Crystal structure analysis of EstA from Arthrobacter sp. Rue61a – an insight into catalytic promiscuity

In this article we analyze the reasons for catalytic promiscuity of a type VIII esterase with β-lactamase fold and the ability to cleave β-lactams. We compared the structure of this enzyme to those of an esterase of the same type without any lactamase ability, an esterase with moderate lactamase ability, and a class C β-lactamase with similar fold. Our results show that for these enzymes, the difference in the substrate specificity is sterically driven.     

基于元基因组学数据集的海洋微生物多样性分析

通过对海洋13个样品元基因组数据的BLAsT搜索,筛选到了16s rRNA基因序列1600条,18s RNA基因序列61条。分类结果显示,细菌在海岸、公开海域深层海水和表层海水3种海洋环境类型中都占优势,其相对百分比分别为98%、59%和91%。相比于海岸和公开海域表层海水,公开海域深层海水中古生菌和Deltaproteobacteria所占的相对含量较高,各31%和27%。海岸检测到的古生菌主要为Euryarcheata,公开海域深层海水检测到的古生菌主要为Crenarchaaeota(93%为与氨氧化相关的MGI纲)。结果表明,氨氧化相关古生菌在深海生态系统中的作用可能较大。     

Human oral viruses are personal,persistent and gender-consistent

Viruses are the most abundant members of the human oral microbiome, yet relatively little is known about their biodiversity in humans. To improve our understanding of the DNA viruses that inhabit the human oral cavity, we examined saliva from a cohort of eight unrelated subjects over a 60-day period. Each subject was examined at 11 time points to characterize longitudinal differences in human oral viruses. Our primary goals were to determine whether oral viruses were specific to individuals and whether viral genotypes persisted over time. We found a subset of homologous viral genotypes across all subjects and time points studied, suggesting that certain genotypes may be ubiquitous among healthy human subjects. We also found significant associations between viral genotypes and individual subjects, indicating that viruses are a highly personalized feature of the healthy human oral microbiome. Many of these oral viruses were not transient members of the oral ecosystem, as demonstrated by the persistence of certain viruses throughout the entire 60-day study period. As has previously been demonstrated for bacteria and fungi, membership in the oral viral community was significantly associated with the sex of each subject. Similar characteristics of personalized, sex-specific microflora could not be identified for oral bacterial communities based on 16S rRNA. Our findings that many viruses are stable and individual-specific members of the oral ecosystem suggest that viruses have an important role in the human oral ecosystem.     

Biodiversity for biocatalysis: A review of the α/β-hydrolase fold superfamily of esterases-lipases discovered in metagenomes

Natural biodiversity undoubtedly inspires biocatalysis research and innovation. Biotransformations of interest also inspire the search for appropriate biocatalysts in nature. Indeed, natural genetic resources have been found to support the hydrolysis and synthesis of not only common but also unusual synthetic scaffolds. The emerging tool of metagenomics has the advantage of allowing straightforward identification of activity directly applicable as biocatalysis. However, new enzymes must not only have outstanding properties in terms of performance but also other properties superior to those of well-established commercial preparations in order to successfully replace the latter. Esterases (EST) and lipases (LIP) from the α/β-hydrolase fold superfamily are among the enzymes primarily used in biocatalysis. Accordingly, they have been extensively examined with metagenomics. Here we provided an updated (October 2015) overview of sequence and functional data sets of 288 EST–LIP enzymes with validated functions that have been isolated in metagenomes and (mostly partially) characterized. Through sequence, biochemical, and reactivity analyses, we attempted to understand the phenomenon of variability and versatility within this group of enzymes and to implement this knowledge to identify sequences encoding EST–LIP which may be useful for biocatalysis. We found that the diversity of described EST–LIP polypeptides was not dominated by a particular type of protein or highly similar clusters of proteins but rather by diverse nonredundant sequences. Purified EST–LIP exhibited a wide temperature activity range of 10–85?°C, although a preferred bias for a mesophilic temperature range (35–40?°C) was observed. At least 60% of the total characterized metagenomics-derived EST–LIP showed outstanding properties in terms of stability (solvent tolerance) and reactivity (selectivity and substrate profile), which are the features of interest in biocatalysis. We hope that, in the future, the search for and utilization of sequences similar to those already encoded and characterized EST–LIP enzymes from metagenomes may be of interest for promoting unresolved biotransformations in the chemical industry. Some examples are discussed in this review.     

Biochemistry and molecular biology of anammox bacteria

Anaerobic ammonium-oxidizing (anammox) bacteria are one of the latest additions to the biogeochemical nitrogen cycle. These bacteria derive their energy for growth from the conversion of ammonium and nitrite into dinitrogen gas in the complete absence of oxygen. These slowly growing microorganisms belong to the order Brocadiales and are affiliated to the Planctomycetes. Anammox bacteria are characterized by a compartmentalized cell architecture featuring a central cell compartment, the “anammoxosome”. Thus far unique “ladderane” lipid molecules have been identified as part of their membrane systems surrounding the different cellular compartments. Nitrogen formation seems to involve the intermediary formation of hydrazine, a very reactive and toxic compound. The genome of the anammox bacterium Kuenenia stuttgartiensis was assembled from a complex microbial community grown in a sequencing batch reactor (74% enriched in this bacterium) using a metagenomics approach. The assembled genome allowed the in silico reconstruction of the anammox metabolism and identification of genes most likely involved in the process. The present anammox pathway is the only one consistent with the available experimental data, thermodynamically and biochemically feasible, and consistent with Ockham’s razor: it invokes minimum biochemical novelty and requires the fewest number of biochemical reactions. The worldwide presence of anammox bacteria has now been established in many oxygen-limited marine and freshwater systems, including oceans, seas, estuaries, marshes, rivers and large lakes. In the marine environment over 50% of the N₂ gas released may be produced by anammox bacteria. Application of the anammox process offers an attractive alternative to current wastewater treatment systems for the removal of ammonia-nitrogen. Currently, at least five full scale reactor systems are operational.     

Novel Biocatalysts by Identification and Design

Enzymes produced from bacteria and eukaryotic organisms are presently being used for a large variety of different biotechnological applications. The rapidly increasing demand for enzymes which are active towards novel and often non-natural substrates has triggered the development of novel molecular biological methods of enzyme isolation and design. The metagenome approach is a cultivation-independent method which allows the direct cloning and expression of environmental DNA thereby providing access to a wealth of so-far unknown biocatalysts. Additionally, newly identified or existing biocatalysts can be further optimized by different methods of directed evolution. Here, the principle of the metagenome approach is outlined and a strategy is presented for the optimization of a bacterial lipase using a combination of rational design and directed evolution.     

Cloning and Heterologous Expression of the Vibrioferrin Biosynthetic Gene Cluster from a Marine Metagenomic Library

A biosynthetic gene cluster of siderophore consisting of five open reading frames (ORFs) was cloned by functional screening of a metagenomic library constructed from tidal-flat sediment. Expression of the cloned biosynthetic genes in Escherichia coli led to the production of vibrioferrin, a siderophore originally reported for the marine bacterium Vibrio parahaemolyticus. To the best of our knowledge, this is the first example of heterologous production of a siderophore by biosynthetic genes cloned from a metagenomic library. The cloned cluster was one of the largest of the clusters obtained by functional screening. In this study, we demonstrated and extended the possibility of function-based metagenomic research.     

Metabolic stratification driven by surface and subsurface interactions in a terrestrial mud volcano

Terrestrial mud volcanism represents the prominent surface geological feature, where fluids and hydrocarbons are discharged along deeply rooted structures in tectonically active regimes. Terrestrial mud volcanoes (MVs) directly emit the major gas phase, methane, into the atmosphere, making them important sources of greenhouse gases over geological time. Quantification of methane emission would require detailed insights into the capacity and efficiency of microbial metabolisms either consuming or producing methane in the subsurface, and establishment of the linkage between these methane-related metabolisms and other microbial or abiotic processes. Here we conducted geochemical, microbiological and genetic analyses of sediments, gases, and pore and surface fluids to characterize fluid processes, community assemblages, functions and activities in a methane-emitting MV of southwestern Taiwan. Multiple lines of evidence suggest that aerobic/anaerobic methane oxidation, sulfate reduction and methanogenesis are active and compartmentalized into discrete, stratified niches, resembling those in marine settings. Surface evaporation and oxidation of sulfide minerals are required to account for the enhanced levels of sulfate that fuels subsurface sulfate reduction and anaerobic methanotrophy. Methane flux generated by in situ methanogenesis appears to alter the isotopic compositions and abundances of thermogenic methane migrating from deep sources, and to exceed the capacity of microbial consumption. This metabolic stratification is sustained by chemical disequilibria induced by the mixing between upward, anoxic, methane-rich fluids and downward, oxic, sulfate-rich fluids.     

新型海洋微生物β-葡萄糖苷酶基因的克隆、表达及重组酶性质

通过功能筛选方法,从中国南海海洋表层海水微生物元基因组文库筛选得到了6个β-葡萄糖苷酶阳性克隆。对其中的一个阳性克隆pSB47B2进一步亚克隆和序列分析,获得一新型β-葡萄糖苷酶基因(命名为bgl1B)开放阅读框。以pET22b(+)为载体、Escherichia coli BL21(DE3)为宿主菌,bgl1B被高效活性重组表达。通过Ni-NTA亲和层析柱纯化了重组Bgl1B(rBgl1B)。纯化的rBgl1B催化pNPG水解反应的最适pH为6.5,最适温度为40oC。在最适反应条件下,rBgl1B水解pNPG的活性达到39.7U/mg,Km和Vmax分别为0.288mmol/L、36.9μmol/min。纤维二糖是rBgl1B的有效作用底物,其Km和Vmax分别为0.173mmol/L、35μmol/min。但rBgl1B不能催化转化蔗糖、乳糖、麦芽糖以及CMC。rBgl1B催化pNPG的水解反应对高浓度的Na+有较好的耐受性,而低浓度的Ca2+、Mn2+对该酶活有一定促进作用。不同于许多来源于真菌的酸性β-葡萄糖苷酶,rBgl1B在pH7.0～9.0范围内具有比较高的酶活力并具有较好的稳定性。