Search

In recent years, CRISPR-Cas toolboxes for Streptomyces editing have rapidly accelerated natural product discovery and engineering. However, Cas efficiencies are oftentimes strain-dependent, and the commonly used Streptococcus pyogenes Cas9 (SpCas9) is notorious for having high levels of off-target toxicity effects. Thus, a variety of Cas proteins is required for greater flexibility of genetic manipulation within a wider range of Streptomyces strains. This study explored the first use of Acidaminococcus sp. Cas12j, a hypercompact Cas12 subfamily, for genome editing in Streptomyces and its potential in activating silent biosynthetic gene clusters (BGCs) to enhance natural product synthesis. While the editing efficiencies of Cas12j were not as high as previously reported efficiencies of Cas12a and Cas9, Cas12j exhibited higher transformation efficiencies compared to SpCas9. Furthermore, Cas12j demonstrated significantly improved editing efficiencies compared to Cas12a in activating BGCs in Streptomyces sp. A34053, a strain wherein both SpCas9 and Cas12a faced limitations in accessing the genome. Overall, this study expanded the repertoire of Cas proteins for genome editing in actinomycetes and highlighted not only the potential of recently characterized Cas12j in Streptomyces but also the importance of having an extensive genetic toolbox for improving the editing success of these beneficial microbes. [ABSTRACT FROM AUTHOR]

Marine cyanobacterial bioactives, due to their diverse chemistry and promising pharmacological properties, hold significant potential as therapeutic agents. Here, we comprehensively review the scientific state‐of‐the‐art relating to marine cyanobacterial bioactives, including bioprospecting, preclinical and clinical studies, mechanisms of action, safety and toxicity considerations, sustainability, and conservation. We discuss challenges in and opportunities for developing marine cyanobacterial bioactives as therapeutics; we underscore their potential in treating life‐threatening health ailments, such as cancer and neurodegenerative, inflammatory and viral diseases. We further articulate the significant potential of marine cyanobacterial bioactives for improving human health, and identify future research directions underpinning their development as therapeutics. [ABSTRACT FROM AUTHOR]

Over the last several years, there has been increased interest from academia and the pharmaceutical/biotech industry in the development of vaccine adjuvants for new and emerging vaccine modalities. Despite this, vaccine adjuvant development still has some of the longest timelines in the pharmaceutical space, from discovery to clinical approval. The reasons for this are manyfold and range from complexities in translation from animal to human models, concerns about safety or reactogenicity, to challenges in sourcing the necessary raw materials at scale. In this review, we will describe the current state of the art for many adjuvant technologies and how they should be approached or applied in the development of new vaccine products. We postulate that there are many factors to be considered and tools to be applied earlier on in the vaccine development pipeline to improve the likelihood of clinical success. These recommendations may require a modified approach to some of the common practices in new product development but would result in more accessible and practical adjuvant-containing products. [ABSTRACT FROM AUTHOR]

Cycads, which primarily consist of the families Cycadaceae and Zamiaceae, possess intrinsic therapeutic attributes that are prominently expressed across their morphological spectrum, including roots, leaves, flowers, and seeds. In Chinese traditional medicine, the leaves of cycads are particularly revered for their profound healing capabilities. This meticulous review engages with existing literature on cycads and presents insightful avenues for future research. Over 210 phytoconstituents have been isolated and identified from various cycad tissues, including flavonoids, azoxy metabolites, sterols, lignans, non-proteogenic amino acids, terpenoids, and other organic constituents. The contemporary pharmacological discourse highlights the antineoplastic, antimicrobial, and antidiabetic activities inherent in these ancient plants, which are of particular importance to the field of oncology. Despite the prevalent focus on crude extracts and total flavonoid content, our understanding of the nuanced pharmacodynamics of cycads lags considerably behind. The notoriety of cycads derived toxicity, notably within the context of Guam's neurological disease cluster, has precipitated an established emphasis on toxicological research within this field. As such, this critical review emphasizes nascent domains deserving of academic and clinical pursuit, whilst nested within the broader matrix of current scientific understanding. The systematic taxonomy, traditional applications, phytochemical composition, therapeutic potential, and safety profile of cycads are holistically interrogated, assimilating an indispensable repository for future scholarly inquiries. In conclusion, cycads stand as a veritable treasure trove of pharmacological virtue, displaying remarkable therapeutic prowess and holding vast promise for ongoing scientific discovery and clinical utilization. Cycads represent a rich, therapeutic resource, exhibiting exceptional medicinal efficacy and enormous potential for continued exploration and application. [Display omitted] • Up to now, more than 210 phytochemicals have been identified from cycads. • Cycads is a double-edged sword, and its toxicity should not be overlooked. • Cycads has great medical development and application value. [ABSTRACT FROM AUTHOR]

Dehydroalanine (Dha) and dehydrobutyrine (Dhb) display considerable flexibility in a variety of chemical and biological reactions. Natural products containing Dha and/or Dhb residues are often found to display diverse biological activities. While the (Z) geometry is predominant in nature, only a handful of metabolites containing (E)‐Dhb have been found thus far. Here we report discovery of a new antimicrobial peptide, albopeptide, through NMR analysis and chemical synthesis, which contains two contiguous unsaturated residues, Dha‐(E)‐Dhb. It displays narrow‐spectrum activity against vancomycin‐resistant Enterococcus faecium. In‐vitro biochemical assays show that albopeptide originates from a noncanonical NRPS pathway featuring dehydration processes and catalysed by unusual condensation domains. Finally, we provide evidence of the occurrence of a previously untapped group of short unsaturated peptides in the bacterial kingdom, suggesting an important biological function in bacteria. [ABSTRACT FROM AUTHOR]

Dehydroalanine (Dha) and dehydrobutyrine (Dhb) display considerable flexibility in a variety of chemical and biological reactions. Natural products containing Dha and/or Dhb residues are often found to display diverse biological activities. While the (Z) geometry is predominant in nature, only a handful of metabolites containing (E)‐Dhb have been found thus far. Here we report discovery of a new antimicrobial peptide, albopeptide, through NMR analysis and chemical synthesis, which contains two contiguous unsaturated residues, Dha‐(E)‐Dhb. It displays narrow‐spectrum activity against vancomycin‐resistant Enterococcus faecium. In‐vitro biochemical assays show that albopeptide originates from a noncanonical NRPS pathway featuring dehydration processes and catalysed by unusual condensation domains. Finally, we provide evidence of the occurrence of a previously untapped group of short unsaturated peptides in the bacterial kingdom, suggesting an important biological function in bacteria. [ABSTRACT FROM AUTHOR]

Type I polyketide synthases (T1PKSs) are one of the most extensively studied PKSs, which can act either iteratively or via an assembly-line mechanism. Domains in the T1PKSs can readily be predicted by computational tools based on their highly conserved sequences. However, to distinguish between iterative and noniterative at the module level remains an overwhelming challenge, which may account for the seemingly biased distribution of T1PKSs in fungi and bacteria: small iterative monomodular T1PKSs that are responsible for the enormously diverse fungal natural products exist almost exclusively in fungi. Here we report the discovery of iterative T1PKSs that are unexpectedly both abundant and widespread in Streptomyces. Seven of 11 systematically selected T1PKS monomodules from monomodular T1PKS biosynthetic gene clusters (BGCs)were experimentally confirmed to be iteratively acting, synthesizing diverse branched/nonbranched linear intermediates, and two of them produced bioactive allenic polyketides and citreodiols as end products, respectively. This study indicates the huge potential of iterative T1PKS BGCs from streptomycetes in the discovery of novel polyketides. [ABSTRACT FROM AUTHOR]

The quest for novel natural products has recently focused on the marine environment as a source for novel microorganisms. Although isolation of marine-derived actinomycete strains is now common, understanding their distribution in the oceans and their adaptation to this environment can be helpful in the selection of isolates for further novel secondary metabolite discovery. This study explores the taxonomic diversity of marine-derived actinomycetes from distinct environments in the coastal areas of the Yucatan Peninsula and their adaptation to the marine environment as a first step towards novel natural product discovery. The use of simple ecological principles, for example, phylogenetic relatedness to previously characterized actinomycetes or seawater requirements for growth, to recognize isolates with adaptations to the ocean in an effort to select for marine-derived actinomycete to be used for further chemical studies. Marine microbial environments are an important source of novel bioactive natural products and, together with methods such as genome mining for detection of strains with biotechnological potential, ecological strategies can bring useful insights in the selection and identification of marine-derived actinomycetes for novel natural product discovery. [ABSTRACT FROM AUTHOR]

2,2′-Bipyridine (2,2′-BP) is the unique molecular scaffold of the bioactive natural products represented by caerulomycins (CAEs) and collismycins (COLs). CAEs and COLs are highly similar in the chemical structures in which their 2,2′-BP cores typically contain a di- or tri-substituted ring A and an unmodified ring B. Here, we summarize the CAE and COL-type 2,2′-BP natural products known or hypothesized to date: (1) isolated using methods traditional for natural product characterization, (2) created by engineering the biosynthetic pathways of CAEs or COLs, and (3) predicted upon bioinformatics-guided genome mining. The identification of these CAE and COL-type 2,2′-BP natural products not only demonstrates the development of research techniques and methods in the field of natural product chemistry but also reflects the general interest in the discovery of CAE and COL-type 2,2′-BP natural products. [ABSTRACT FROM AUTHOR]

Since the onset of microbiology in the late 19th century, scientists have been growing microorganisms almost exclusively as pure cultures, resulting in a limited and biased view of the microbial world. Only a paradigm shift in cultivation techniques – from axenic to mixed cultures – can allow a full comprehension of the (chemical) communication of microorganisms, with profound consequences for natural product discovery, microbial ecology, symbiosis, and pathogenesis, to name a few areas. Three main technical advances during the last decade are fueling the realization of this revolution in microbiology: microfluidics, next-generation 3D-bioprinting, and single-cell metabolomics. These technological advances can be implemented for large-scale, systematic cocultivation studies involving three or more microorganisms. In this review, we present recent trends in microbiology tools and discuss how these can be employed to decode the chemical language that microorganisms use to communicate. [ABSTRACT FROM AUTHOR]

The biosynthetically well-studied landomycin A cluster has been used to demonstrate the unbalancing of gene transcription as an efficient method for the generation of new compounds. Landomycin A structural genes were decoupled from the native regulators LanI and LanK and placed under the control of a single synthetic promoter and expressed in a heterologous host Streptomyces albus J1074. In contrast to their native quantitative and temporal regulation, these genes were transcribed as a single polycistronic mRNA leading to the production of four novel and two known compounds. No glycosylated landomycins were detected though the entire biosynthetic cluster was transcribed, showing the crucial role of the balanced gene expression for the production of landomycin A. Two new compounds, fridamycin F and G, isolated in this study were shown to originate from the interplay between the expressed biosynthetic pathway and metabolic network of the heterologous host. Structure activity studies of the isolated compounds as well as results of transcriptome sequencing are discussed in this article. [ABSTRACT FROM AUTHOR]

Microbial natural products are an evolved resource of bioactive small molecules, which form the foundation of many modern therapeutic regimes. Ribosomally synthesized and posttranslationally modified peptides (RiPPs) represent a class of natural products which have attracted extensive interest for their diverse chemical structures and potent biological activities. Genome sequencing has revealed that the vast majority of genetically encoded natural products remain unknown. Many bioinformatic resources have therefore been developed to predict the chemical structures of natural products, particularly nonribosomal peptides and polyketides, from sequence data. However, the diversity and complexity of RiPPs have challenged systematic investigation of RiPP diversity, and consequently the vast majority of genetically encoded RiPPs remain chemical "dark matter." Here, we introduce an algorithm to catalog RiPP biosynthetic gene clusters and chart genetically encoded RiPP chemical space. A global analysis of 65,421 prokaryotic genomes revealed 30,261 RiPP clusters, encoding 2,231 unique products. We further leverage the structure predictions generated by our algorithm to facilitate the genome-guided discovery of a molecule from a rare family of RiPPs. Our results provide the systematic investigation of RiPP genetic and chemical space, revealing the widespread distribution of RiPP biosynthesis throughout the prokaryotic tree of life, and provide a platform for the targeted discovery of RiPPs based on genome sequencing. [ABSTRACT FROM AUTHOR]

Background: With thousands of fungal genomes being sequenced, each genome containing up to 70 secondary metabolite (SM) clusters 30-80 kb in size, breakthrough techniques are needed to characterize this SM wealth. Results: Here we describe a novel system-level methodology for unbiased cloning of intact large SM clusters from a single fungal genome for one-step transformation and expression in a model host. All 56 intact SM clusters from Aspergillus terreus were individually captured in self-replicating fungal artificial chromosomes (FACs) containing both the E. coli F replicon and an Aspergillus autonomously replicating sequence (AMA1). Candidate FACs were successfully shuttled between E. coli and the heterologous expression host A. nidulans. As proof-of-concept, an A. nidulans FAC strain was characterized in a novel liquid chromatography-high resolution mass spectrometry (LC-HRMS) and data analysis pipeline, leading to the discovery of the A. terreus astechrome biosynthetic machinery. Conclusion: The method we present can be used to capture the entire set of intact SM gene clusters and/or pathways from fungal species for heterologous expression in A. nidulans and natural product discovery. [ABSTRACT FROM AUTHOR]

Cyanobacteria produce a wide variety of cyclic peptides, including the widespread hepatotoxins microcystins and nodularins. Another class of peptides, cyclic glycosylated lipopeptides called hassallidins, show antifungal activity. Previously, two hassallidins (A and B) were reported from an epilithic cyanobacterium Hassallia sp. and found to be active against opportunistic human pathogenic fungi. Bioinformatic analysis of the Anabaena sp. 90 genome identified a 59-kb cryptic inactive nonribosomal peptide synthetase gene cluster proposed to be responsible for hassallidin biosynthesis. Here we describe the hassallidin biosynthetic pathway from Anabaena sp. SYKE748A, as well as the large chemical variation and common occurrence of hassallidins in filamentous cyanobacteria. Analysis demonstrated that 20 strains of the genus Anabaena carry hassallidin synthetase genes and produce a multitude of hassallidin variants that exhibit activity against Candida albicans. The compounds discovered here were distinct from previously reported hassallidins A and B. The IC50 of hassallidin D was 0.29-1.0 μM against Candida strains. A large variation in amino acids, sugars, their degree of acetylation, and fatty acid side chain length was detected. In addition, hassallidins were detected in other cyanobacteria including Aphanizomenon, Cylindrospermopsis raciborskii, Nostoc, and Tolypothrix. These compounds may protect some of the most important bloom-forming and globally distributed cyanobacteria against attacks by parasitic fungi. [ABSTRACT FROM AUTHOR]

Microbial natural products have played a key role in the development of clinical agents in nearly all therapeutic areas. Recent advances in genome sequencing have revealed that there is an incredible wealth of new polyketide and non-ribosomal peptide natural product diversity to be mined from genetic data. The diversity and complexity of polyketide and non-ribosomal peptide biosynthesis has required the development of unique bioinformatics tools to identify, annotate, and predict the structures of these natural products from their biosynthetic gene clusters. This review highlights and evaluates web-based bioinformatics tools currently available to the natural product community for genome mining to discover new polyketides and non-ribosomal peptides. [ABSTRACT FROM AUTHOR]

Burkholderia species have emerged as a new source of diverse natural products. This mini-review covers all of the natural products discovered in recent years from Burkholderia sp. by genome-guided approaches-these refer to the use of bacterial genome sequence as an entry point for in silico structural prediction, wet lab experimental design, and execution. While reliable structural prediction based on cryptic biosynthetic gene cluster sequence was not always possible due to noncanonical domains and/or module organization of a deduced biosynthetic pathway, a molecular genetic method was often employed to detect or alter the expression level of the gene cluster to achieve an observable phenotype, which facilitated downstream natural product purification and identification. Those examples of natural product discovery from Burkholderia sp. provide practical guidance for future exploration of Gram-negative bacteria as a new source of natural products. [ABSTRACT FROM AUTHOR]

Actinomycetes produce many pharmaceutically useful compounds through type I polyketide biosynthetic pathways. Soil has traditionally been an important source for these actinomycete-derived pharmaceuticals. As the rate of antibiotic discovery has decreased and the incidence of antibiotic resistance has increased, researchers have looked for alternatives to soil for bioprospecting. Street sediment, where actinomycetes make up a larger fraction of the bacterial population than in soil, is one such alternative environment. To determine if these differences in actinomycetal community structure are reflected in type I polyketide synthases (PKSI) distribution, environmental DNA from soils and street sediments was characterized by sequencing amplicons of PKSI-specific PCR primers. Amplicons covered two domains: the last 80 amino acids of the ketosynthase (KS) domain and the first 240 amino acids of the acyltransferase (AT) domain. One hundred and ninety clones from ten contrasting soils from six regions and nine street sediments from six cities were sequenced. Twenty-five clones from two earthworm-affected samples were also sequenced. UniFrac lineage-specific analysis identified two clades that clustered with actinomycetal GenBank matches that were street sediment-specific, one similar to the PKSI segment of the mycobactin siderophore involved in mycobacterial virulence. A clade of soil-specific sequences clustered with GenBank matches from the ambruticin and jerangolid pathways of Sorangium cellulosum. All three of these clades were found in sites >700 km apart. Street sediments are enriched in actinomycetal PKSIs. Non-actinomycetal PKSI pathways may be more chemically diverse than actinomycetal PKSIs. Common soil and street sediment PKIs are globally distributed. [ABSTRACT FROM AUTHOR]

Functional metagenomics, the study of the collective genome of a microbial community by expressing it in a foreign host, is an emerging field in biotechnology. Over the past years, the possibility of novel product discovery through metagenomics has developed rapidly . Thus, metagenomics has been heralded as a promising mining strategy of resources for the biotechnological and pharmaceutical industry. However, in spite of innovative work in the field of functional genomics in recent years, yields from function-based metagenomics studies still fall short of producing significant amounts of new products that are valuable for biotechnological processes. Thus, a new set of strategies is required with respect to fostering gene expression in comparison to the traditional work. These new strategies should address a major issue, that is, how to successfully express a set of unknown genes of unknown origin in a foreign host in high throughput. This article is an opinionating review of functional metagenomic screening of natural microbial communities, with a focus on the optimization of new product discovery. It first summarizes current major bottlenecks in functional metagenomics and then provides an overview of the general metagenomic assessment strategies, with a focus on the challenges that are met in the screening for, and selection of, target genes in metagenomic libraries. To identify possible screening limitations, strategies to achieve optimal gene expression are reviewed, examining the molecular events all the way from the transcription level through to the secretion of the target gene product. [ABSTRACT FROM AUTHOR]

Numerous marine organisms, including macroalgae, produce bioactive compounds. As chemical extraction processes do not distinguish between compounds originating from the alga and its associated microflora, a key step immediately prior to isolation of molecules, is elimination of epibionts from the alga studied. In chemical, ecological and biotechnological contexts, a clear identification of the source organism, when symbiotic interactions occurs, is crucial. We aimed to improve understanding of specific secondary metabolite production by macroalgae through development of a metho-dology for removing microbial epibionts immediately prior to chemical extraction without damaging algal cell surfaces (to avoid extraction of endogenous macroalgal compounds at this stage). Various solutions were tested: non-polar solvents (dichloromethane and hexane), alcohols (methanol, ethanol and isopropanol), oxidants (sodium hypochlorite, hydrogen peroxide and iodine) and a natural product (tea tree oil) at various concentrations and contact times. Five macroalgal species were employed: Chondrus crispus, Fucus serratus, Palmaria palmata, Saccharina latissima and Ulva lactuca. Scanning electron microscopy was used to evaluate the surface integrity. The best epibiont removal rate without damaging tissue was obtained with mixtures of ethanol (40-50%) and sodium hypochlorite (1%). Interestingly, macroalgal moisture content influenced treatment efficacy. [ABSTRACT FROM AUTHOR]

A widely accepted species concept for bacteria has yet to be established. As a result, species designations are inconsistently applied and tied to what can be considered arbitrary metrics. Increasing access to DNA sequence data and clear evidence that bacterial genomes are dynamic entities that include large numbers of horizontally acquired genes have added a new level of insight to the ongoing species concept debate. Despite uncertainties over how to apply species concepts to bacteria, there is clear evidence that sequence-based approaches can be used to resolve cohesive groups that maintain the properties of species. This cohesion is clearly evidenced in the genus Salinispora, where three species have been discerned despite very close relationships based on 16S rRNA sequence analysis. The major phenotypic differences among the three species are associated with secondary metabolite production, which occurs in species-specific patterns. These patterns are maintained on a global basis and provide evidence that secondary metabolites have important ecological functions. These patterns also suggest that an effective strategy for natural product discovery is to target the cultivation of new Salinispora taxa. Alternatively, bioinformatic analyses of biosynthetic genes provide opportunities to predict secondary metabolite novelty and reduce the redundant isolation of well-known metabolites. Although much remains to be learned about the evolutionary relationships among bacteria and how fundamental units of diversity can be resolved, genus and species descriptions remain the most effective method of scientific communication. [ABSTRACT FROM AUTHOR]

[Display omitted] Continual efforts to unearth new natural product chemistry expands our understanding of the scope of chemical space. Here we report the two low-abundance piperazic acid-containing linear peptides dentigerumycins F (1) and G (2), discovered through a combined genome-mining/15N NMR-based discovery method. Dentigerumycins F and G demonstrate a dynamic equilibrium in their oxane ring systems, which complicated structure elucidation. The application of various NMR solvents and conditions revealed the constitution of these natural products. [ABSTRACT FROM AUTHOR]

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a structurally diverse group of natural products. They feature a wide range of intriguing post-translational modifications, as exemplified by the biarylitides. These are a family of cyclic tripeptides found in Planomonospora, carrying a biaryl linkage between two aromatic amino acids. Recent genomic analyses revealed that the minimal biosynthetic prerequisite of biarylitide biosynthesis consists of only one ribosomally synthesized pentapeptide precursor as the substrate and a modifying cytochrome-P450-dependent enzyme. In silico analyses revealed that minimal biarylitide RiPP clusters are widespread among natural product producers across phylogenetic borders, including myxobacteria. We report here the genome-guided discovery of the first myxobacterial biarylitide MeYLH, termed Myxarylin, from Pyxidicoccus fallax An d48. Myxarylin was found to be an N-methylated tripeptide that surprisingly exhibits a C–N biaryl crosslink. In contrast to Myxarylin, previously isolated biarylitides are N-acetylated tripeptides that feature a C–C biaryl crosslink. Furthermore, the formation of Myxarylin was confirmed by the heterologous expression of the identified biosynthetic genes in Myxococcus xanthus DK1622. These findings expand the structural and biosynthetic scope of biarylitide-type RiPPs and emphasize the distinct biochemistry found in the myxobacterial realm. [ABSTRACT FROM AUTHOR]

Myxobacteria represent a viable source of chemically diverse and biologically active secondary metabolites. The myxochelins are a well-studied family of catecholate-type siderophores produced by various myxobacterial strains. Here, we report the discovery, isolation, and structure elucidation of three new myxochelins N1–N3 from the terrestrial myxobacterium Corallococcus sp. MCy9049, featuring an unusual nicotinic acid moiety. Precursor-directed biosynthesis (PDB) experiments and total synthesis were performed in order to confirm structures, improve access to pure compounds for bioactivity testing, and to devise a biosynthesis proposal. The combined evaluation of metabolome and genome data covering myxobacteria supports the notion that the new myxochelin congeners reported here are in fact frequent side products of the known myxochelin A biosynthetic pathway in myxobacteria. [ABSTRACT FROM AUTHOR]