Microbial Biosynthetic Gene Clusters Research Articles

New insights into the enteric methane production based on the archaeal genome map of ruminant gastrointestinal tract

IntroductionAs one of the important components of ruminant gastrointestinal tract (GIT) microbiome, archaea are involved in many biological processes, especially methanogenesis. However, the diversity and individual metabolic characteristics of archaea in this habitat remain largely understudied, partly due to the lack of a unified reference genome catalog. ObjectivesThis study aimed to construct a unified genome map for taxonomic and functional exploration of ruminant GIT archaea in the future. MethodsWe collected archaeal genomes from public sources and new data of this study. We performed phylogenetic analysis, functional genomics analyses, prophages identification based on the genomes. Using collected genomes as a reference, we conducted metagenomic and metatranscriptomic analyses on rumen fluid samples from 18 dairy cows with different methane (CH4) production. ResultsWe constructed the ruminant GIT archaeal genomes (RGAG) by compiling 405 strain-level (160 species) non-redundant archaeal genomes from more than 10 ruminant species. The functional heterogeneity and methanogenic structure within RGAG was investigated. RGAG possessed 1,124 (99.5 %) unknown microbial biosynthetic gene clusters. A survey of RGAG-borne prophages identified 63 prophages with 122 host-beneficial genes and 18 auxiliary metabolic genes (AMGs). The pipeline for both metagenomics and metatranscriptomics generated in the study revealed the roles of archaeal genomes under-assessed in general analyses of muti-omics. The highly expressed genus Methanosphaera was negatively correlated with CH4 production. ConclusionA unified genome map of ruminant GIT archaea is constructed in the study. Functional genomics indicates that the multifaceted functions of RGAG remains undiscovered. Multi-omics analyses reveals the advantages of metatranscriptomics over metagenomics in studying rumen archaeal communities. Differences in rumen archaeal community structure among cattle with different CH4 production may reflect the balance between rumen hydrogen production and methanogenesis. Our work provides new understanding of archaeal functions in the ruminant GIT and potential targets for future CH4 reduction.

Identification and characterization of the siderochelin biosynthetic gene cluster via coculture.

Many microbial biosynthetic gene clusters (BGCs) are inactive under standard laboratory conditions, making characterization of their products difficult. Silent BGCs are likely activated by specific cues in their natural environment, such as the presence of competitors. Growth conditions such as coculture with other microbes, which more closely mimic natural environments, are practical strategies for inducing silent BGCs. Here, we utilize coculture to activate BGCs in nine actinobacteria strains. We observed increased production of the ferrous siderophores siderochelin A and B during coculture of Amycolatopsis strain WAC04611 and Tsukamurella strain WAC06889b. Furthermore, we identified the siderochelin BGC in WAC04611 and discovered that the GntR-family transcription factor sidR3 represses siderochelin production. Deletion of the predicted aminotransferase sidA abolished production of the carboxamides siderochelin A/B and led to the accumulation of the carboxylate siderochelin D. Finally, we deleted the predicted hydroxylase sidB and established that it is essential for siderochelin production. Our findings show that microbial coculture can successfully activate silent BGCs and lead to the discovery and characterization of unknown BGCs for molecules like siderochelin.IMPORTANCESiderophores are vital iron-acquisition elements required by microbes for survival in a variety of environments. Furthermore, many siderophores are essential for the virulence of various human pathogens, making them a possible target for antibacterials. The significance of our work is in the identification and characterization of the previously unknown BGC for the siderophore siderochelin. Our work adds to the growing knowledge of siderophore biosynthesis, which may aid in the future development of siderophore-targeting pharmaceuticals and inform on the ecological roles of these compounds. Furthermore, our work demonstrates that combining microbial coculture with metabolomics is a valuable strategy for identifying upregulated compounds and their BGCs.

Hibiscus acid and hydroxycitric acid dimethyl esters from Hibiscus flowers induce production of dithiolopyrrolone antibiotics by Streptomyces Strain MBN2-2

Plants and microbes are closely associated with each other in their ecological niches. Much has been studied about plant–microbe interactions, but little is known about the effect of phytochemicals on microbes at the molecular level. To access the products of cryptic biosynthetic gene clusters in bacteria, we incorporated an organic extract of hibiscus flowers into the culture media of different Actinobacteria isolated from plant rhizospheres. This approach led to the production of broad-spectrum dithiolopyrrolone (DTP) antibiotics, thiolutin (1) and aureothricin (2), by Streptomyces sp. MBN2-2. The compounds from the hibiscus extract responsible for triggering the production of these two DTPs were found to be hibiscus acid dimethyl ester (3) and hydroxycitric acid 1,3-dimethyl ester (4). It was subsequently found that the addition of either Fe2+ or Fe3+ to culture media induced the production of 1 and 2. The Chrome Azurol S (CAS) assay revealed that 3 and 4 can chelate iron, and therefore, the mechanism leading to the production of thiolutin and aureothricin appears to be related to changes in iron concentration levels. This work supports the idea that phytochemicals can be used to activate the production of cryptic microbial biosynthetic gene clusters and further understand plant–microbe interactions.Graphical

Biosynthetic Gene Clusters from Swine Gut Microbiome.

The abuse of antibiotics has become a serious health challenge in the veterinary field. It creates environmental selection pressure on bacteria and facilitates the rapid spread of antibiotic resistance genes. The speed of discovery and application of cost-effective alternatives to antibiotics is slow in pig production. Natural products from biosynthetic gene clusters (BGCs) represent promising therapeutic agents for animal and human health and have attracted extraordinary passion from researchers due to their ability to participate in biofilm inhibition, stress resistance, and the killing of competitors. In this study, we detected the presence of diverse secondary metabolite genes in porcine intestines through sequence alignment in the antiSMASH database. After comparing variations in microbial BGCs' composition between the ileum and the colon, it was found that the abundance of the resorcinol gene cluster was elevated in the ileal microbiome, whereas the gene cluster of arylpolyene was enriched in the colonic microbiome. The investigation of BGCs' diversity and composition differences between the ileal and colonic microbiomes provided novel insights into further utilizing BGCs in livestock. The importance of BGCs in gut microbiota deserves more attention for promoting healthy swine production.

TaxiBGC: a Taxonomy-Guided Approach for Profiling Experimentally Characterized Microbial Biosynthetic Gene Clusters and Secondary Metabolite Production Potential in Metagenomes.

Biosynthetic gene clusters (BGCs) in microbial genomes encode bioactive secondary metabolites (SMs), which can play important roles in microbe-microbe and host-microbe interactions. Given the biological significance of SMs and the current profound interest in the metabolic functions of microbiomes, the unbiased identification of BGCs from high-throughput metagenomic data could offer novel insights into the complex chemical ecology of microbial communities. Currently available tools for predicting BGCs from shotgun metagenomes have several limitations, including the need for computationally demanding read assembly, predicting a narrow breadth of BGC classes, and not providing the SM product. To overcome these limitations, we developed taxonomy-guided identification of biosynthetic gene clusters (TaxiBGC), a command-line tool for predicting experimentally characterized BGCs (and inferring their known SMs) in metagenomes by first pinpointing the microbial species likely to harbor them. We benchmarked TaxiBGC on various simulated metagenomes, showing that our taxonomy-guided approach could predict BGCs with much-improved performance (mean F1 score, 0.56; mean PPV score, 0.80) compared with directly identifying BGCs by mapping sequencing reads onto the BGC genes (mean F1 score, 0.49; mean PPV score, 0.41). Next, by applying TaxiBGC on 2,650 metagenomes from the Human Microbiome Project and various case-control gut microbiome studies, we were able to associate BGCs (and their SMs) with different human body sites and with multiple diseases, including Crohn's disease and liver cirrhosis. In all, TaxiBGC provides an in silico platform to predict experimentally characterized BGCs and their SM production potential in metagenomic data while demonstrating important advantages over existing techniques. IMPORTANCE Currently available bioinformatics tools to identify BGCs from metagenomic sequencing data are limited in their predictive capability or ease of use to even computationally oriented researchers. We present an automated computational pipeline called TaxiBGC, which predicts experimentally characterized BGCs (and infers their known SMs) in shotgun metagenomes by first considering the microbial species source. Through rigorous benchmarking techniques on simulated metagenomes, we show that TaxiBGC provides a significant advantage over existing methods. When demonstrating TaxiBGC on thousands of human microbiome samples, we associate BGCs encoding bacteriocins with different human body sites and diseases, thereby elucidating a possible novel role of this antibiotic class in maintaining the stability of microbial ecosystems throughout the human body. Furthermore, we report for the first time gut microbial BGC associations shared among multiple pathologies. Ultimately, we expect our tool to facilitate future investigations into the chemical ecology of microbial communities across diverse niches and pathologies.

A single amino acid residue controls acyltransferase activity in a polyketide synthase from Toxoplasma gondii

SummaryType I polyketide synthases (PKSs) are multidomain, multimodule enzymes capable of producing complex polyketide metabolites. These modules contain an acyltransferase (AT) domain, which selects acyl-CoA substrates to be incorporated into the metabolite scaffold. Herein, we reveal the sequences of three AT domains from a polyketide synthase (TgPKS2) from the apicomplexan parasite Toxoplasma gondii. Phylogenic analysis indicates these ATs (AT1, AT2, and AT3) are distinct from domains in well-characterized microbial biosynthetic gene clusters. Biochemical investigations revealed that AT1 and AT2 hydrolyze malonyl-CoA but the terminal AT3 domain is non-functional. We further identify an “on-off switch” residue that controls activity such that a single amino acid change in AT3 confers hydrolysis activity while the analogous mutation in AT2 eliminates activity. This biochemical analysis of AT domains from an apicomplexan PKS lays the foundation for further molecular and structural studies on PKSs from T. gondii and other protists.

Microbial metabolites: cause or consequence in gastrointestinal disease?

Systems biology studies have established that changes in gastrointestinal microbiome composition and function can adversely impact host physiology. Notable diseases synonymously associated with dysbiosis include inflammatory bowel diseases, cancer, metabolic disorders, and opportunistic and recurrent pathogen infections. However, there is a scarcity of mechanistic data that advances our understanding of taxonomic correlations with pathophysiological host-microbiome interactions. Generally, to survive a hostile gut environment, microbes are highly metabolically active and produce trans-kingdom signaling molecules to interact with competing microorganisms and the host. These specialized metabolites likely play important homeostatic roles, and identifying disease-specific taxa and their effector pathways can provide better strategies for diagnosis, treatment, and prevention, as well as the discovery of innovative therapeutics. The signaling role of microbial biotransformation products such as bile acids, short-chain fatty acids, polysaccharides, and dietary tryptophan is increasingly recognized, but little is known about the identity and function of metabolites that are synthesized by microbial biosynthetic gene clusters, including ribosomally synthesized and posttranslationally modified peptides (RiPPs), nonribosomal peptides (NRPs), polyketides (PKs), PK-NRP hybrids, and terpenes. Here we consider how bioactive natural products directly encoded by the human microbiome can contribute to the pathophysiology of gastrointestinal disease, cancer, autoimmune, antimicrobial-resistant bacterial and viral infections (including COVID-19). We also present strategies used to discover these compounds and the biological activities they exhibit, with consideration of therapeutic interventions that could emerge from understanding molecular causation in gut microbiome research.

Long-read metagenomics of soil communities reveals phylum-specific secondary metabolite dynamics

Microbial biosynthetic gene clusters (BGCs) encoding secondary metabolites are thought to impact a plethora of biologically mediated environmental processes, yet their discovery and functional characterization in natural microbiomes remains challenging. Here we describe deep long-read sequencing and assembly of metagenomes from biological soil crusts, a group of soil communities that are rich in BGCs. Taking advantage of the unusually long assemblies produced by this approach, we recovered nearly 3,000 BGCs for analysis, including 712 full-length BGCs. Functional exploration through metatranscriptome analysis of a 3-day wetting experiment uncovered phylum-specific BGC expression upon activation from dormancy, elucidating distinct roles and complex phylogenetic and temporal dynamics in wetting processes. For example, a pronounced increase in BGC transcription occurs at night primarily in cyanobacteria, implicating BGCs in nutrient scavenging roles and niche competition. Taken together, our results demonstrate that long-read metagenomic sequencing combined with metatranscriptomic analysis provides a direct view into the functional dynamics of BGCs in environmental processes and suggests a central role of secondary metabolites in maintaining phylogenetically conserved niches within biocrusts.

Deciphering the Microbial Taxonomy and Functionality of Two Diverse Mangrove Ecosystems and Their Potential Abilities To Produce Bioactive Compounds.

Mangroves, as important and special ecosystems, create unique ecological environments for examining the microbial gene capacity and potential for producing bioactive compounds. However, little is known about the biogeochemical implications of microbiomes in mangrove ecosystems, especially the variations between pristine and anthropogenic mangroves. To elucidate this, we investigated the microbial taxonomic and functional shifts of the mangrove microbiomes and their potential for bioactive compounds in two different coastal mangrove ecosystems in southern China. A gene catalogue, including 87 million unique genes, was constructed, based on deep shotgun metagenomic sequencing. Differentially enriched bacterial and archaeal taxa between pristine mangroves (Guangxi) and anthropogenic mangroves (Shenzhen) were found. The Nitrospira and ammonia-oxidizing archaea, specifically, were more abundant in Shenzhen mangroves, while sulfate-reducing bacteria and methanogens were more abundant in Guangxi mangroves. The results of functional analysis were consistent with the taxonomic results, indicating that the Shenzhen mangrove microbiome has a higher abundance of genes involved in nitrogen metabolism while the Guangxi mangrove microbiome has a higher capacity for sulfur metabolism and methanogenesis. Biosynthetic gene clusters were identified in the metagenome data and in hundreds of de novo reconstructed nonredundant microbial genomes, respectively. Notably, we found different biosynthetic potential in different taxa, and we identified three high quality and novel Acidobacteria genomes with a large number of BGCs. In total, 67,278 unique genes were annotated with antibiotic resistance, indicating the prevalence and persistence in multidrug-resistant genes in the mangrove microbiome.IMPORTANCE This study comprehensively described the taxonomy and functionality of mangrove microbiomes, including their capacity for secondary metabolite biosynthesis and their ability to resist antibiotics. The microbial taxonomic and functional characteristics differed between geographical locations, corresponding to the environmental condition of two diverse mangrove regions. A large number of microbial biosynthetic gene clusters encoding novel bioactivities were found, and this can serve as a valuable resource to guide novel bioactive compound discovery for potential clinical uses.

Computational identification of co-evolving multi-gene modules in microbial biosynthetic gene clusters

The biosynthetic machinery responsible for the production of bacterial specialised metabolites is encoded by physically clustered group of genes called biosynthetic gene clusters (BGCs). The experimental characterisation of numerous BGCs has led to the elucidation of subclusters of genes within BGCs, jointly responsible for the same biosynthetic function in different genetic contexts. We developed an unsupervised statistical method able to successfully detect a large number of modules (putative functional subclusters) within an extensive set of predicted BGCs in a systematic and automated manner. Multiple already known subclusters were confirmed by our method, proving its efficiency and sensitivity. In addition, the resulting large collection of newly defined modules provides new insights into the prevalence and putative biosynthetic role of these modular genetic entities. The automated and unbiased identification of hundreds of co-evolving group of genes is an essential breakthrough for the discovery and biosynthetic engineering of high-value compounds.

Epigenetic modulations of mycoendophytes for novel bioactive molecules

Abstract The need for new analogues with higher efficacy to combat the multidrug-resistant microbes as well as other health implications is the need of the hour. Medicinal chemistry has been focused highly in this regard by researchers, due to labour and redundancy of new skeletons in the conventional mode of natural product discovery. However, the less toxicity imposed by natural products would definitely provoke an interest in developing new analogues from natural sources. Microbes especially endophytes having high metabolic diversity have been an aid to attending the prerequisite clinical challenges. The insights of biosynthetic gene clusters of endophytes have proven to be more potential in drug lead discovery. The epigenetic changes in microbial biosynthetic gene clusters would envision new possible skeletal structural diversities. This review is focused on the modulation as well as activation strategies of the biosynthetic gene clusters for biosynthesizing products with pharmaceutical importance along with possible regulatory elements involved in the expression.

Broad-Host-Range Expression Reveals Native and Host Regulatory Elements That Influence Heterologous Antibiotic Production in Gram-Negative Bacteria.

ABSTRACTHeterologous expression has become a powerful tool for studying microbial biosynthetic gene clusters (BGCs). Here, we extend the transformation-associated recombination cloning and heterologous expression platform for microbial BGCs to include Gram-negative proteobacterial expression hosts. Using a broad-host-range expression platform, we test the implicit assumption that biosynthetic pathways are more successfully expressed in more closely related heterologous hosts. Cloning and expression of the violacein BGC from Pseudoalteromonas luteoviolacea 2ta16 revealed robust production in two proteobacterial hosts, Pseudomonas putida KT2440 and Agrobacterium tumefaciens LBA4404, but very little production of the antibiotic in various laboratory strains of Escherichia coli, despite their closer phylogenetic relationship. We identified a nonclustered LuxR-type quorum-sensing receptor from P. luteoviolacea 2ta16, PviR, that increases pathway transcription and violacein production in E. coli by ∼60-fold independently of acyl-homoserine lactone autoinducers. Although E. coli harbors the most similar homolog of PviR identified from all of the hosts tested, overexpression of various E. coli transcription factors did not result in a statistically significant increase in violacein production, while overexpression of two A. tumefaciens PviR homologs significantly increased production. Thus, this work not only introduces a new genetic platform for the heterologous expression of microbial BGCs, it also challenges the assumption that host phylogeny is an accurate predictor of host compatibility.

Plant-like biosynthesis of isoquinoline alkaloids in Aspergillus fumigatus.

Natural product discovery efforts have focused primarily on microbial biosynthetic gene clusters (BGCs) containing large multi-modular PKSs and NRPSs; however, sequencing of fungal genomes has revealed a vast number of BGCs containing smaller NRPS-like genes of unknown biosynthetic function. Using comparative metabolomics, we show that a BGC in the human pathogen Aspergillus fumigatus named fsq, which contains an NRPS-like gene lacking a condensation domain, produces several novel isoquinoline alkaloids, the fumisoquins. These compounds derive from carbon-carbon bond formation between two amino acid-derived moieties followed by a sequence that is directly analogous to isoquinoline alkaloid biosynthesis in plants. Fumisoquin biosynthesis requires the N-methyltransferase FsqC and the FAD-dependent oxidase FsqB, which represent functional analogs of coclaurine N-methyltransferase and berberine bridge enzyme in plants. Our results show that BGCs containing incomplete NRPS modules may reveal new biosynthetic paradigms and suggest that plant-like isoquinoline biosynthesis occurs in diverse fungi.

Linking Biosynthetic Gene Clusters to their Metabolites via Pathway- Targeted Molecular Networking.

The connection of microbial biosynthetic gene clusters to the small molecule metabolites they encode is central to the discovery and characterization of new metabolic pathways with ecological and pharmacological potential. With increasing microbial genome sequence information being deposited into publicly available databases, it is clear that microbes have the coding capacity for many more biologically active small molecules than previously realized. Of increasing interest are the small molecules encoded by the human microbiome, as these metabolites likely mediate a variety of currently uncharacterized human-microbe interactions that influence health and disease. In this mini-review, we describe the ongoing biosynthetic, structural, and functional characterizations of the genotoxic colibactin pathway in gut bacteria as a thematic example of linking biosynthetic gene clusters to their metabolites. We also highlight other natural products that are produced through analogous biosynthetic logic and comment on some current disconnects between bioinformatics predictions and experimental structural characterizations. Lastly, we describe the use of pathway-targeted molecular networking as a tool to characterize secondary metabolic pathways within complex metabolomes and to aid in downstream metabolite structural elucidation efforts.