A Mechanism‐Based Approach to Screening Metagenomic Libraries for Discovery of Unconventional Glycosidases

Functional metagenomics has opened new opportunities for enzyme discovery. To exploit the full potential of this new tool, the design of selective screens is essential, especially when searching for rare enzymes. To identify novel glycosidases that employ cleavage strategies other than the conventional Koshland mechanisms, a suitable screen was needed. Focusing on the unsaturated glucuronidases (UGLs), it was found that use of simple aryl glycoside substrates did not allow sufficient discrimination against β‐glucuronidases, which are widespread in bacteria. While conventional glycosidases cannot generally hydrolyze thioglycosides efficiently, UGLs follow a distinct mechanism that allows them to do so. Thus, fluorogenic thioglycoside substrates featuring thiol‐based self‐immolative linkers were synthesized and assessed as selective substrates. The generality of the approach was validated with another family of unconventional glycosidases, the GH4 enzymes. Finally, the utility of these substrates was tested by screening a small metagenomic library.     

近期工业微生物关键技术和应用

近年来,随着宏基因组学、蛋白质组学和代谢组学等技术的发展,工业微生物技术在资源、医药和手性合成等领域已经成为热点技术,并开拓了电子和纳米技术等新的应用领域.本文综述了几项最新的工业微生物技术,主要包括:微生物环氧化水解酶催化合成手性二醇、微生物甲酸脱氢酶用于再生氧化还原反应的辅因子、通过噬菌体展示技术得到纳米级金属丝、代谢网络改造和重建用于传统发酵生产以及有机溶剂耐受菌和宏基因组技术的应用.     

Strategies for Natural Products Discovery from Uncultured Microorganisms

Microorganisms are highly regarded as a prominent source of natural products that have significant importance in many fields such as medicine, farming, environmental safety, and material production. Due to this, only tiny amounts of microorganisms can be cultivated under standard laboratory conditions, and the bulk of microorganisms in the ecosystems are still unidentified, which restricts our knowledge of uncultured microbial metabolism. However, they could hypothetically provide a large collection of innovative natural products. Culture-independent metagenomics study has the ability to address core questions in the potential of NP production by cloning and analysis of microbial DNA derived directly from environmental samples. Latest advancements in next generation sequencing and genetic engineering tools for genome assembly have broadened the scope of metagenomics to offer perspectives into the life of uncultured microorganisms. In this review, we cover the methods of metagenomic library construction, and heterologous expression for the exploration and development of the environmental metabolome and focus on the function-based metagenomics, sequencing-based metagenomics, and single-cell metagenomics of uncultured microorganisms.     

Archaeal and Bacterial Content in a Two-Stage Anaerobic System for Efficient Energy Production from Agricultural Wastes

Anaerobic digestion (AD) is a microbially-driven process enabling energy production. Microorganisms are the core of anaerobic digesters and play an important role in the succession of hydrolysis, acidogenesis, acetogenesis, and methanogenesis processes. The diversity of participating microbial communities can provide new information on digester performance for biomass valorization and biofuel production. In this study anaerobic systems were used, operating under mesophilic conditions that realized biodegradation processes of waste wheat straw pretreated with NaOH—a renewable source for hydrogen and methane production. These processes could be managed and optimized for hydrogen and methane separately but combining them in a two-stage system can lead to higher yields and a positive energy balance. The aim of the study was to depict a process of biohydrogen production from lignocellulosic waste followed by a second one leading to the production of biomethane. Archaeal and bacterial consortia in a two-stage system operating with wheat straw were identified for the first time and the role of the most important representatives was elucidated. The mixed cultures were identified by the molecular-biological methods of metagenomics. The results showed that biohydrogen generation is most probably due to the presence of Proteiniphilum saccharofermentans, which was 28.2% to 45.4% of the microbial community in the first and the second bioreactor, respectively. Archaeal representatives belonging to Methanobacterium formicicum (0.71% of the community), Methanosarcina spelaei (0.03%), Methanothrix soehngenii (0.012%), and Methanobacterium beijingense (0.01%) were proven in the methane-generating reactor. The correlation between substrate degradation and biogas accumulation was calculated, together with the profile of fatty acids as intermediates produced during the processes. The hydrogen concentration in the biogas reached 14.43%, and the Methane concentration was 69%. Calculations of the energy yield during the two-stage process showed 1195.89 kWh·t⁻¹ compared to a 361.62 kWh·t⁻¹ cumulative yield of energy carrier for a one-stage process.     

From cellulosomes to cellulosomics

Cellulosomes are intricate multienzyme systems produced by several cellulolytic bacteria, the first example of which was discovered in the anaerobic thermophilic bacterium, Clostridium thermocellum. Cellulosomes are designed for efficient degradation of plant cell wall polysaccharides, notably cellulose—the most abundant renewable polymer on earth. The component parts of the multicomponent complex are integrated by virtue of a unique family of integrating modules, the cohesins and the dockerins, whose distribution and specificity dictate the overall cellulosome architecture. A full generation of research has elapsed since the original publications that documented the cellulosome concept. In this review, we provide a personal account on the discovery process, while describing how divergent cellulosome systems were identified and investigated, culminating in the collaboration of several labs worldwide to tackle together the challenging field of cellulosome genomics and metagenomics. © 2008 The Japan Chemical Journal Forum and Wiley Periodicals, Inc. Chem Rec 8: 364–377; 2008: Published online in Wiley InterScience ( www.interscience.wiley.com ) DOI 10.1002/tcr.20160     

A resampling strategy for studying robustness in virus detection pipelines

Next-generation sequencing is regularly used to identify viral sequences in DNA or RNA samples of infected hosts. A major step of most pipelines for virus detection is to map sequence reads against known virus genomes. Due to small differences between the sequences of related viruses, and due to several biological or technical errors, mapping underlies uncertainties. As a consequence, the resulting list of detected viruses can lack robustness.A new approach for generating artificial sequencing reads together with a strategy of resampling from the original findings is proposed that can help to assess the robustness of the originally identified list of viruses. From the original mapping result in form of a SAM file, a set of statistical distributions are derived. These are used in the resampling pipeline to generate new artificial reads which are again mapped versus the reference genomes. By summarizing the resampling procedure, the analyst receives information about whether the presence of a particular virus in the sample gains or losses evidence, and thus about the robustness of the original mapping list but also that of individual viruses in this list. To judge robustness, several indicators are derived from the resampling procedure such as the correlation between original and resampling read counts, or the statistical detection of outliers in the differences of read counts. Additionally, graphical illustrations of read count shifts via Sankey diagrams are provided.To demonstrate the use of the new approach, the resampling approach is applied to three real-world data samples, one of them with laboratory-confirmed Influenza sequences, and to artificially generated data where virus sequences have been spiked into the sequencing data of a host. By applying the resampling pipeline, several viruses drop from the original list while new viruses emerge, showing robustness of those viruses that remain in the list.The evaluation of the new approach shows that the resampling approach is helpful to analyze the viral content of a biological sample, to rate the robustness of original findings and to better show the overall distribution of findings. The method is also applicable to other virus detection pipelines based on read mapping.     

式根岛海绵活性物质及功能宏基因组学的应用

式根岛海绵及其高度复杂的共生体系是生物活性物质的丰富重要来源, 产生以calyculins为主要成分的毒性物质. 活性物质含量低, 使得直接从海绵分离大量活性物质不可取. 海绵共生菌是活性物质的主要生产者, 但是大部分的共生菌是不可培养的, 使得单纯的分离培养不能发挥很好的作用. 功能宏基因组学避开了培养的瓶颈, 可以获取独特的活性物质及其功能基因. 对式根岛海绵2013年2月以前分离出的活性物质作一综述, 并对非培养依赖型的功能宏基因组学, 在蕴含大量不可培养微生物的式根岛海绵中的应用进行综述. 探讨功能宏基因组在研究开发式根岛海绵及其他海绵的活性物质及有趣功能基因中的应用潜能, 为研究国内海绵的丰富活性物质和有趣的基因提供一种思路和方法.