Metagenomic gene discovery: How far have we moved into novel sequence space?

Metagenomics emerged in the late 1990s as a tool for accessing and studying the collective microbial genetic material in the environment. The advent of the technology generated great excitement, as it has provided new opportunities and technologies for studying the wealth of microbial genetic diversity in the environment. Metagenomics has been widely predicted to access new dimensions of protein sequence space. A decade on, we review how far we have actually moved into new sequence space (and other aspects of protein space) using metagenomic tools. While several novel enzyme activities and protein structures have been identified through metagenomic strategies, the greatest advancement has been made in the isolation of novel protein sequences, some of which have no close relatives, form deeply branched lineages and even represent novel families. This is particularly true for glycosyl hydrolases and lipase/esterases, despite the fact that these activities are frequently screened for in metagenomic studies. However, there is much room for improvement in the methods employed and they will need to be addressed so that access to novel biocatalytic activities can be widened.     

Marine Dadabacteria exhibit genome streamlining and phototrophy-driven niche partitioning

The remineralization of organic material via heterotrophy in the marine environment is performed by a diverse and varied group of microorganisms that can specialize in the type of organic material degraded and the niche they occupy. The marine Dadabacteria are cosmopolitan in the marine environment and belong to a candidate phylum for which there has not been a comprehensive assessment of the available genomic data to date. Here in, we assess the functional potential of the marine pelagic Dadabacteria in comparison to members of the phylum that originate from terrestrial, hydrothermal, and subsurface environments. Our analysis reveals that the marine pelagic Dadabacteria have streamlined genomes, corresponding to smaller genome sizes and lower nitrogen content of their DNA and predicted proteome, relative to their phylogenetic counterparts. Collectively, the Dadabacteria have the potential to degrade microbial dissolved organic matter, specifically peptidoglycan and phospholipids. The marine Dadabacteria belong to two clades with apparent distinct ecological niches in global metagenomic data: a clade with the potential for photoheterotrophy through the use of proteorhodopsin, present predominantly in surface waters up to 100 m depth; and a clade lacking the potential for photoheterotrophy that is more abundant in the deep photic zone.Subject terms: Water microbiology, Marine microbiology, Metagenomics, Microbial ecology     

Ammonia-oxidising Crenarchaeota: important players in the nitrogen cycle?

Cultivation-independent molecular surveys show that members of the kingdom Crenarchaeota within the domain Archaea represent a substantial component of microbial communities in aquatic and terrestrial environments. Recently, metagenomic studies have revealed that such Crenarchaeota contain and express genes related to those of bacterial ammonia monooxygenases. Furthermore, a marine chemolithoautotrophic strain was isolated that uses ammonia as a sole energy source. Considering the ubiquity and abundance of Crenarchaeota, these findings considerably challenge the accepted view of the microbial communities involved in global nitrogen cycling. However, the quantitative contribution of Archaea to nitrification in marine and terrestrial environments still remains to be elucidated.     

Achievements and new knowledge unraveled by metagenomic approaches

Metagenomics has paved the way for cultivation-independent assessment and exploitation of microbial communities present in complex ecosystems. In recent years, significant progress has been made in this research area. A major breakthrough was the improvement and development of high-throughput next-generation sequencing technologies. The application of these technologies resulted in the generation of large datasets derived from various environments such as soil and ocean water. The analyses of these datasets opened a window into the enormous phylogenetic and metabolic diversity of microbial communities living in a variety of ecosystems. In this way, structure, functions, and interactions of microbial communities were elucidated. Metagenomics has proven to be a powerful tool for the recovery of novel biomolecules. In most cases, functional metagenomics comprising construction and screening of complex metagenomic DNA libraries has been applied to isolate new enzymes and drugs of industrial importance. For this purpose, several novel and improved screening strategies that allow efficient screening of large collections of clones harboring metagenomes have been introduced.     

Metagenomics: Is it a powerful tool to obtain lipases for application in biocatalysis?

In recent years, metagenomic strategies have been widely used to isolate and identify new enzymes from uncultivable components of microbial communities. Among these enzymes, various lipases have been obtained from metagenomic libraries from different environments and characterized. Although many of these lipases have characteristics that could make them interesting for application in biocatalysis, relatively little work has been done to evaluate their potential to catalyze industrially important reactions. In the present article, we highlight the latest research on lipases obtained through metagenomic tools, focusing on studies of activity and stability and investigations of application in biocatalysis. We also discuss the challenges of metagenomic approaches for the bioprospecting of new lipases.     

海洋微生物宏基因组工程进展与展望

据初步统计,生活于海洋环境包括大洋深处的微生物有100万种以上,构成了一个动态的遗传基因库,其中绝大多数微生物或者从来没有经过实验室培养,或者至今无法培养,因而其分类地位及其生态学功能尚未为人类所认识。随着16S rRNA序列分析与系统分类学的广泛应用,海洋微生物多样性研究领域已经发生了很可观的改变,这些变化极大的丰富了人们对的微生物多样性及其生态功能的认识和理解。这里结合笔者近十年来的工作实践,讨论近年来在海洋微生物资源开发利用方面的研究进展,提出一个带有自动化特征的宏基因组功能表达平台,探讨海洋微生物资源利用的新途径。可以预见在不久的将来,海洋环境宏基因组工程研究将在一定程度上使得传统未培养海洋微生物基因资源及其功能产物能够为人类所开发和利用。     

Extracellular enzymes in terrestrial, freshwater, and marine environments: perspectives on system variability and common research needs

Extracellular enzymes produced by heterotrophic microbial communities are major drivers of carbon and nutrient cycling in terrestrial, freshwater, and marine environments. Although carbon and nutrient cycles are coupled on global scales, studies of extracellular enzymes associated with terrestrial, freshwater, and marine microbial communities are not often compared across ecosystems. In part, this disconnect arises because the environmental parameters that control enzyme activities in terrestrial and freshwater systems, such as temperature, pH, and moisture content, have little explanatory power for patterns of enzyme activities in marine systems. Instead, factors such as the functional diversity of microbial communities may explain varying patterns of enzyme activities observed in the ocean to date. In any case, many studies across systems focus on similar issues that highlight the commonalities of microbial community organization. Examples include the effective lifetime of enzymes released into the environment; the extent to which microbial communities coordinate enzyme expression to decompose complex organic substrates; and the influence of microbial community composition on enzyme activities and kinetics. Here we review the often-disparate research foci in terrestrial, freshwater, and marine environments. We consider the extent to which environmental factors may regulate extracellular enzyme activities within each ecosystem, and highlight commonalities and current methodological challenges to identify research questions that may aid in integrating cross-system perspectives in the future.     

Bacterial Exopolysaccharides from Extreme Marine Environments with Special Consideration of the Southern Ocean, Sea Ice, and Deep-Sea Hydrothermal Vents: A Review

Exopolysaccharides (EPSs) are high molecular weight carbohydrate polymers that make up a substantial component of the extracellular polymers surrounding most microbial cells in the marine environment. EPSs constitute a large fraction of the reduced carbon reservoir in the ocean and enhance the survival of marine bacteria by influencing the physicochemical environment around the bacterial cell. Microbial EPSs are abundant in the Antarctic marine environment, for example, in sea ice and ocean particles, where they may assist microbial communities to endure extremes of temperature, salinity, and nutrient availability. The microbial biodiversity of Antarctic ecosystems is relatively unexplored. Deep-sea hydrothermal vent environments are characterized by high pressure, extreme temperature, and heavy metals. The commercial value of microbial EPSs from these habitats has been established recently. Extreme environments offer novel microbial biodiversity that produces varied and promising EPSs. The biotechnological potential of these biopolymers from hydrothermal vent environments as well as from Antarctic marine ecosystems remains largely untapped.     

Advances in recovery of novel biocatalysts from metagenomes

Metagenomics has accelerated the process of discovery of novel biocatalysts by enabling scientists to tap directly into the entire diversity of enzymes held within natural microbial populations. Their characterization has revealed a great deal of valuable information about enzymatic activity in terms of factors which influence their stability and activity under a wide range of conditions. Many of the biocatalysts have particular properties making them suitable for biotechnological applications. A diverse array of strategies has been developed to optimize each step of the process of generating and screening metagenomic libraries for novel biocatalysts. This review covers the diversity of metagenome-derived enzymes characterized to date, and the strategies currently being developed to optimize discovery of novel metagenomic biocatalysts.     

Environmental constraints on life histories in Antarctic ecosystems: tempos, timings and predictability

Knowledge of Antarctic biotas and environments has increased dramatically in recent years. There has also been a rapid increase in the use of novel technologies. Despite this, some fundamental aspects of environmental control that structure physiological, ecological and life-history traits in Antarctic organisms have received little attention. Possibly the most important of these is the timing and availability of resources, and the way in which this dictates the tempo or pace of life. The clearest view of this effect comes from comparisons of species living in different habitats. Here, we (i) show that the timing and extent of resource availability, from nutrients to colonisable space, differ across Antarctic marine, intertidal and terrestrial habitats, and (ii) illustrate that these differences affect the rate at which organisms function. Consequently, there are many dramatic biological differences between organisms that live as little as 10 m apart, but have gaping voids between them ecologically.Identifying the effects of environmental timing and predictability requires detailed analysis in a wide context, where Antarctic terrestrial and marine ecosystems are at one extreme of the continuum of available environments for many characteristics including temperature, ice cover and seasonality. Anthropocentrically, Antarctica is harsh and as might be expected terrestrial animal and plant diversity and biomass are restricted. By contrast, Antarctic marine biotas are rich and diverse, and several phyla are represented at levels greater than global averages. There has been much debate on the relative importance of various physical factors that structure the characteristics of Antarctic biotas. This is especially so for temperature and seasonality, and their effects on physiology, life history and biodiversity. More recently, habitat age and persistence through previous ice maxima have been identified as key factors dictating biodiversity and endemism. Modern molecular methods have also recently been incorporated into many traditional areas of polar biology. Environmental predictability dictates many of the biological characters seen in all of these areas of Antarctic research.     

Recent Advances in Function-based Metagenomic Screening

Metagenomes from uncultured microorganisms are rich resources for novel enzyme genes. The methods used to screen the metagenomic libraries fall into two categories, which are based on sequence or function of the enzymes. The sequence-based approaches rely on the known sequences of the target gene families. In contrast, the function-based approaches do not involve the incorporation of metagenomic sequencing data and, therefore, may lead to the discovery of novel gene sequences with desired functions. In this review, we discuss the function-based screening strategies that have been used in the identification of enzymes from metagenomes. Because of its simplicity, agar plate screening is most commonly used in the identification of novel enzymes with diverse functions. Other screening methods with higher sensitivity are also employed, such as microtiter plate screening. Furthermore, several ultra-high-throughput methods were developed to deal with large metagenomic libraries. Among these are the FACS-based screening, droplet-based screening, and the in vivo reporter-based screening methods. The application of these novel screening strategies has increased the chance for the discovery of novel enzyme genes.     

Mobile learning applications to improve invertebrate zoology online teaching

The use of new technologies including personal mobile devices has become an indispensable tool in our daily lives, and thus its presence in education is becoming ever more ubiquitous. In the current scenario imposed by the COVID‐19 pandemic, in which in‐person presence in classrooms has been enormously reduced at all educational levels, the use of mobile learning and cutting‐edge methods can greatly improve the way students learn and enhance their online‐learning experience. Mobile applications, combined with extended reality technologies such as virtual reality (VR) and augmented reality (AR), are powerful tools that connect real and virtual environments and allow higher interaction for the user. We have leveraged the advantages of mobile learning and extended reality technologies to develop a series of mobile applications and associated educational activities for university‐level courses involving invertebrate zoology field work. In particular, we have developed (a) a VR SCUBA diving video to explore the diversity of a marine protected area; (b) an AR mobile app to visualize 3D models of marine invertebrates; and (c) a mobile‐based catalogue to explore the terrestrial biodiversity of one of the most diverse regions of Spain. Here we provide detailed information describing the design and creation of these tools, as well as their application in class, to facilitate and encourage their use in higher education. Despite the relatively recent application of these technologies in education, they have an enormous potential: they improve student motivation and learning, can be adapted to different learning styles, reduce social inequalities, and facilitate inclusiveness and diversity practices in the classroom.     

Biodiversity and potentials of marine-derived microorganisms

The marine environment is a prolific resource for the isolation of less exploited microorganisms. As a matter of fact, in the sea, untapped habitats exist with unique characteristics. In addition, the potential contribution of marine sources to the discovery of new bioactive molecules was recently recognized. Biosearch Italia possesses a collection of about 40 000 microorganisms, isolated from different ecological niches. In the search of new bioactive entities, investigations were expanded to marine habitats including marine sediments and organisms. More then 800 microorganisms have been isolated. About half belong to fungal genera, the others being actinomycetes. The frequency of antibiotic activities produced by these marine strains has been determined. Initial data are encouraging: marine isolates produce antibiotic activities with frequencies comparable to terrestrial ones. These activities probably represent a mixture of novel metabolites and known products previously discovered from terrestrial isolates. Further investigations are ongoing to assess the novelty of these observed microbiological activities.     

RiboFR-Seq: a novel approach to linking 16S rRNA amplicon profiles to metagenomes

16S rRNA amplicon analysis and shotgun metagenome sequencing are two main culture-independent strategies to explore the genetic landscape of various microbial communities. Recently, numerous studies have employed these two approaches together, but downstream data analyses were performed separately, which always generated incongruent or conflict signals on both taxonomic and functional classifications. Here we propose a novel approach, RiboFR-Seq (Ribosomal RNA gene flanking region sequencing), for capturing both ribosomal RNA variable regions and their flanking protein-coding genes simultaneously. Through extensive testing on clonal bacterial strain, salivary microbiome and bacterial epibionts of marine kelp, we demonstrated that RiboFR-Seq could detect the vast majority of bacteria not only in well-studied microbiomes but also in novel communities with limited reference genomes. Combined with classical amplicon sequencing and shotgun metagenome sequencing, RiboFR-Seq can link the annotations of 16S rRNA and metagenomic contigs to make a consensus classification. By recognizing almost all 16S rRNA copies, the RiboFR-seq approach can effectively reduce the taxonomic abundance bias resulted from 16S rRNA copy number variation. We believe that RiboFR-Seq, which provides an integrated view of 16S rRNA profiles and metagenomes, will help us better understand diverse microbial communities.     

Metagenomics

The total number of prokaryotic cells on earth has been estimated to be approximately 4–6 × 10³⁰, with the majority of these being uncharacterized. This diversity represents a vast genetic bounty that may be exploited for the discovery of novel genes, entire metabolic pathways and potentially valuable end‐products thereof. Metagenomics constitutes the functional and sequence‐based analysis of the collective microbial genomes (microbiome) in a particular environment or environmental niche. Herein, we review the most recent sequence‐based metagenomic analyses of some of the most microbiologically diverse locations on earth; including soil, marine water and the insect and human gut. Such studies have helped to uncover several previously unknown facts; from the true microbial diversity of extreme environments to the actual extent of symbiosis that exists in the insect and human gut. In this respect, metagenomics has and will continue to play an essential part in the new and evolving area of microbial systems biology.     

Targeted metagenomics: a high-resolution metagenomics approach for specific gene clusters in complex microbial communities

A major research goal in microbial ecology is to understand the relationship between gene organization and function involved in environmental processes of potential interest. Given that more than an estimated 99% of microorganisms in most environments are not amenable to culturing, methods for culture-independent studies of genes of interest have been developed. The wealth of metagenomic approaches allows environmental microbiologists to directly explore the enormous genetic diversity of microbial communities. However, it is extremely difficult to obtain the appropriate sequencing depth of any particular gene that can entirely represent the complexity of microbial metagenomes and be able to draw meaningful conclusions about these communities. This review presents a summary of the metagenomic approaches that have been useful for collecting more information about specific genes. Specific subsets of metagenomes that focus on sequence analysis were selected in each metagenomic studies. This 'targeted metagenomics' approach will provide extensive insight into the functional, ecological and evolutionary patterns of important genes found in microorganisms from various ecosystems.     

Identification of carbohydrate degrading bacteria in sub-tropical regions

Several bacteria from the islands of Puerto Rico, Vieques and Trinidad were isolated for their carbohydrate degrading activities. These terrestrial and marine bacterium were collected from pineapple agricultural lands, tropical rain forests, coastlands and mangrove swamps. Organisms were screened for activity using chromogenic substrates (AZCL Megazyme International Ltd., Ireland). The media composition for the effective culturing of some of the marine organisms has also been standardized. Gram-negative organisms were identified by sequence analysis of the PCR- amplified partial small subunit rRNA gene. Results indicate that the majority of the marine organisms tested belong to the genera Vibrio and Pseudoalteromonas and in the terrestrial environments Chryseobacterium predominated. These experiments reveal that sub-tropical environments are potentially good sources of microorganisms with novel carbohydrase activities.