Identification of clusters of secondary metabolite biosynthetic genes in the Camelina sativa genome.

Multidrug-resistant pathogens pose an earnest risk to human health. Therefore, new antibiotics need to be developed quickly. Most of the antibiotics we use today are derived from secondary metabolites, which are produced by plants. Genome mining tools allow us to detect biosynthetic gene clusters (BGCs) responsible for the production of secondary metabolites. Focusing on the most promising BGCs-coding antibiotics with unique pathways is currently a challenge. In silico approach like genome mining are used to visualise the action of these bioactive chemicals. Camelina sativa is a well-known medicinal plant and it would be interesting to study its secondary metabolites. In this work, we found seven bioactive compounds in this plant using the genome mining approach. Further, the clusters of genes involved in the biosynthesis of these compounds were analysed with their metabolic pathways. This work illuminates new ground on the evolution of BGCs for the nutritional improvement of C. sativa.

Comparative genomics of Metarhizium brunneum strains V275 and ARSEF 4556: unraveling intraspecies diversity.

Entomopathogenic fungi belonging to the Order Hypocreales are renowned for their ability to infect and kill insect hosts, while their endophytic mode of life and the beneficial rhizosphere effects on plant hosts have only been recently recognized. Understanding the molecular mechanisms underlying their different lifestyles could optimize their potential as both biocontrol and biofertilizer agents, as well as the wider appreciation of niche plasticity in fungal ecology. This study describes the comprehensive whole genome sequencing and analysis of one of the most effective entomopathogenic and endophytic EPF strains, Metarhizium brunneum V275 (commercially known as Lalguard Met52), achieved through Nanopore and Illumina reads. Comparative genomics for exploring intraspecies variability and analyses of key gene sets were conducted with a second effective EPF strain, M. brunneum ARSEF 4556. The search for strain- or species-specific genes was extended to M. brunneum strain ARSEF 3297 and other species of genus Metarhizium, to identify molecular mechanisms and putative key genome adaptations associated with mode of life differences. Genome size differed significantly, with M. brunneum V275 having the largest genome amongst M. brunneum strains sequenced to date. Genome analyses revealed an abundance of plant-degrading enzymes, plant colonization-associated genes, and intriguing intraspecies variations regarding their predicted secondary metabolic compounds and the number and localization of Transposable Elements. The potential significance of the differences found between closely related endophytic and entomopathogenic fungi, regarding plant growth-promoting and entomopathogenic abilities, are discussed, enhancing our understanding of their diverse functionalities and putative applications in agriculture and ecology.

Advancing cyanobacterial biotechnology: new frontiers in natural product discovery and production.

Cyanobacteria, as oxygenic phototrophs, offer significant potential for sustainable biotechnology applications. Cyanobacterial natural products, with antimicrobial, anticancer, and plant growth-promoting properties, hold promise in pharmaceuticals, agriculture, and environmental remediation. By leveraging advanced technologies, cyanobacteria can significantly impact various industries, supporting the green biotechnology agenda. Recent advancements in integrated omics, orphan gene cluster activation, genetic manipulation, and chemo-enzymatic methods are expanding their biotechnological relevance. Omics technologies revolutionize cyanobacterial natural product research by facilitating biosynthetic gene cluster identification. Heterologous expression and pathway reconstitution enable complex natural product production, while high-titer strategies like metabolic engineering enhance yields. Interdisciplinary research and technological progress position cyanobacteria as valuable sources of bioactive compounds, driving sustainable biotechnological practices forward.

High-Throughput Mining of Novel Compounds from Known Microbes: A Boost to Natural Product Screening.

Advanced techniques can accelerate the pace of natural product discovery from microbes, which has been lagging behind the drug discovery era. Therefore, the present review article discusses the various interdisciplinary and cutting-edge techniques to present a concrete strategy that enables the high-throughput screening of novel natural compounds (NCs) from known microbes. Recent bioinformatics methods revealed that the microbial genome contains a huge untapped reservoir of silent biosynthetic gene clusters (BGC). This article describes several methods to identify the microbial strains with hidden mines of silent BGCs. Moreover, antiSMASH 5.0 is a free, accurate, and highly reliable bioinformatics tool discussed in detail to identify silent BGCs in the microbial genome. Further, the latest microbial culture technique, HiTES (high-throughput elicitor screening), has been detailed for the expression of silent BGCs using 500-1000 different growth conditions at a time. Following the expression of silent BGCs, the latest mass spectrometry methods are highlighted to identify the NCs. The recently emerged LAESI-IMS (laser ablation electrospray ionization-imaging mass spectrometry) technique, which enables the rapid identification of novel NCs directly from microtiter plates, is presented in detail. Finally, various trending 'dereplication' strategies are emphasized to increase the effectiveness of NC screening.

Histone Deacetylase UvHST2 Is a Global Regulator of Secondary Metabolism in Ustilaginoidea virens.

Ustilaginoidea virens, the causal agent of rice false smut, produces a large amount of mycotoxins, including ustilaginoidins and sorbicillinoids. However, little is known about the regulatory mechanism of mycotoxin biosynthesis inU. virens. Here, we demonstrate that the NAD+-dependent histone deacetylase UvHST2 negatively regulates ustilaginoidin biosynthesis. UvHst2 knockout caused retarded hypha growth and reduced conidiation and pathogenicity inU. virens. Transcriptome analysis revealed that the transcription factor genes, transporter genes, and other tailoring genes in eight biosynthetic gene clusters (BGCs) including ustilaginoidin and sorbicillinoid BGCs were upregulated in ΔUvhst2. Interestingly, the UvHst2 deletion affects alternative splicing. Metabolomics revealed that UvHST2 negatively regulates the biosynthesis of various mycotoxins including ustilaginoidins, sorbicillin, ochratoxin B, zearalenone, and O-M-sterigmatocystin. Combined transcriptome and metabolome analyses uncover that UvHST2 positively regulates pathogenicity but negatively modulates the expression of BGCs involved in secondary metabolism. Collectively, UvHST2 functions as a global regulator of secondary metabolism inU. virens.

Fungal BGCs for Production of Secondary Metabolites: Main Types, Central Roles in Strain Improvement, and Regulation According to the Piano Principle.

Filamentous fungi are one of the most important producers of secondary metabolites. Some of them can have a toxic effect on the human body, leading to diseases. On the other hand, they are widely used as pharmaceutically significant drugs, such as antibiotics, statins, and immunosuppressants. A single fungus species in response to various signals can produce 100 or more secondary metabolites. Such signaling is possible due to the coordinated regulation of several dozen biosynthetic gene clusters (BGCs), which are mosaically localized in different regions of fungal chromosomes. Their regulation includes several levels, from pathway-specific regulators, whose genes are localized inside BGCs, to global regulators of the cell (taking into account changes in pH, carbon consumption, etc.) and global regulators of secondary metabolism (affecting epigenetic changes driven by velvet family proteins, LaeA, etc.). In addition, various low-molecular-weight substances can have a mediating effect on such regulatory processes. This review is devoted to a critical analysis of the available data on the "turning on" and "off" of the biosynthesis of secondary metabolites in response to signals in filamentous fungi. To describe the ongoing processes, the model of "piano regulation" is proposed, whereby pressing a certain key (signal) leads to the extraction of a certain sound from the "musical instrument of the fungus cell", which is expressed in the production of a specific secondary metabolite.

Charting the Lipopeptidome of Nonpathogenic Pseudomonas.

A major source of pseudomonad-specialized metabolites is the nonribosomal peptide synthetases (NRPSs) assembling siderophores and lipopeptides. Cyclic lipopeptides (CLPs) of the Mycin and Peptin families are frequently associated with, but not restricted to, phytopathogenic species. We conducted an in silico analysis of the NRPSs encoded by lipopeptide biosynthetic gene clusters in nonpathogenic Pseudomonas genomes, covering 13 chemically diversified families. This global assessment of lipopeptide production capacity revealed it to be confined to the Pseudomonas fluorescens lineage, with most strains synthesizing a single type of CLP. Whereas certain lipopeptide families are specific for a taxonomic subgroup, others are found in distant groups. NRPS activation domain-guided peptide predictions enabled reliable family assignments, including identification of novel members. Focusing on the two most abundant lipopeptide families (Viscosin and Amphisin), a portion of their uncharted diversity was mapped, including characterization of two novel Amphisin family members (nepenthesin and oakridgin). Using NMR fingerprint matching, known Viscosin-family lipopeptides were identified in 15 (type) species spread across different taxonomic groups. A bifurcate genomic organization predominates among Viscosin-family producers and typifies Xantholysin-, Entolysin-, and Poaeamide-family producers but most families feature a single NRPS gene cluster embedded between cognate regulator and transporter genes. The strong correlation observed between NRPS system phylogeny and rpoD-based taxonomic affiliation indicates that much of the structural diversity is linked to speciation, providing few indications of horizontal gene transfer. The grouping of most NRPS systems in four superfamilies based on activation domain homology suggests extensive module dynamics driven by domain deletions, duplications, and exchanges. IMPORTANCE Pseudomonas species are prominent producers of lipopeptides that support proliferation in a multitude of environments and foster varied lifestyles. By genome mining of biosynthetic gene clusters (BGCs) with lipopeptide-specific organization, we mapped the global Pseudomonas lipopeptidome and linked its staggering diversity to taxonomy of the producers, belonging to different groups within the major Pseudomonas fluorescens lineage. Activation domain phylogeny of newly mined lipopeptide synthetases combined with previously characterized enzymes enabled assignment of predicted BGC products to specific lipopeptide families. In addition, novel peptide sequences were detected, showing the value of substrate specificity analysis for prioritization of BGCs for further characterization. NMR fingerprint matching proved an excellent tool to unequivocally identify multiple lipopeptides bioinformatically assigned to the Viscosin family, by far the most abundant one in Pseudomonas and with stereochemistry of all its current members elucidated. In-depth analysis of activation domains provided insight into mechanisms driving lipopeptide structural diversification.

Genome mining, antimicrobial and plant growth-promoting potentials of halotolerant Bacillus paralicheniformis ES-1 isolated from salt mine.

Salinity severely affects crop yield by hindering nitrogen uptake and reducing plant growth. Plant growth-promoting bacteria (PGPB) are capable of providing cross-protection against biotic/abiotic stresses and facilitating plant growth. Genome-level knowledge of PGPB is necessary to translate the knowledge into a product as efficient biofertilizers and biocontrol agents. The current study aimed to isolate and characterize indigenous plant growth-promoting strains with the potential to promote plant growth under various stress conditions. In this regard, 72 bacterial strains were isolated from various saline-sodic soil/lakes; 19 exhibited multiple in vitro plant growth-promoting traits, including indole 3 acetic acid production, phosphate solubilization, siderophore synthesis, lytic enzymes production, biofilm formation, and antibacterial activities. To get an in-depth insight into genome composition and diversity, whole-genome sequence and genome mining of one promising Bacillus paralicheniformis strain ES-1 were performed. The strain ES-1 genome carries 12 biosynthetic gene clusters, at least six genomic islands, and four prophage regions. Genome mining identified plant growth-promoting conferring genes such as phosphate solubilization, nitrogen fixation, tryptophan production, siderophore, acetoin, butanediol, chitinase, hydrogen sulfate synthesis, chemotaxis, and motility. Comparative genome analysis indicates the region of genome plasticity which shapes the structure and function of B. paralicheniformis and plays a crucial role in habitat adaptation. The strain ES-1 has a relatively large accessory genome of 649 genes (~ 19%) and 180 unique genes. Overall, these results provide valuable insight into the bioactivity and genomic insight into B. paralicheniformis strain ES-1 with its potential use in sustainable agriculture.

OSMAC-Based Discovery and Biosynthetic Gene Clusters Analysis of Secondary Metabolites from Marine-Derived Streptomyces globisporus SCSIO LCY30.

The one strain many compounds (OSMAC) strategy is an effective method for activating silent gene clusters by cultivating microorganisms under various conditions. The whole genome sequence of the marine-derived strain Streptomyces globisporus SCSIO LCY30 revealed that it contains 30 biosynthetic gene clusters (BGCs). By using the OSMAC strategy, three types of secondary metabolites were activated and identified, including three angucyclines, mayamycin A (1), mayamycin B (2), and rabolemycin (3); two streptophenazines (streptophenazin O (4) and M (5)); and a macrolide dimeric dinactin (6), respectively. The biosynthetic pathways of the secondary metabolites in these three families were proposed based on the gene function prediction and structural information. The bioactivity assays showed that angucycline compounds 1-3 exhibited potent antitumor activities against 11 human cancer cell lines and antibacterial activities against a series of Gram-positive bacteria. Mayamycin (1) selectively exhibited potent cytotoxicity activity against triple-negative breast cancer (TNBC) cell lines such as MDA-MB-231, MDA-MB-468, and Bt-549, with IC50 values of 0.60-2.22 µM.

Marine Actinobacteria a New Source of Antibacterial Metabolites to Treat Acne Vulgaris Disease-A Systematic Literature Review.

Acne vulgaris is a multifactorial disease that remains under-explored; up to date it is known that the bacterium Cutibacterium acnes is involved in the disease occurrence, also associated with a microbial dysbiosis. Antibiotics have become a mainstay treatment generating the emergence of antibiotic-resistant bacteria. In addition, there are some reported side effects of alternative treatments, which indicate the need to investigate a different therapeutic approach. Natural products continue to be an excellent option, especially those extracted from actinobacteria, which represent a prominent source of metabolites with a wide range of biological activities, particularly the marine actinobacteria, which have been less studied than their terrestrial counterparts. Therefore, this systematic review aimed to identify and evaluate the potential anti-infective activity of metabolites isolated from marine actinobacteria strains against bacteria related to the development of acne vulgaris disease. It was found that there is a variety of compounds with anti-infective activity against Staphylococcus aureus and Staphylococcus epidermidis, bacteria closely related to acne vulgaris development; nevertheless, there is no report of a compound with antibacterial activity or quorum-sensing inhibition toward C. acnes, which is a surprising result. Since two of the most widely used antibiotics for the treatment of acne targeting C. acnes were obtained from actinobacteria of the genus Streptomyces, this demonstrates a great opportunity to pursue further studies in this field, considering the potential of marine actinobacteria to produce new anti-infective compounds.

Deep Functional Profiling of Wild Animal Microbiomes Reveals Probiotic Bacillus pumilus Strains with a Common Biosynthetic Fingerprint.

The biodiversity of microorganisms is maintained by intricate nets of interactions between competing species. Impaired functionality of human microbiomes correlates with their reduced biodiversity originating from aseptic environmental conditions and antibiotic use. Microbiomes of wild animals are free of these selective pressures. Microbiota provides a protecting shield from invasion by pathogens in the wild, outcompeting their growth in specific ecological niches. We applied ultrahigh-throughput microfluidic technologies for functional profiling of microbiomes of wild animals, including the skin beetle, Siberian lynx, common raccoon dog, and East Siberian brown bear. Single-cell screening of the most efficient killers of the common human pathogen Staphylococcus aureus resulted in repeated isolation of Bacillus pumilus strains. While isolated strains had different phenotypes, all of them displayed a similar set of biosynthetic gene clusters (BGCs) encoding antibiotic amicoumacin, siderophore bacillibactin, and putative analogs of antimicrobials including bacilysin, surfactin, desferrioxamine, and class IId cyclical bacteriocin. Amicoumacin A (Ami) was identified as a major antibacterial metabolite of these strains mediating their antagonistic activity. Genome mining indicates that Ami BGCs with this architecture subdivide into three distinct families, characteristic of the B. pumilus, B. subtilis, and Paenibacillus species. While Ami itself displays mediocre activity against the majority of Gram-negative bacteria, isolated B. pumilus strains efficiently inhibit the growth of both Gram-positive S. aureus and Gram-negative E. coli in coculture. We believe that the expanded antagonistic activity spectrum of Ami-producing B. pumilus can be attributed to the metabolomic profile predetermined by their biosynthetic fingerprint. Ultrahigh-throughput isolation of natural probiotic strains from wild animal microbiomes, as well as their metabolic reprogramming, opens up a new avenue for pathogen control and microbiome remodeling in the food industry, agriculture, and healthcare.

Genome Mining and Comparative Genome Analysis Revealed Niche-Specific Genome Expansion in Antibacterial Bacillus pumilus Strain SF-4.

The present study reports the isolation of antibacterial exhibiting Bacillus pumilus (B. pumilus) SF-4 from soil field. The genome of this strain SF-4 was sequenced and analyzed to acquire in-depth genomic level insight related to functional diversity, evolutionary history, and biosynthetic potential. The genome of the strain SF-4 harbor 12 Biosynthetic Gene Clusters (BGCs) including four Non-ribosomal peptide synthetases (NRPSs), two terpenes, and one each of Type III polyketide synthases (PKSs), hybrid (NRPS/PKS), lipopeptide, ß-lactone, and bacteriocin clusters. Plant growth-promoting genes associated with de-nitrification, iron acquisition, phosphate solubilization, and nitrogen metabolism were also observed in the genome. Furthermore, all the available complete genomes of B. pumilus strains were used to highlight species boundaries and diverse niche adaptation strategies. Phylogenetic analyses revealed local diversification and indicate that strain SF-4 is a sister group to SAFR-032 and 150a. Pan-genome analyses of 12 targeted strains showed regions of genome plasticity which regulate function of these strains and proposed direct strain adaptations to specific habitats. The unique genome pool carries genes mostly associated with "biosynthesis of secondary metabolites, transport, and catabolism" (Q), "replication, recombination and repair" (L), and "unknown function" (S) clusters of orthologous groups (COG) categories. Moreover, a total of 952 unique genes and 168 exclusively absent genes were prioritized across the 12 genomes. While newly sequenced B. pumilus SF-4 genome consists of 520 accessory, 59 unique, and seven exclusively absent genes. The current study demonstrates genomic differences among 12 B. pumilus strains and offers comprehensive knowledge of the respective genome architecture which may assist in the agronomic application of this strain in future.

Current Approaches for Advancement in Understanding the Molecular Mechanisms of Mycotoxin Biosynthesis.

Filamentous fungi are able to synthesise a remarkable range of secondary metabolites, which play various key roles in the interaction between fungi and the rest of the biosphere, determining their ecological fitness. Many of them can have a beneficial activity to be exploited, as well as negative impact on human and animal health, as in the case of mycotoxins contaminating large quantities of food, feed, and agricultural products worldwide and posing serious health and economic risks. The elucidation of the molecular aspects of mycotoxin biosynthesis has been greatly sped up over the past decade due to the advent of next-generation sequencing technologies, which greatly reduced the cost of genome sequencing and related omic analyses. Here, we briefly highlight the recent progress in the use and integration of omic approaches for the study of mycotoxins biosynthesis. Particular attention has been paid to genomics and transcriptomic approaches for the identification and characterisation of biosynthetic gene clusters of mycotoxins and the understanding of the regulatory pathways activated in response to physiological and environmental factors leading to their production. The latest innovations in genome-editing technology have also provided a more powerful tool for the complete explanation of regulatory and biosynthesis pathways. Finally, we address the crucial issue of the interpretation of the combined omics data on the biology of the mycotoxigenic fungi. They are rapidly expanding and require the development of resources for more efficient integration, as well as the completeness and the availability of intertwined data for the research community.

Multifarious Elicitors: Invoking Biosynthesis of Various Bioactive Secondary Metabolite in Fungi.

Natural products are considered to be the lifeline treatment for several diseases where their structural complexity makes them a source of potential lead molecules. As a producer of antibiotics, food colorants, enzymes, and nutritious food, fungi are beneficial to humans. Fungi, as a source of novel natural products, draw attention of scientists. However, redundant isolation of metabolite retards the rate of discovery. So, apart from the standard conditions for the production of secondary metabolites, certain induction strategies are used to trigger biosynthetic genes in fungi. Advancement in the computational tools helps in connecting gene clusters and their metabolite production. Therefore, modern analytical tools and the genomic era in hand leads to the identification of manifold of cryptic metabolites. The cryptic biosynthetic gene cluster (BGC) has become a treasure hunt for new metabolites representing biosynthetic pathways, regulatory mechanisms, and other factors. This review includes the use of chemical inducers/epigenetic modifiers and co-culture (species interaction) techniques to induce these BGCs. Furthermore, it cites a detailed representation of molecules isolated using these strategies. Since the induction occurs on the genomic molecular DNA and histones, this together brings a significant exploration of the biosynthetic pathways.Graphical Abstract.

Isolation, Genomic and Metabolomic Characterization of Streptomyces tendae VITAKN with Quorum Sensing Inhibitory Activity from Southern India.

Streptomyces are among the most promising genera in terms of production ability to biosynthesize a variety of bioactive secondary metabolites with pharmaceutical interest. Coinciding with the increase in genomic sequencing of these bacteria, mining of their genomes for biosynthetic gene clusters (BGCs) has become a routine component of natural product discovery. Herein, we describe the isolation and characterization of a Streptomyces tendae VITAKN with quorum sensing inhibitory (QSI) activity that was isolated from southern coastal part of India. The nearly complete genome consists of 8,621,231bp with a GC content of 72.2%. Sequence similarity networks of the BGCs detected from this strain against the Minimum Information about a Biosynthetic Gene Cluster (MIBiG) database and 3365 BGCs predicted by antiSMASH analysis of publicly available complete Streptomyces genomes were generated through the BiG-SCAPE-CORASON platform to evaluate its biosynthetic novelty. Crude extract analysis using high-performance liquid chromatography connected to high resolution tandem mass spectrometry (HPLC-HRMS/MS) and dereplication through the Global Natural Product Social Molecular Networking (GNPS) online workflow resulted in the identification of cyclic dipeptides (2, 5-diketopiperazines, DKPs) in the extract, which are known to possess QSI activity. Our results highlight the potential of genome mining coupled with LC-HRMS/MS and in silico tools (GNPS) as a valid approach for the discovery of novel QSI lead compounds. This study also provides the biosynthetic diversity of BGCs and an assessment of the predicted chemical space yet to be discovered.

Engineered Streptomyces lividans Strains for Optimal Identification and Expression of Cryptic Biosynthetic Gene Clusters.

Streptomyces lividans is a suitable host for the heterologous expression of biosynthetic gene clusters (BGCs) from actinomycetes to discover "cryptic" secondary metabolites. To improve the heterologous expression of BGCs, herein we optimized S. lividans strain SBT5 via the stepwise integration of three global regulatory genes and two codon-optimized multi-drug efflux pump genes and deletion of a negative regulatory gene, yielding four engineered strains. All optimization steps were observed to promote the heterologous production of polyketides, non-ribosomal peptides, and hybrid antibiotics. The production increments of these optimization steps were additional, so that the antibiotic yields were several times or even dozens of times higher than the parent strain SBT5 when the final optimized strain, S. lividans LJ1018, was used as the heterologous expression host. The heterologous production of these antibiotics in S. lividans LJ1018 and GX28 was also much higher than in the strains from which the BGCs were isolated. S. lividans LJ1018 and GX28 markedly promoted the heterologous production of secondary metabolites, without requiring manipulation of gene expression components such as promoters on individual gene clusters. Therefore, these strains are well-suited as heterologous expression hosts for secondary metabolic BGCs. In addition, we successfully conducted high-throughput library expression and functional screening (LEXAS) of one bacterial artificial chromosome library and two cosmid libraries of three Streptomyces genomes using S. lividans GX28 as the library-expression host. The LEXAS experiments identified clones carrying intact BGCs sufficient for the heterologous production of piericidin A1, murayaquinone, actinomycin D, and dehydrorabelomycin. Notably, due to lower antibiotic production, the piericidin A1 BGC had been overlooked in a previous LEXAS screening using S. lividans SBT5 as the expression host. These results demonstrate the feasibility and superiority of S. lividans GX28 as a host for high-throughput screening of genomic libraries to mine cryptic BGCs and bioactive compounds.