfer fucose to pNP-Fuc (self-condensation) or to lactose. With the α-L-fucosidase from Thermotoga maritima and the metagenome-derived Mfuc5, different fucosyllactose variants including the principal fucosylated HMO 2’-fucosyllactose were synthesised in yields of up to ~6.4%. Mfuc5 was able to release fucose from xyloglucan and could also use it as a fucosyl-donor for synthesis of fucosyllactose. This is the first study describing the use of glycosyl hydrolases for the synthesis of genuine fucosylated human milk oligosaccharides.     

High-throughput siRNA-based functional target validation

The drug discovery process pursued by major pharmaceutical companies for many years starts with target identification followed by high-throughput screening (HTS) with the goal of identifying lead compounds. To accomplish this goal, significant resources are invested into automation of the screening process or HTS. Robotic systems capable of handling thousands of data points per day are implemented across the pharmaceutical sector. Many of these systems are amenable to handling cell-based screening protocols as well. On the other hand, as companies strive to develop innovative products based on novel mechanisms of action(s), one of the current bottlenecks of the industry is the target validation process. Traditionally, bioinformatics and HTS groups operate separately at different stages of the drug discovery process. The authors describe the convergence and integration of HTS and bioinformatics to perform high-throughput target functional identification and validation. As an example of this approach, they initiated a project with a functional cell-based screen for a biological process of interest using libraries of small interfering RNA (siRNA) molecules. In this protocol, siRNAs function as potent gene-specific inhibitors. siRNA-mediated knockdown of the target genes is confirmed by TaqMan analysis, and genes with impacts on biological functions of interest are selected for further analysis. Once the genes are confirmed and further validated, they may be used for HTS to yield lead compounds.     

Bioprospecting metagenomes: glycosyl hydrolases for converting biomass

Throughout immeasurable time, microorganisms evolved and accumulated remarkable physiological and functional heterogeneity, and now constitute the major reserve for genetic diversity on earth. Using metagenomics, namely genetic material recovered directly from environmental samples, this biogenetic diversification can be accessed without the need to cultivate cells. Accordingly, microbial communities and their metagenomes, isolated from biotopes with high turnover rates of recalcitrant biomass, such as lignocellulosic plant cell walls, have become a major resource for bioprospecting; furthermore, this material is a major asset in the search for new biocatalytics (enzymes) for various industrial processes, including the production of biofuels from plant feedstocks. However, despite the contributions from metagenomics technologies consequent upon the discovery of novel enzymes, this relatively new enterprise requires major improvements. In this review, we compare function-based metagenome screening and sequence-based metagenome data mining, discussing the advantages and limitations of both methods. We also describe the unusual enzymes discovered via metagenomics approaches, and discuss the future prospects for metagenome technologies.     

溶解性多糖单加氧酶的研究进展

木质纤维素是地球上储藏量最为丰富的可再生生物资源。将木质纤维素酶解成寡糖或单糖是生物质利用的关键。然而,传统的糖苷水解酶很难对其进行有效降解。溶解性多糖单加氧酶是一种全新的生物质降解酶,丰富了生物质降解的模式。它以氧化方式作用于糖链,产生更多的还原端以便糖苷水解酶能进一步进行催化。本文综述了LPMO的发现历史、分类、作用机制与活性测定方法,并讨论了LPMO在饲料添加剂、功能性食品与生物能源等领域的应用前景。     

Evidence that the xylanase activity from <Emphasis Type="Italic">Sulfolobus solfataricus</Emphasis> Oα is encoded by the endoglucanase precursor gene (<Emphasis Type="Italic">sso1354</Emphasis>) and characterization of the associated cellulase activity

Sulfolobus solfataricus strain Oalpha was previously isolated for its ability to grow on minimal medium supplemented with xylan as a carbon source. The strain exhibited thermostable xylanase activity but several attempts to identify the gene encoding for the activity failed. Further studies showed that the xylanase displayed activity on carboxymethylcellulose (CMC) and the new activity was characterized. It exhibited an optimal temperature and pH of 95 degrees C and 3.5, respectively, and a half-life of 53 min at 95 degrees C. The enzyme, which was demonstrated to be glycosylated, hydrolyzed CMC in an endo-manner releasing cellobiose and other cello-oligomers. Analysis of the tryptic fragments by tandem mass spectrometry led to identification of the endoglucanase precursor, encoded by the sso1354 gene, as the protein possessing dual activity. The efficiency of the SSO1354 protein in degrading cellulosic and hemicellulosic fractions contained in agronomic residues was tested at low pH and high temperature. Cellulose and xylan were degraded to glucose and xylose at 90 degrees C, pH 4 by an enzyme mix consisting of SSO1354 and additional glycosyl hydrolases from S. solfataricus Oalpha. Given its role in saccharification processes requiring high temperatures and acidic environments, SSO1354 represents an interesting candidate for the utilization of agro-industrial waste for fuel production.     

The expanding diversity of serine hydrolases

Serine hydrolases use a hydroxyl of a serine, assisted by one or more other residues, to cleave peptide bonds. They belong to several different families whose general mechanism is well known. However, the subtle structural differences that have recently been observed across a variety of families shed light on their functional diversity, including variations in mechanism of action, differences in the modes of substrate binding, and substrate-assisted orientation of catalytic residues. Of particular interest are the Rhomboid family serine proteinases that are active within the plasma membrane, for which several new structures have been reported. Because these enzymes are involved in biological and pathological processes, many are becoming important targets of drug design.     

厌氧氨氧化菌驱动脱氮途径的研究进展

厌氧氨氧化菌(anaerobic ammonia-oxidizing bacteria, AnAOB)的代谢多样性,使得该菌群能够在海洋、湿地和陆地等不同的自然生态系统中广泛分布,甚至在一些极热和极寒环境中也检测到了该菌群的存在。本文回顾并总结了厌氧氨氧化菌在不同生态系统中的发现、分布及脱氮贡献等方面的研究,分析了厌氧氨氧化菌分布的主要环境影响因素。该综述将帮助我们更好地理解全球氮循环中厌氧氨氧化菌的实际角色和功能,并基于厌氧氨氧化(anaerobicammoniaoxidation,anammox)过程,探究能与其进行协作的新型生物脱氮工艺,以期为这些工艺的研发和推广提供生态学基础和新的思考,从而实现脱氮工艺的技术变革。     

Mechanism of the quorum-quenching lactonase (AiiA) from Bacillus thuringiensis. 1. Product-bound structures

Enzymes capable of hydrolyzing N-acyl- l-homoserine lactones (AHLs) used in some bacterial quorum-sensing pathways are of considerable interest for their ability to block undesirable phenotypes. Most known AHL hydrolases that catalyze ring opening (AHL lactonases) are members of the metallo-beta-lactamase enzyme superfamily and rely on a dinuclear zinc site for catalysis and stability. Here we report the three-dimensional structures of three product complexes formed with the AHL lactonase from Bacillus thuringiensis. Structures of the lactonase bound with two different concentrations of the ring-opened product of N-hexanoyl- l-homoserine lactone are determined at 0.95 and 1.4 A resolution and exhibit different product configurations. A structure of the ring-opened product of the non-natural N-hexanoyl- l-homocysteine thiolactone at 1.3 A resolution is also determined. On the basis of these product-bound structures, a substrate-binding model is presented that differs from previous proposals. Additionally, the proximity of the product to active-site residues and observed changes in protein conformation and metal coordination provide insight into the catalytic mechanism of this quorum-quenching metalloenzyme.     

A Proton Wire and Water Channel Revealed in the Crystal Structure of Isatin Hydrolase

The high resolution crystal structures of isatin hydrolase from Labrenzia aggregata in the apo and the product state are described. These are the first structures of a functionally characterized metal-dependent hydrolase of this fold. Isatin hydrolase converts isatin to isatinate and belongs to a novel family of metalloenzymes that include the bacterial kynurenine formamidase. The product state, mimicked by bound thioisatinate, reveals a water molecule that bridges the thioisatinate to a proton wire in an adjacent water channel and thus allows the proton released by the reaction to escape only when the product is formed. The functional proton wire present in isatin hydrolase isoform b represents a unique catalytic feature common to all hydrolases is here trapped and visualized for the first time. The local molecular environment required to coordinate thioisatinate allows stronger and more confident identification of orthologous genes encoding isatin hydrolases within the prokaryotic kingdom. The isatin hydrolase orthologues found in human gut bacteria raise the question as to whether the indole-3-acetic acid degradation pathway is present in human gut flora.     

A fluorometric assay for high-throughput screening targeting nicotinamide phosphoribosyltransferase

Nicotinamide adenine dinucleotide (NAD) plays a crucial role in many cellular processes. As the rate-limiting enzyme of the predominant NAD biosynthesis pathway in mammals, nicotinamide phosphoribosyltransferase (Nampt) regulates the cellular NAD level. Tumor cells are more sensitive to the NAD levels, making them more susceptible to Nampt inhibition than their nontumorigenic counterparts. Experimental evidence has indicated that Nampt might have proangiogenic activity and supports the growth of some tumors, so Nampt inhibitors may be promising as antitumor agents. However, only four Nampt inhibitors have been reported, and no high-throughput screening (HTS) strategy for Nampt has been proposed to date, largely limiting the drug discovery targeting Nampt. Therefore, the development of a robust HTS strategy for Nampt is both imperative and significant. Here we developed a fluorometric method for a Nampt activity assay by measuring the fluorescence of nicotinamide mononucleotide (NMN) derivative resulting from the enzymatic product NMN through simple chemical reactions. Then we set up an HTS system after thorough optimizations of this method and validated that it is feasible and effective through a pilot screening on a small library. This HTS system should expedite the discovery of Nampt inhibitors as antitumor drug candidates.     

Unraveling the cellulolytic and hemicellulolytic potential of two novel <Emphasis Type="Italic">Streptomyces</Emphasis> strains

The Streptomyces spp. are notorious plant biomass decomposers in soil environments, but only few strains were biochemically and genetically characterized. Here, we employed functional screening along with genomic sequencing for identification of novel lignocellulolytic Streptomyces strains. Streptomyces strains isolated from soil were functional screened based on their cellulolytic and hemicellulolytic capacities by enzymatic plate assays containing carboxymethylcellulose (CMC) and beechwood xylan as sole carbon source. Subsequently, genomes of Streptomyces strains were sequenced, annotated, and interpreted to correlate their genetic contents with biochemical properties. Among the 80 bacterial isolates that were screened for enzymatic activity, two Streptomyces strains (named as F1 and F7) exhiting higher endoglucanase and endoxylanase activities were selected for biochemical and genomic characterization. After cultivation on steam-pretreated sugarcane bagasse-based medium, the supernatant of the strains F1 and F7 exhibited enzymatic activity against different substrates, such as arabinan, rye arabinoxylan, β-glucan, starch, CMC, xylan, and chitin. Furthermore, strain F7 was able to degrade pectin, mannan, and lichenan. The genomic analysis of both strains revealed a diversity of carbohydrate-active enzymes. The F1 and F7 genomes encode 33 and 44 different types of glycosyl hydrolases families, respectively. Moreover, the genomic analysis also identified genes related to degradation of lignin-derived aromatic compounds. Collectively, the study revealed two novel Streptomyces strains and further insights on the degradation capability of lignocellulolytic bacteria, from which a number of technologies can arise, such as saccharification processes.     

Discovering functional novelty in metagenomes: examples from light-mediated processes

The emerging coverage of diverse habitats by metagenomic shotgun data opens new avenues of discovering functional novelty using computational tools. Here, we apply three different concepts for predicting novel functions within light-mediated microbial pathways in five diverse environments. Using phylogenetic approaches, we discovered two novel deep-branching subfamilies of photolyases (involved in light-mediated repair) distributed abundantly in high-UV environments. Using neighborhood approaches, we were able to assign seven novel functional partners in luciferase synthesis, nitrogen metabolism, and quorum sensing to BLUF domain-containing proteins (involved in light sensing). Finally, by domain analysis, for RcaE proteins (involved in chromatic adaptation), we predict 16 novel domain architectures that indicate novel functionalities in habitats with little or no light. Quantification of protein abundance in the various environments supports our findings that bacteria utilize light for sensing, repair, and adaptation far more widely than previously thought. While the discoveries illustrate the opportunities in function discovery, we also discuss the immense conceptual and practical challenges that come along with this new type of data.     

Metagenomics and the protein universe

Metagenomics sequencing projects have dramatically increased our knowledge of the protein universe and provided over one-half of currently known protein sequences; they have also introduced a much broader phylogenetic diversity into the protein databases. The full analysis of metagenomic datasets is only beginning, but it has already led to the discovery of thousands of new protein families, likely representing novel functions specific to given environments. At the same time, a deeper analysis of such novel families, including experimental structure determination of some representatives, suggests that most of them represent distant homologs of already characterized protein families, and thus most of the protein diversity present in the new environments are due to functional divergence of the known protein families rather than the emergence of new ones.     

Natural Products in High Throughput Screening: Automated High-Quality Sample Preparation

At present, compound libraries from combinatorial chemistry are the major source for high throughput screening (HTS) programs in drug discovery. On the other hand, nature has been proven to be an outstanding source for new and innovative drugs. Secondary metabolites from plants, animals, and microorganisms show a striking structural diversity that supplements chemically synthesized compounds or libraries in drug discovery programs. Unfortunately, extracts from natural sources are usually complex mixtures of compounds, often generated in time-consuming and, for the most part, manual processes. Because quality and quantity of the provided samples play a pivotal role in the success of HTS programs, this poses serious problems. In order to make samples of natural origin competitive with synthetic compound libraries, we devised a novel, automated sample preparation procedure based on solid-phase extraction (SPE). By making use of modified Zymark (Hopkinton, MA) RapidTrace? SPE workstations, we developed an easy-to-handle and effective fractionation method that generates high-quality samples from natural origin, fulfilling the requirements for an integration in high throughput drug discovery programs.     

土壤宏基因组学技术及其应用

传统的基于培养的研究方法只能反映土壤中少数(0.1%～10 %)微生物的信息,而大部分微生物目前还不能培养,因而这部分微生物资源尚难以被有效地开发利用.宏基因组学是分子生物学技术应用于环境微生物生态学研究而形成的一个新概念,主要技术包括土壤DNA的提取、文库的构建和目标基因克隆的筛选.它可为揭示微生物生态功能及其分子基础提供更全面的遗传信息,并已在微生物新功能基因筛选、活性物质开发和微生物多样性研究等方面取得了显著成果.本文对土壤宏基因组学技术的方法和应用作了详细介绍.     

Functional Screening of Metagenome and Genome Libraries for Detection of Novel Flavonoid-Modifying Enzymes

The functional detection of novel enzymes other than hydrolases from metagenomes is limited since only a very few reliable screening procedures are available that allow the rapid screening of large clone libraries. For the discovery of flavonoid-modifying enzymes in genome and metagenome clone libraries, we have developed a new screening system based on high-performance thin-layer chromatography (HPTLC). This metagenome extract thin-layer chromatography analysis (META) allows the rapid detection of glycosyltransferase (GT) and also other flavonoid-modifying activities. The developed screening method is highly sensitive, and an amount of 4 ng of modified flavonoid molecules can be detected. This novel technology was validated against a control library of 1,920 fosmid clones generated from a single Bacillus cereus isolate and then used to analyze more than 38,000 clones derived from two different metagenomic preparations. Thereby we identified two novel UDP glycosyltransferase (UGT) genes. The metagenome-derived gtfC gene encoded a 52-kDa protein, and the deduced amino acid sequence was weakly similar to sequences of putative UGTs from Fibrisoma and Dyadobacter. GtfC mediated the transfer of different hexose moieties and exhibited high activities on flavones, flavonols, flavanones, and stilbenes and also accepted isoflavones and chalcones. From the control library we identified a novel macroside glycosyltransferase (MGT) with a calculated molecular mass of 46 kDa. The deduced amino acid sequence was highly similar to sequences of MGTs from Bacillus thuringiensis. Recombinant MgtB transferred the sugar residue from UDP-glucose effectively to flavones, flavonols, isoflavones, and flavanones. Moreover, MgtB exhibited high activity on larger flavonoid molecules such as tiliroside.