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Fungal Genetics and Biology : FG & B Jun 2024The soil and indoor fungus Stachybotrys chartarum can induce respiratory disorders, collectively referred to as stachybotryotoxicosis, owing to its prolific production...
The soil and indoor fungus Stachybotrys chartarum can induce respiratory disorders, collectively referred to as stachybotryotoxicosis, owing to its prolific production of diverse bioactive secondary metabolites (SMs) or mycotoxins. Although many of these toxins responsible for the harmful effects on animals and humans have been identified in the genus Stachybotrys, however a number of SMs remain elusive. Through in silico analyses, we have identified 37 polyketide synthase (PKS) genes, highlighting that the chemical profile potential of Stachybotrys is far from being fully explored. Additionally, by leveraging phylogenetic analysis of known SMs produced by non-reducing polyketide synthases (NR-PKS) in other filamentous fungi, we showed that Stachybotrys possesses a rich reservoir of untapped SMs. To unravel natural product biosynthesis in S. chartarum, genetic engineering methods are crucial. For this purpose, we have developed a reliable protocol for the genetic transformation of S. chartarum and applied it to the ScPKS14 biosynthetic gene cluster. This cluster is homologous to the already known Claviceps purpurea CpPKS8 BGC, responsible for the production of ergochromes. While no novel SMs were detected, we successfully applied genetic tools, such as the generation of deletionand overexpression strains of single cluster genes. This toolbox can now be readily employed to unravel not only this particular BGC but also other candidate BGCs present in S. chartarum, making this fungus accessible for genetic engineering.
Topics: Stachybotrys; Multigene Family; Polyketide Synthases; Mycotoxins; Phylogeny; Biosynthetic Pathways; Genetic Engineering; Secondary Metabolism; Fungal Proteins
PubMed: 38636782
DOI: 10.1016/j.fgb.2024.103892 -
Phytochemistry Jun 2024Bafilomycins are macrocyclic polyketides with intriguing structures and therapeutic value. Genomic analysis of Streptomyces sp. SCSIO 66814 revealed a type I polyketide...
Genome-guided discovery of two undescribed 6,6-spiroketal polyketides and stereochemical correction of bafilomycins P and Q from the marine-derived Streptomyces sp. SCSIO 66814.
Bafilomycins are macrocyclic polyketides with intriguing structures and therapeutic value. Genomic analysis of Streptomyces sp. SCSIO 66814 revealed a type I polyketide synthase biosynthetic gene cluster (BGC), namely blm, which encoded bafilomycins and featured rich post-modification genes. The One strain many compounds (OSMAC) strategy led to the discovery of six compounds related to the blm BGC from the strain, including two previously undescribed 6,6-spiroketal polyketides, streptospirodienoic acids D (1) and E (2), and four known bafilomycins, bafilomycins P (3), Q (4), D (5), and G (6). The structures of 1 and 2 were determined by extensive spectroscopic analysis, quantum calculation, and biosynthetic analysis. Additionally, the absolute configurations of the 6/5/5 tricyclic ring moiety containing six consecutive chiral carbons in the putative structures of 3 and 4 were corrected through NOE analysis, DP4+ calculation, and single-crystal X-ray diffraction data. Bioinformatic analysis uncovered a plausible biosynthetic pathway for compounds 1-6, indicating that both streptospirodienoic acids and bafilomycins were derived from the same blm BGC. Additionally, sequence analysis revealed that the KR domains of module 2 from blm BGC was B1-type, further supporting the configurations of 1-4. Notably, compounds 3 and 4 displayed significant cytotoxic activities against A-549 human non-small cell lung cancer cells and HCT-116 human colon cancer cells.
Topics: Streptomyces; Polyketides; Humans; Stereoisomerism; Drug Screening Assays, Antitumor; Molecular Structure; Antineoplastic Agents; Macrolides; Cell Proliferation; Spiro Compounds; Structure-Activity Relationship; Polyketide Synthases; Cell Line, Tumor; Genome, Bacterial; Multigene Family
PubMed: 38636687
DOI: 10.1016/j.phytochem.2024.114101 -
Microbial Genomics Apr 2024are prolific producers of secondary metabolites from which many clinically useful compounds have been derived. They inhabit diverse habitats but have rarely been...
are prolific producers of secondary metabolites from which many clinically useful compounds have been derived. They inhabit diverse habitats but have rarely been reported in vertebrates. Here, we aim to determine to what extent the ecological source (bat host species and cave sites) influence the genomic and biosynthetic diversity of bacteria. We analysed draft genomes of 132 isolates sampled from 11 species of insectivorous bats from six cave sites in Arizona and New Mexico, USA. We delineated 55 species based on the genome-wide average nucleotide identity and core genome phylogenetic tree. isolates that colonize the same bat species or inhabit the same site exhibit greater overall genomic similarity than they do with from other bat species or sites. However, when considering biosynthetic gene clusters (BGCs) alone, BGC distribution is not structured by the ecological or geographical source of the that carry them. Each genome carried between 19-65 BGCs (median=42.5) and varied even among members of the same species. Nine major classes of BGCs were detected in ten of the 11 bat species and in all sites: terpene, non-ribosomal peptide synthetase, polyketide synthase, siderophore, RiPP-like, butyrolactone, lanthipeptide, ectoine, melanin. Finally, genomes carry multiple hybrid BGCs consisting of signature domains from two to seven distinct BGC classes. Taken together, our results bring critical insights to understanding -bat ecology and BGC diversity that may contribute to bat health and in augmenting current efforts in natural product discovery, especially from underexplored or overlooked environments.
Topics: Animals; Chiroptera; Phylogeny; Genomics; Arizona; Bacteria
PubMed: 38625724
DOI: 10.1099/mgen.0.001238 -
Journal of Biotechnology Jun 2024The 2-pyrone moiety is present in a wide range of structurally diverse natural products with various biological activities. The plant biosynthetic routes towards these...
The 2-pyrone moiety is present in a wide range of structurally diverse natural products with various biological activities. The plant biosynthetic routes towards these compounds mainly depend on the activity of either type III polyketide synthase-like 2-pyrone synthases or hydroxylating 2-oxoglutarate dependent dioxygenases. In the present study, the substrate specificity of these enzymes is investigated by a systematic screening using both natural and artificial substrates with the aims of efficiently forming (new) products and understanding the underlying catalytic mechanisms. In this framework, we focused on the in vitro functional characterization of a 2-pyrone synthase Gh2PS2 from Gerbera x hybrida and two dioxygenases AtF6'H1 and AtF6'H2 from Arabidopsis thaliana using a set of twenty aromatic and aliphatic CoA esters as substrates. UHPLC-ESI-HRMS based analyses of reaction intermediates and products revealed a broad substrate specificity of the enzymes, enabling the facile "green" synthesis of this important class of natural products and derivatives in a one-step/one-pot reaction in aqueous environment without the need for halogenated or metal reagents and protective groups. Using protein modeling and substrate docking we identified amino acid residues that seem to be important for the observed product scope.
Topics: Pyrones; Esters; Arabidopsis; Substrate Specificity; Coenzyme A; Molecular Docking Simulation; Biological Products; Dioxygenases
PubMed: 38616039
DOI: 10.1016/j.jbiotec.2024.04.006 -
BMC Genomics Apr 2024Pseudoalteromonas viridis strain BBR56 was isolated from seawater at Dutungan Island, South Sulawesi, Indonesia. Bacterial DNA was isolated using Promega Genomic DNA...
Pseudoalteromonas viridis strain BBR56 was isolated from seawater at Dutungan Island, South Sulawesi, Indonesia. Bacterial DNA was isolated using Promega Genomic DNA TM050. DNA purity and quantity were assessed using NanoDrop spectrophotometers and Qubit fluorometers. The DNA library and sequencing were prepared using Oxford Nanopore Technology GridION MinKNOW 20.06.9 with long read, direct, and comprehensive analysis. High accuracy base calling was assessed with Guppy version 4.0.11. Filtlong and NanoPlot were used for filtering and visualizing the FASTQ data. Flye (2.8.1) was used for de novo assembly analysis. Variant calls and consensus sequences were created using Medaka. The annotation of the genome was elaborated by DFAST. The assembled genome and annotation were tested using Busco and CheckM. Herein, we found that the highest similarity of the BBR56 isolate was 98.37% with the 16 S rRNA gene sequence of P. viridis G-1387. The genome size was 5.5 Mb and included chromosome 1 (4.2 Mbp) and chromosome 2 (1.3 Mbp), which encoded 61 pseudogenes, 4 noncoding RNAs, 113 tRNAs, 31 rRNAs, 4,505 coding DNA sequences, 4 clustered regularly interspaced short palindromic repeats, 4,444 coding genes, and a GC content of 49.5%. The sequence of the whole genome of P. viridis BBR56 was uploaded to GenBank under the accession numbers CP072425-CP072426, biosample number SAMN18435505, and bioproject number PRJNA716373. The sequence read archive (SRR14179986) was successfully obtained from NCBI for BBR56 raw sequencing reads. Digital DNA-DNA hybridization results showed that the genome of BBR56 had the potential to be a new species because no other bacterial genomes were similar to the sample. Biosynthetic gene clusters (BGCs) were assessed using BAGEL4 and the antiSMASH bacterial version. The genome harbored diverse BGCs, including genes that encoded polyketide synthase, nonribosomal peptide synthase, RiPP-like, NRP-metallophore, hydrogen cyanide, betalactone, thioamide-NRP, Lant class I, sactipeptide, and prodigiosin. Thus, BBR56 has considerable potential for further exploration regarding the use of its secondary metabolite products in the human and fisheries sectors.
Topics: Humans; Pseudoalteromonas; Pseudogenes; Gene Library; DNA, Bacterial
PubMed: 38615000
DOI: 10.1186/s12864-024-10266-6 -
Plant Physiology and Biochemistry : PPB May 20242-(2-Phenylethyl) chromone (PEC) and its derivatives are markers of agarwood formation and are also related to agarwood quality. However, the biosynthetic and regulatory...
2-(2-Phenylethyl) chromone (PEC) and its derivatives are markers of agarwood formation and are also related to agarwood quality. However, the biosynthetic and regulatory mechanisms of PECs still remain mysterious. Several studies suggested that type III polyketide synthases (PKSs) contribute to PEC biosynthesis in Aquilaria sinensis. Furthermore, systematic studies on the evolution of PKSs in A. sinensis have rarely been reported. Herein, we comprehensively analyzed PKS genes from 12 plant genomes and characterized the AsPKSs in detail. A unique branch contained only AsPKS members was identified through evolutionary analysis, including AsPKS01 that was previously indicated to participate in PEC biosynthesis. AsPKS07 and AsPKS08, two tandem-duplicated genes of AsPKS01 and lacking orthologous genes in evolutionary models, were selected for their transient expression in the leaves of Nicotiana benthamiana. Subsequently, PECs were detected in the extracts of N. benthamiana leaves, suggesting that AsPKS07 and AsPKS08 promote PEC biosynthesis. The interaction between the promoters of AsPKS07, AsPKS08 and five basic leucine zippers (bZIPs) from the S subfamily indicated that their transcripts could be regulated by these transcription factors (TFs) and might further contribute to PECs biosynthesis in A. sinensis. Our findings provide valuable insights into the molecular evolution of the PKS gene family in A. sinensis and serve as a foundation for advancing PEC production through the bioengineering of gene clusters. Ultimately, this contribution is expected to shed light on the mechanism underlying agarwood formation.
Topics: Thymelaeaceae; Evolution, Molecular; Phylogeny; Multigene Family; Plant Proteins; Gene Expression Regulation, Plant; Nicotiana; Polyketide Synthases
PubMed: 38604011
DOI: 10.1016/j.plaphy.2024.108571 -
MicrobiologyOpen Apr 2024Microbial communities from various environments have been studied in the quest for new natural products with a broad range of applications in medicine and biotechnology....
Microbial communities from various environments have been studied in the quest for new natural products with a broad range of applications in medicine and biotechnology. We employed an enrichment method and genome mining tools to examine the biosynthetic potential of microbial communities in the sediments of a coastal sinkhole within the karst ecosystem of the Yucatán Peninsula, Mexico. Our investigation led to the detection of 203 biosynthetic gene clusters (BGCs) and 55 secondary metabolites (SMs) within 35 high-quality metagenome-assembled genomes (MAGs) derived from these subcommunities. The most abundant types of BGCs were Terpene, Nonribosomal peptide-synthetase, and Type III polyketide synthase. Some of the in silico identified BGCs and SMs have been previously reported to exhibit biological activities against pathogenic bacteria and fungi. Others could play significant roles in the sinkhole ecosystem, such as iron solubilization and osmotic stress protection. Interestingly, 75% of the BGCs showed no sequence homology with bacterial BGCs previously reported in the MiBIG database. This suggests that the microbial communities in this environment could be an untapped source of genes encoding novel specialized compounds. The majority of the BGCs were identified in pathways found in the genus Virgibacillus, followed by Sporosarcina, Siminovitchia, Rhodococcus, and Halomonas. The latter, along with Paraclostridium and Lysinibacillus, had the highest number of identified BGC types. This study offers fresh insights into the potential ecological role of SMs from sediment microbial communities in an unexplored environment, underscoring their value as a source of novel natural products.
Topics: Bacteria; Metagenome; Microbiota; Multigene Family; Bacillaceae; Biological Products; Biosynthetic Pathways
PubMed: 38593340
DOI: 10.1002/mbo3.1407 -
Archives of Razi Institute Oct 2023Antibiotic resistance is rising dramatically worldwide, and thus the production of new antibiotics is indispensable. Recent scientific initiatives have focused on the...
Antibiotic resistance is rising dramatically worldwide, and thus the production of new antibiotics is indispensable. Recent scientific initiatives have focused on the bioprospecting of microorganisms' secondary metabolites, with a particular focus on the look for natural products with antimicrobial properties derived from endophytes. All plant species, regardless of their type, are thought to anchor endophytic bacteria (EB). There are many potential uses for the natural therapeutic compounds made by EB in medicine, agriculture, and the pharmaceutical industry. To investigate antibacterial properties in this study, (formerly, ) were isolated from Boiss., identified, and underwent bioprospecting by morphological and molecular methods. Samples were collected from Ilam, Iran, and then divided into roots, leaves, stems, and flowers. After disinfection, they were cut into 2 mm pieces, cultured on casein agar culture medium, and incubated at 28ºC for up to four weeks. was identified using the polymerase chain reaction method targeting the 16S rRNA gene. To evaluate the antibacterial properties of the isolated , the agar diffusion method was used. In parallel, the frequencies of biosynthetic gene clusters, including polyketide synthase ( and ) and nonribosomal peptide synthetase () genes, were determined in the isolated . Ninety bacteria were isolated from different parts of flowers. Thirty-eight (42.2%) of these bacteria belonged to the phylum , and out of these 38, 15 isolates (39.5%) had antibacterial properties. Of these, 11 isolates (73.3%) exhibited antibacterial effects against aureus, 2 (13.3%) against , 3 (20%) against , and two isolates (13.3%) against sub-species of serovar Typhimurium. The results of the molecular analysis of , , and genes showed that out of 38 isolated strains, 23 isolates (60.5%) carried gene, 6 (15.8%) harbored gene, and 20 isolates (52.6%) had gene. This study indicates that Boiss. has a number of active that produce secondary metabolites with antibacterial properties.
Topics: Animals; Plants, Medicinal; Anthemis; RNA, Ribosomal, 16S; Agar; Bacteria; Anti-Bacterial Agents
PubMed: 38590676
DOI: 10.22092/ARI.2023.78.5.1638 -
Beilstein Journal of Organic Chemistry 2024An isotopic labelling method was developed to investigate substrate binding by ketosynthases, exemplified by the second ketosynthase of the polyketide synthase BaeJ...
An isotopic labelling method was developed to investigate substrate binding by ketosynthases, exemplified by the second ketosynthase of the polyketide synthase BaeJ involved in bacillaene biosynthesis (BaeJ-KS2). For this purpose, both enantiomers of a C-labelled -acetylcysteamine thioester (SNAC ester) surrogate of the proposed natural intermediate of BaeJ-KS2 were synthesised, including an enzymatic step with glutamate decarboxylase, and incubated with BaeJ-KS2. Substrate binding was demonstrated through C NMR analysis of the products against the background of various control experiments.
PubMed: 38590531
DOI: 10.3762/bjoc.20.67 -
Zoological Science Apr 2024Sea urchins have been used as model organisms in developmental biology research and the genomes of several sea urchin species have been sequenced. Recently, genome...
Sea urchins have been used as model organisms in developmental biology research and the genomes of several sea urchin species have been sequenced. Recently, genome editing technologies have become available for sea urchins, and methods for gene knockout using the CRISPRCas9 system have been established. is an important marine fishery resource with edible gonads. Although has been used as a biological research material, its genome has not yet been published, and it is a non-model sea urchin for molecular biology research. However, as recent advances in genome editing technology have facilitated genome modification in non-model organisms, we applied genome editing using the CRISPR-Cas9 system to . In this study, we targeted genes encoding ETS transcription factor () and pigmentation-related polyketide synthase (). Gene fragments were isolated using primers designed by inter-specific sequence comparisons within Echinoidea. When gene was targeted using two sgRNAs, one successfully introduced mutations and impaired skeletogenesis. In the gene knockout, when two sgRNAs targeting the close vicinity of the site corresponding to the target site that showed 100% mutagenesis efficiency of the gene in , mutagenesis was not observed. However, two other sgRNAs targeting distant sites efficiently introduced mutations. In addition, knockout exhibited an albino phenotype in the pluteus larvae and adult sea urchins after metamorphosis. This indicates that the CRISPRCas9 system can be used to modify the genome of the non-model sea urchin .
Topics: Animals; Anthocidaris; CRISPR-Cas Systems; RNA, Guide, CRISPR-Cas Systems; Gene Knockout Techniques; Sea Urchins; Gene Editing
PubMed: 38587910
DOI: 10.2108/zs230052