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Toxicon : Official Journal of the... May 2024Microcystins (MCs) are a family of chemically diverse toxins produced by numerous distantly related cyanobacteria. They are potent inhibitors of eukaryotic protein...
Microcystins (MCs) are a family of chemically diverse toxins produced by numerous distantly related cyanobacteria. They are potent inhibitors of eukaryotic protein phosphatases 1 and 2A and are responsible for the toxicosis and death of wild and domestic animals around the world. Microcystins are synthesized on large enzyme complexes comprised of peptide synthetases, polyketide synthases, and additional modifying enzymes. Bioinformatic analysis identified the presence of an additional uncharacterized enzyme in the microcystin (mcy) biosynthetic gene cluster in Fischerella sp. PCC 9339, which we named McyK, that lacked a clearly defined role in the biosynthesis of microcystin. Further bioinformatic analysis suggested that McyK belongs to the inosamine-phosphate amidinotransferase family and could be involved in synthesizing homo amino acids. Quadrupole time-of-flight tandem mass spectrometry (Q-TOFMS/MS) analysis confirmed that Fischerella sp. PCC 9339 produces MC-Leucine-Homoarginine(MC-LHar) and [Aspartic acid]MC-Leucine-Homoarginine ([Asp]MC-LHar) as the dominant chemical variants. We hypothesized that the McyK enzyme might be involved in the production of microcystin variants containing homoarginine (Har) in the strain. Heterologous expression of a codon-optimized mcyK gene in Escherichia coli confirmed that McyK is responsible for the synthesis of L-Har. These results confirm the production of MC-LHar, a novel microcystin chemical variant [Asp]MC-LHar, and a new microcystin biosynthetic enzyme involved in supply of the rare homo-amino acid Har to the microcystin biosynthetic pathway in Fischerella sp. PCC 9339. This study provides new insights into the logic underpinning the biosynthesis of microcystin chemical variants and broadens our knowledge of structural diversity of the microcystin family of toxins.
Topics: Microcystins; Homoarginine; Biosynthetic Pathways; Multigene Family; Cyanobacteria; Tandem Mass Spectrometry
PubMed: 38670499
DOI: 10.1016/j.toxicon.2024.107733 -
ACS Synthetic Biology May 2024spp. are "nature's antibiotic factories" that produce valuable bioactive metabolites, such as the cytotoxic anthracycline polyketides. While the anthracyclines have...
spp. are "nature's antibiotic factories" that produce valuable bioactive metabolites, such as the cytotoxic anthracycline polyketides. While the anthracyclines have hundreds of natural and chemically synthesized analogues, much of the chemical diversity stems from enzymatic modifications to the saccharide chains and, to a lesser extent, from alterations to the core scaffold. Previous work has resulted in the generation of a BioBricks synthetic biology toolbox in M1152Δ that could produce aklavinone, 9--aklavinone, auramycinone, and nogalamycinone. In this work, we extended the platform to generate oxidatively modified analogues two crucial strategies. (i) We swapped the ketoreductase and first-ring cyclase enzymes for the aromatase cyclase from the mithramycin biosynthetic pathway in our polyketide synthase (PKS) cassettes to generate 2-hydroxylated analogues. (ii) Next, we engineered several multioxygenase cassettes to catalyze 11-hydroxylation, 1-hydroxylation, 10-hydroxylation, 10-decarboxylation, and 4-hydroxyl regioisomerization. We also developed improved plasmid vectors and M1152Δ expression hosts to produce anthracyclinones. This work sets the stage for the combinatorial biosynthesis of bespoke anthracyclines using recombinant hosts.
Topics: Polyketide Synthases; Anthracyclines; Streptomyces coelicolor; Streptomyces; Biosynthetic Pathways; Hydroxylation; Anti-Bacterial Agents
PubMed: 38662967
DOI: 10.1021/acssynbio.4c00043 -
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 -
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 -
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 -
Frontiers in Fungal Biology 2024Fungal polyketides are a large group of secondary metabolites, valuable due to their diverse spectrum of pharmacological activities. Polyketide biosynthesis in...
Fungal polyketides are a large group of secondary metabolites, valuable due to their diverse spectrum of pharmacological activities. Polyketide biosynthesis in filamentous fungi presents some challenges: small yield and low-purity titers. To tackle these issues, we switched to the yeast , an easily cultivable heterologous host. As an oleaginous yeast, displays a high flux of acetyl- and malonyl-CoA precursors used in lipid synthesis. Likewise, acetyl- and malonyl-CoA are the building blocks of many natural polyketides, and we explored the possibility of redirecting this flux toward polyketide production. Despite its promising prospect, has so far only been used for heterologous expression of simple type III polyketide synthases (PKSs) from plants. Therefore, we decided to evaluate the potential of by targeting the more complex fungal polyketides synthesized by type I PKSs. We employed a CRISPR-Cas9-mediated genome editing method to achieve markerless gene integration of the genes responsible for bostrycoidin biosynthesis in Fusarium solani (, , and ) and 6-methylsalicylic acid (6-MSA) biosynthesis in Aspergillus hancockii (6MSAS). Moreover, we attempted titer optimization through metabolic engineering by overexpressing two enzymes, TGL4 and AOX2, involved in lipid β-oxidation, but we did not observe an effect on polyketide production. With maximum titers of 403 mg/L 6-MSA and 35 mg/L bostrycoidin, the latter being substantially higher than our previous results in (2.2 mg/L), this work demonstrates the potential of as a platform for heterologous production of complex fungal polyketides.
PubMed: 38586602
DOI: 10.3389/ffunb.2024.1327777