Microbial Secondary Metabolism Research Articles

Succession of microbial community composition and secondary metabolism during marine biofilm development.

In nature, secondary metabolites mediate interactions between microorganisms residing in complex microbial communities. However, the degree to which community dynamics can be linked to secondary metabolite potential remains largely unknown. In this study, we address the relationship between community succession and secondary metabolism variation. We used 16S and 18S rRNA gene and adenylation domain amplicon sequencing, genome-resolved metagenomics, and untargeted metabolomics to track the taxons, biosynthetic gene clusters, and metabolome dynamics in situ of microorganisms during marine biofilm succession over 113days. Two phases were identified during the community succession, with a clear shift around Day 29, where the alkaloid secondary metabolites, pseudanes, were also detected. The microbial secondary metabolite potential changed between the phases, and only a few community members, including Myxococotta spp., were responsible for the majority of the biosynthetic gene cluster potential in the early succession phase. In the late phase, bryozoans and benthic copepods were detected, and the microbial nonribosomal peptide potential drastically decreased in association with a reduction in the relative abundance of the prolific secondary metabolite producers. Conclusively, this study provides evidence that the early succession of the marine biofilm community favors prokaryotes with high nonribosomal peptide synthetase potential. In contrast, the late succession is dominated by multicellular eukaryotes and a reduction in bacterial nonribosomal peptide synthetase potential.

Multi-integrated approach for unraveling small open reading frames potentially associated with secondary metabolism in Streptomyces.

Small open reading frames (smORFs) are widely distributed in various living organisms. However, their functions remain largely unexplored. In addition, annotation and detection of smORFs are limited using existing methods and hindered by their specific properties. In this study, we systematically investigated smORFs and smORF-encoded peptides (SEPs) in Streptomyces, which are well-known bacterial producers of diverse bioactive secondary metabolites. We established a peptidogenomic workflow based on multi-integrated comprehensive database search and database-independent de novo sequencing to identify smORFs in Streptomyces xinghaiensis NRRL B-24674T (S187). In addition, we described SEPome related to the secondary metabolism, which include 68 novel SEPs and 79 common smORFs with Streptomyces coelicolor A3 (2). Functional analysis of universal smORFs revealed enrichment in biosynthetic processes, stress response, ribosomes, and nucleic acid binding. Meanwhile, 5 Cryptic smORF-encoded Peptides (CSEPs) distributed in non-annotated regions of the genome, and non-coding RNAs could encode for CSEPs. A total of 66 new RNAs, including 32 non-coding RNAs (ncRNAs) were revealed, and 4 ncRNA-encoded peptides were identified. Furthermore, an investigation of carbon metabolism showed that NagE functions in spore formation and secondary metabolism in Streptomyces. Particularly, NagE was observed to function in the biosynthesis of anti-complement agents in S. xinghaiensis, suggesting a novel role of the phosphoenolpyruvate phosphotransferase system in microbial secondary metabolism. We thus provide an effective strategy for analyzing public data sets of model strains to identify smORFs for non-model species. The ncRNAs and SEPs present rich sources for engineering streptomycetes to produce bioactive compounds. IMPORTANCE Due to their small size and special chemical features, small open reading frame (smORF)-encoding peptides (SEPs) are often neglected. However, they may play critical roles in regulating gene expression, enzyme activity, and metabolite production. Studies on bacterial microproteins have mainly focused on pathogenic bacteria, which are importance to systematically investigate SEPs in streptomycetes and are rich sources of bioactive secondary metabolites. Our study is the first to perform a global identification of smORFs in streptomycetes. We established a peptidogenomic workflow for non-model microbial strains and identified multiple novel smORFs that are potentially linked to secondary metabolism in streptomycetes. Our multi-integrated approach in this study is meaningful to improve the quality and quantity of the detected smORFs. Ultimately, the workflow we established could be extended to other organisms and would benefit the genome mining of microproteins with critical functions for regulation and engineering useful microorganisms.

Biosurfactant from a thermo-halophilic strain of Bacillus alveayuensis isolated from a Brazilian oil reservoir: Production, chemical characterization, antimicrobial activity, and efficiency in wettability reversal and oil removal from oil-soaked sand.

Biosurfactants (BS) are amphiphilic molecules from microbial secondary metabolism capable of reducing surface and interfacial tension between non-miscible phases, thus having a wide range of industrial and environmental applications. In this context, the present work aimed to study the biosurfactant produced by the bacterial strain Ar70C7-2, isolated from rocky samples of a deep offshore Brazilian oil reservoir, to evaluate its application in the oil industry. Its optimized production conditions, chemical structure, stability in extreme conditions, surface-active properties in porous media, and antibacterial action were evaluated. Strain Ar70C7-2 showed 100% similarity to Bacillus alveayuensis and was able to produce 0.27 g/L of crude BS in the Mineral Medium with glycerin and NH4NO3 at a ratio of 2, under fixed conditions of 70 g/L NaCl, incubation at 55 °C, and no stirring. The semi-purified BS presented emulsification index (E24) above 55%, surface tension of 31.8 mN/m, interfacial tension (against n-hexadecane) of 17.6 mN/m and CMC of 65 mg/L. The BS belongs to the class of lipopeptides, with a protein composition of 7% and fatty acid chain ranging from C14 to C18, with homologue C16 as the most abundant. Mass spectra of BS indicate the presence of surfactin, iturin, and fengycin families. Semi-purified BS was able to remove approximately 30% of the motor oil (10W40) present in oil-soaked sand, as tested by the contaminated sand washing method; it also promoted the alteration of carbonate wettability, as demonstrated in the flotation test and by the Washburn method. BS inhibited growth of the sulfate-reducing bacteria (SRB) Desulfovibrio marinus BRS-1 at concentrations of 2250 μg/mL and showed bactericidal action at concentrations of 2500 μg/mL. These results demonstrate that the biosurfactant produced by strain Ar70C7-2 has the potential for application in MEOR, bioremediation, and controlling SRB growth, encouraging further studies to validate its prospect in laboratory and field conditions.

Flux balance analysis-based metabolic modeling of microbial secondary metabolism: Current status and outlook.

In microorganisms, different from primary metabolism for cellular growth, secondary metabolism is for ecological interactions and stress responses and an important source of natural products widely used in various areas such as pharmaceutics and food additives. With advancements of sequencing technologies and bioinformatics tools, a large number of biosynthetic gene clusters of secondary metabolites have been discovered from microbial genomes. However, due to challenges from the difficulty of genome-scale pathway reconstruction and the limitation of conventional flux balance analysis (FBA) on secondary metabolism, the quantitative modeling of secondary metabolism is poorly established, in contrast to that of primary metabolism. This review first discusses current efforts on the reconstruction of secondary metabolic pathways in genome-scale metabolic models (GSMMs), as well as related FBA-based modeling techniques. Additionally, potential extensions of FBA are suggested to improve the prediction accuracy of secondary metabolite production. As this review posits, biosynthetic pathway reconstruction for various secondary metabolites will become automated and a modeling framework capturing secondary metabolism onset will enhance the predictive power. Expectedly, an improved FBA-based modeling workflow will facilitate quantitative study of secondary metabolism and in silico design of engineering strategies for natural product production.

Identification and Analysis of the Biosynthetic Gene Cluster for the Hydrazide-Containing Aryl Polyene Spinamycin.

Natural products containing nitrogen-nitrogen (N-N) bonds have attracted much attention because of their bioactivities and chemical features. Several recent studies have revealed the nitrous acid-dependent N-N bond-forming machinery. However, the catalytic mechanisms of hydrazide synthesis using nitrous acid remain unknown. Herein, we focused on spinamycin, a hydrazide-containing aryl polyene produced by Streptomyces albospinus JCM3399. In the S. albospinus genome, we discovered a putative spinamycin biosynthetic gene (spi) cluster containing genes that encode a type II polyketide synthase and genes for the secondary metabolism-specific nitrous acid biosynthesis pathway. A gene inactivation experiment showed that this cluster was responsible for spinamycin biosynthesis. A feeding experiment using stable isotope-labeled sodium nitrite and analysis of nitrous acid-synthesizing enzymes in vitro strongly indicated that one of the nitrogen atoms of the hydrazide group was derived from nitrous acid. In vitro substrate specificity analysis of SpiA3, which is responsible for loading a starter substrate onto polyketide synthase, indicated that N-N bond formation occurs after starter substrate loading. In vitro analysis showed that the AMP-dependent ligase SpiA7 catalyzes the diazotization of an amino group on a benzene ring without a hydroxy group, resulting in a highly reactive diazo intermediate, which may be the key step in hydrazide group formation. Therefore, we propose the overall biosynthetic pathway of spinamycin. This study expands our knowledge of N-N bond formation in microbial secondary metabolism.

Evolution and regulation of microbial secondary metabolism.

Microbes have disproportionate impacts on the macroscopic world. This is in part due to their ability to grow to large populations that collectively secrete massive amounts of secondary metabolites and alter their environment. Yet, the conditions favoring secondary metabolism despite the potential costs for primary metabolism remain unclear. Here we investigated the biosurfactants that the bacterium Pseudomonas aeruginosa makes and secretes to decrease the surface tension of surrounding liquid. Using a combination of genomics, metabolomics, transcriptomics, and mathematical modeling we show that the ability to make surfactants from glycerol varies inconsistently across the phylogenetic tree; instead, lineages that lost this ability are also worse at reducing the oxidative stress of primary metabolism on glycerol. Experiments with different carbon sources support a link with oxidative stress that explains the inconsistent distribution across the P. aeruginosa phylogeny and suggests a general principle: P. aeruginosa lineages produce surfactants if they can reduce the oxidative stress produced by primary metabolism and have excess resources, beyond their primary needs, to afford secondary metabolism. These results add a new layer to the regulation of a secondary metabolite unessential for primary metabolism but important to change physical properties of the environments surrounding bacterial populations.

Environmental structure impacts microbial composition and secondary metabolism

Determining the drivers of microbial community assembly is a central theme of microbial ecology, and chemical ecologists seek to characterize how secondary metabolites mediate these assembly patterns. Environmental structure affects how communities assemble and what metabolic pathways aid in that assembly. Here, we bridged these two perspectives by addressing the chemical drivers of community assembly within a spatially structured landscape with varying oxygen availability. We hypothesized that structured environments would favor higher microbial diversity and metabolite diversity. We anticipated that the production of a compound would be more advantageous in a structured environment (less mixing) compared to an unstructured environment (more mixing), where the molecule would have a diminished local effect. We observed this to be partially true in our experiments: structured environments had similar microbial diversity compared to unstructured environments but differed significantly in the metabolites produced. We also found that structured environments selected for communities with higher evenness, rather than communities with higher richness. This supports the idea that when characterizing the drivers of community assembly, it matters less about who is there and more about what they are doing. Overall, these data contribute to a growing effort to approach microbial community assembly with interdisciplinary tools and perspectives.

Microbial Secondary Metabolism and Biotechnology.

In recent decades scientific research has demonstrated that the microbial world is infinitely richer and more surprising than we could have imagined. Every day, new molecules produced by microorganisms are discovered, and their incredible diversity has not yet delivered all of its messages. The current challenge of research is to select from the wide variety of characterized microorganisms and compounds, those which could provide rapid answers to crucial questions about human or animal health or more generally relating to society’s demands for medicine, pharmacology, nutrition or everyday well-being.

Fermentative production of alternative antimicrobial peptides and enzymes

Conventional broad-spectrum antibiotics synthesized through microbial secondary metabolism with non-degradable residues can often accumulate in the environments, lead to the selection of antibiotic resistance genes, and contribute to the development of antibiotic resistant bacterial strains . In the light of emergent antibiotic resistance, developing new antibacterial agents is essential. In recent decades, reported infections of hospital patients by superbugs, which are resistant to multiple types of antibiotics, have appeared life-threatening and untreatable. It has been more serious with ongoing increasing incidences of these infections. New alternative antimicrobial strategies are needed urgently more than ever. Among many diversified discoveries, seeking biodegradable antibacterial polypeptides/proteins/enzymes as alternative antimicrobial agents have drawn a great deal of attention and resulted many new publications in recent years. In this mini-review, we only focus on recent discoveries and development related to specific bacteriocins and endolysins that target on specific pathogens found in biorefinery and ag-related applications. We will discuss the use of genetic engineering technologies with different recombinant hosts to effectively produce selected antibacterial agents as well as fermentative productions and potential applications of the biodegradable antibacterial agents and emerging technologies. • Brief history of Penicillin. • Gram-positive bacteriocins. • Microcin expression. • Recombinant production of endolysins.

Investigating the Role of Vanadium-Dependent Haloperoxidase Enzymology in Microbial Secondary Metabolism and Chemical Ecology.

ABSTRACTThe chemical diversity of natural products is established by an elegant network of biosynthetic machinery and controlled by a suite of intracellular and environmental cues. Advances in genomics, transcriptomics, and metabolomics have provided useful insight to understand how organisms respond to abiotic and biotic factors to adjust their chemical output; this has permitted researchers to begin asking bigger-picture questions regarding the ecological significance of these molecules to the producing organism and its community. Our lab is motivated by understanding how select microbes construct and manipulate bioactive molecules by utilizing vanadium-dependent haloperoxidase (VHPO) enzymology. This commentary will give perspective into our efforts to understand the unique VHPO-catalyzed conversions which modulate the activities within two ecologically relevant natural product families. Through enhancing our knowledge of microbial natural product biosynthesis, we can understand how and why these bioactive molecules are created.

Comprehensive prediction of secondary metabolite structure and biological activity from microbial genome sequences

Novel antibiotics are urgently needed to address the looming global crisis of antibiotic resistance. Historically, the primary source of clinically used antibiotics has been microbial secondary metabolism. Microbial genome sequencing has revealed a plethora of uncharacterized natural antibiotics that remain to be discovered. However, the isolation of these molecules is hindered by the challenge of linking sequence information to the chemical structures of the encoded molecules. Here, we present PRISM 4, a comprehensive platform for prediction of the chemical structures of genomically encoded antibiotics, including all classes of bacterial antibiotics currently in clinical use. The accuracy of chemical structure prediction enables the development of machine-learning methods to predict the likely biological activity of encoded molecules. We apply PRISM 4 to chart secondary metabolite biosynthesis in a collection of over 10,000 bacterial genomes from both cultured isolates and metagenomic datasets, revealing thousands of encoded antibiotics. PRISM 4 is freely available as an interactive web application at http://prism.adapsyn.com.

A chromatogram-simplified Streptomyces albus host for heterologous production of natural products

Cloning natural product biosynthetic gene clusters from cultured or uncultured sources and their subsequent expression by genetically tractable heterologous hosts is an essential strategy for the elucidation and characterisation of novel microbial natural products. The availability of suitable expression hosts is a critical aspect of this workflow. In this work, we mutagenised five endogenous biosynthetic gene clusters from Streptomyces albus S4, which reduced the complexity of chemical extracts generated from the strain and eliminated antifungal and antibacterial bioactivity. We showed that the resulting quintuple mutant can express foreign biosynthetic gene clusters by heterologously producing actinorhodin, cinnamycin and prunustatin. We envisage that our strain will be a useful addition to the growing suite of heterologous expression hosts available for exploring microbial secondary metabolism.

A pharmaceutical model for the molecular evolution of microbial natural products.

Microbes are talented chemists with the ability to generate tremendously complex and diverse natural products which harbor potent biological activities. Natural products are produced using sets of specialized biosynthetic enzymes encoded by secondary metabolism pathways. Here, we present a two-step evolutionary model to explain the diversification of biosynthetic pathways that account for the proliferation of these molecules. We argue that the appearance of natural product families has been a slow and infrequent process. The first step led to the original emergence of bioactive molecules and different classes of natural products. However, much of the chemical diversity observed today has resulted from the endless modification of the ancestral biosynthetic pathways. The second step rapidly modulates the pre-existing biological activities to increase their potency and to adapt to changing environmental conditions. We highlight the importance of enzyme promiscuity in this process, as it facilitates both the incorporation of horizontally transferred genes into secondary metabolic pathways and the functional differentiation of proteins to catalyze novel chemistry. We provide examples where single point mutations or recombination events have been sufficient for new enzymatic activities to emerge. A unique feature in the evolution of microbial secondary metabolism is that gene duplication is not essential but offers opportunities to synthesize more complex metabolites. Microbial natural products are highly important for the pharmaceutical industry due to their unique bioactivities. Therefore, understanding the natural mechanisms leading to the formation of diverse metabolic pathways is vital for future attempts to utilize synthetic biology for the generation of novel molecules.

IMG-ABC v.5.0: an update to the IMG/Atlas of Biosynthetic Gene Clusters Knowledgebase.

Microbial secondary metabolism is a reservoir of bioactive compounds of immense biotechnological and biomedical potential. The biosynthetic machinery responsible for the production of these secondary metabolites (SMs) (also called natural products) is often encoded by collocated groups of genes called biosynthetic gene clusters (BGCs). High-throughput genome sequencing of both isolates and metagenomic samples combined with the development of specialized computational workflows is enabling systematic identification of BGCs and the discovery of novel SMs. In order to advance exploration of microbial secondary metabolism and its diversity, we developed the largest publicly available database of predicted BGCs combined with experimentally verified BGCs, the Integrated Microbial Genomes Atlas of Biosynthetic gene Clusters (IMG-ABC) (https://img.jgi.doe.gov/abc-public). Here we describe the first major content update of the IMG-ABC knowledgebase, since its initial release in 2015, refreshing the BGC prediction pipeline with the latest version of antiSMASH (v5) as well as presenting the data in the context of underlying environmental metadata sourced from GOLD (https://gold.jgi.doe.gov/). This update has greatly improved the quality and expanded the types of predicted BGCs compared to the previous version.

IMG-ABC: new features for bacterial secondary metabolism analysis and targeted biosynthetic gene cluster discovery in thousands of microbial genomes.

Secondary metabolites produced by microbes have diverse biological functions, which makes them a great potential source of biotechnologically relevant compounds with antimicrobial, anti-cancer and other activities. The proteins needed to synthesize these natural products are often encoded by clusters of co-located genes called biosynthetic gene clusters (BCs). In order to advance the exploration of microbial secondary metabolism, we developed the largest publically available database of experimentally verified and predicted BCs, the Integrated Microbial Genomes Atlas of Biosynthetic gene Clusters (IMG-ABC) (https://img.jgi.doe.gov/abc/). Here, we describe an update of IMG-ABC, which includes ClusterScout, a tool for targeted identification of custom biosynthetic gene clusters across 40 000 isolate microbial genomes, and a new search capability to query more than 700 000 BCs from isolate genomes for clusters with similar Pfam composition. Additional features enable fast exploration and analysis of BCs through two new interactive visualization features, a BC function heatmap and a BC similarity network graph. These new tools and features add to the value of IMG-ABC's vast body of BC data, facilitating their in-depth analysis and accelerating secondary metabolite discovery.

ChemInform Abstract: Biosynthetic and Synthetic Access to Amino Sugars

AbstractReview: 265 refs.

Chassis optimization as a cornerstone for the application of synthetic biology based strategies in microbial secondary metabolism

The increased number of bacterial genome sequencing projects has generated over the last years a large reservoir of genomic information. In silico analysis of this genomic data has renewed the interest in bacterial bioprospecting for bioactive compounds by unveiling novel biosynthetic gene clusters of unknown or uncharacterized metabolites. However, only a small fraction of those metabolites is produced under laboratory-controlled conditions; the remaining clusters represent a pool of novel metabolites that are waiting to be “awaken”. Activation of the biosynthetic gene clusters that present reduced or no expression (known as cryptic or silent clusters) by heterologous expression has emerged as a strategy for the identification and production of novel bioactive molecules. Synthetic biology, with engineering principles at its core, provides an excellent framework for the development of efficient heterologous systems for the expression of biosynthetic gene clusters. However, a common problem in its application is the host-interference problem, i.e., the unpredictable interactions between the device and the host that can hamper the desired output. Although an effort has been made to develop orthogonal devices, the most proficient way to overcome the host-interference problem is through genome simplification. In this review we present an overview on the strategies and tools used in the development of hosts/chassis for the heterologous expression of specialized metabolites biosynthetic gene clusters. Finally, we introduce the concept of specialized host as the next step of development of expression hosts.

IMG-ABC: A Knowledge Base To Fuel Discovery of Biosynthetic Gene Clusters and Novel Secondary Metabolites.

ABSTRACTIn the discovery of secondary metabolites, analysis of sequence data is a promising exploration path that remains largely underutilized due to the lack of computational platforms that enable such a systematic approach on a large scale. In this work, we present IMG-ABC (https://img.jgi.doe.gov/abc), an atlas of biosynthetic gene clusters within the Integrated Microbial Genomes (IMG) system, which is aimed at harnessing the power of “big” genomic data for discovering small molecules. IMG-ABC relies on IMG’s comprehensive integrated structural and functional genomic data for the analysis of biosynthetic gene clusters (BCs) and associated secondary metabolites (SMs). SMs and BCs serve as the two main classes of objects in IMG-ABC, each with a rich collection of attributes. A unique feature of IMG-ABC is the incorporation of both experimentally validated and computationally predicted BCs in genomes as well as metagenomes, thus identifying BCs in uncultured populations and rare taxa. We demonstrate the strength of IMG-ABC’s focused integrated analysis tools in enabling the exploration of microbial secondary metabolism on a global scale, through the discovery of phenazine-producing clusters for the first time in Alphaproteobacteria. IMG-ABC strives to fill the long-existent void of resources for computational exploration of the secondary metabolism universe; its underlying scalable framework enables traversal of uncovered phylogenetic and chemical structure space, serving as a doorway to a new era in the discovery of novel molecules.

AntiSMASH 3.0-a comprehensive resource for the genome mining of biosynthetic gene clusters.

Microbial secondary metabolism constitutes a rich source of antibiotics, chemotherapeutics, insecticides and other high-value chemicals. Genome mining of gene clusters that encode the biosynthetic pathways for these metabolites has become a key methodology for novel compound discovery. In 2011, we introduced antiSMASH, a web server and stand-alone tool for the automatic genomic identification and analysis of biosynthetic gene clusters, available at http://antismash.secondarymetabolites.org. Here, we present version 3.0 of antiSMASH, which has undergone major improvements. A full integration of the recently published ClusterFinder algorithm now allows using this probabilistic algorithm to detect putative gene clusters of unknown types. Also, a new dereplication variant of the ClusterBlast module now identifies similarities of identified clusters to any of 1172 clusters with known end products. At the enzyme level, active sites of key biosynthetic enzymes are now pinpointed through a curated pattern-matching procedure and Enzyme Commission numbers are assigned to functionally classify all enzyme-coding genes. Additionally, chemical structure prediction has been improved by incorporating polyketide reduction states. Finally, in order for users to be able to organize and analyze multiple antiSMASH outputs in a private setting, a new XML output module allows offline editing of antiSMASH annotations within the Geneious software.

Microbial Biodiscovery: Back to the Future

Microbial metabolites are remarkable versatile as potent and selective drug lead candidates, and as in situ molecular probes, capable of interrogating key signalling, transport and developmental pathways. Microbial biodiscovery as a drug discovery paradigm has served science and society extremely well, and with appropriate modernisation and reinvestment is well placed to continue to do so into the future. Advances across many disciplines have revealed an untapped silent microbial secondary metabolism, which promises access to unprecedented bioactive chemical space. This renewed capacity can be further enhanced by recognition of the critical importance of widening the search parameters from narrow single bioassay/indication directed programs, to target both active and (seemingly) inactive metabolites, as well as new and known compounds, and a diversity of non-enzymatic chemical transformation products (all too often dismissed as artefacts). Many of the technical and commercial challenges that confronted microbial biodiscovery late last century have been resolved. The need is great and the time is right to re-plumb microbial biodiscovery back into the drug discovery pipeline.