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Molecules (Basel, Switzerland) Apr 2024Chalkophomycin is a novel chalkophore with antibiotic activities isolated from sp. CB00271, while its potential in studying cellular copper homeostasis makes it an...
Chalkophomycin is a novel chalkophore with antibiotic activities isolated from sp. CB00271, while its potential in studying cellular copper homeostasis makes it an important probe and drug lead. The constellation of -hydroxylpyrrole, 2-oxazoline, diazeniumdiolate, and methoxypyrrolinone functional groups into one compact molecular architecture capable of coordinating cupric ions draws interest to unprecedented enzymology responsible for chalkophomycin biosynthesis. To elucidate the biosynthetic machinery for chalkophomycin production, the biosynthetic gene cluster from . sp. CB00271 was identified, and its involvement in chalkophomycin biosynthesis was confirmed by gene replacement. The cluster was localized to a ~31 kb DNA region, consisting of 19 open reading frames that encode five nonribosomal peptide synthetases (ChmHIJLO), one modular polyketide synthase (ChmP), six tailoring enzymes (ChmFGMNQR), two regulatory proteins (ChmAB), and four resistance proteins (ChmA'CDE). A model for chalkophomycin biosynthesis is proposed based on functional assignments from sequence analysis and structure modelling, and is further supported by analogy to over 100 -type gene clusters in public databases. Our studies thus set the stage to fully investigate chalkophomycin biosynthesis and to engineer chalkophomycin analogues through a synthetic biology approach.
Topics: Multigene Family; Streptomyces; Polyketide Synthases; Peptide Synthases; Bacterial Proteins
PubMed: 38731473
DOI: 10.3390/molecules29091982 -
Metabolites Aug 2023Actinomycetia are known for their ability to produce a wide range of bioactive secondary metabolites having significant therapeutic importance. This study aimed to...
Actinomycetia are known for their ability to produce a wide range of bioactive secondary metabolites having significant therapeutic importance. This study aimed to explore the potential of actinomycetia as a source of bioactive compounds with antimicrobial properties against multi-drug-resistant (MDR) clinical pathogens. A total of 65 actinomycetia were isolated from two unexplored forest ecosystems, namely the Pobitora Wildlife Sanctuary (PWS) and the Deepor Beel Wildlife Sanctuary (DBWS), located in the Indo-Burma mega-biodiversity hotspots of northeast India, out of which 19 isolates exhibited significant antimicrobial activity. 16S rRNA gene sequencing was used for the identification and phylogenetic analysis of the 19 potent actinomycetia isolates. The results reveal that the most dominant genus among the isolates was (84.21%), followed by rare actinomycetia genera such as , , and . Furthermore, seventeen of the isolates tested positive for at least one antibiotic biosynthetic gene, specifically type II polyketide synthase (PKS-II) and nonribosomal peptide synthetases (NRPSs). These genes are associated with the production of bioactive compounds with antimicrobial properties. Among the isolated strains, three actinomycetia strains, namely sp. PBR1, sp. PBR36, and sp. DBR11, demonstrated the most potent antimicrobial activity against seven test pathogens. This was determined through in vitro antimicrobial bioassays and the minimum inhibitory concentration (MIC) values of ethyl acetate extracts. Gas chromatography-mass spectrometry (GS-MS) and whole-genome sequencing (WGS) of the three strains revealed a diverse group of bioactive compounds and secondary metabolite biosynthetic gene clusters (smBGCs), respectively, indicating their high therapeutic potential. These findings highlight the potential of these microorganisms to serve as a valuable resource for the discovery and development of novel antibiotics and other therapeutics with high therapeutic potential.
PubMed: 37623855
DOI: 10.3390/metabo13080911 -
Scientific Reports Nov 2023With the advent of long-term human habitation in space and on the moon, understanding how the built environment microbiome of space habitats differs from Earth habitats,...
Phylogenomics, phenotypic, and functional traits of five novel (Earth-derived) bacterial species isolated from the International Space Station and their prevalence in metagenomes.
With the advent of long-term human habitation in space and on the moon, understanding how the built environment microbiome of space habitats differs from Earth habitats, and how microbes survive, proliferate and spread in space conditions, is becoming more important. The microbial tracking mission series has been monitoring the microbiome of the International Space Station (ISS) for almost a decade. During this mission series, six unique strains of Gram-stain-positive bacteria, including two spore-forming and three non-spore-forming species, were isolated from the environmental surfaces of the ISS. The analysis of their 16S rRNA gene sequences revealed > 99% similarities with previously described bacterial species. To further explore their phylogenetic affiliation, whole genome sequencing was undertaken. For all strains, the gyrB gene exhibited < 93% similarity with closely related species, which proved effective in categorizing these ISS strains as novel species. Average nucleotide identity and digital DNA-DNA hybridization values, when compared to any known bacterial species, were < 94% and <50% respectively for all species described here. Traditional biochemical tests, fatty acid profiling, polar lipid, and cell wall composition analyses were performed to generate phenotypic characterization of these ISS strains. A study of the shotgun metagenomic reads from the ISS samples, from which the novel species were isolated, showed that only 0.1% of the total reads mapped to the novel species, supporting the idea that these novel species are rare in the ISS environments. In-depth annotation of the genomes unveiled a variety of genes linked to amino acid and derivative synthesis, carbohydrate metabolism, cofactors, vitamins, prosthetic groups, pigments, and protein metabolism. Further analysis of these ISS-isolated organisms revealed that, on average, they contain 46 genes associated with virulence, disease, and defense. The main predicted functions of these genes are: conferring resistance to antibiotics and toxic compounds, and enabling invasion and intracellular resistance. After conducting antiSMASH analysis, it was found that there are roughly 16 cluster types across the six strains, including β-lactone and type III polyketide synthase (T3PKS) clusters. Based on these multi-faceted taxonomic methods, it was concluded that these six ISS strains represent five novel species, which we propose to name as follows: Arthrobacter burdickii IIF3SC-B10 (= NRRL B-65660 = DSM 115933), Leifsonia virtsii F6_8S_P_1A (= NRRL B-65661 = DSM 115931), Leifsonia williamsii F6_8S_P_1B (= NRRL B-65662 = DSM 115932), Paenibacillus vandeheii F6_3S_P_1C (= NRRL B-65663 = DSM 115940), and Sporosarcina highlanderae F6_3S_P_2 (= NRRL B-65664 = DSM 115943). Identifying and characterizing the genomes and phenotypes of novel microbes found in space habitats, like those explored in this study, is integral for expanding our genomic databases of space-relevant microbes. This approach offers the only reliable method to determine species composition, track microbial dispersion, and anticipate potential threats to human health from monitoring microbes on the surfaces and equipment within space habitats. By unraveling these microbial mysteries, we take a crucial step towards ensuring the safety and success of future space missions.
Topics: Humans; Metagenome; Phylogeny; RNA, Ribosomal, 16S; Prevalence; Phenotype; Paenibacillus; Fatty Acids; DNA; DNA, Bacterial; Sequence Analysis, DNA; Bacterial Typing Techniques
PubMed: 37932283
DOI: 10.1038/s41598-023-44172-w -
Metabolic Engineering Nov 2023DHA is a marine PUFA of commercial value, given its multiple health benefits. The worldwide emerging shortage in DHA supply has increased interest in microbial cell...
Multi-omics view of recombinant Yarrowia lipolytica: Enhanced ketogenic amino acid catabolism increases polyketide-synthase-driven docosahexaenoic production to high selectivity at the gram scale.
DHA is a marine PUFA of commercial value, given its multiple health benefits. The worldwide emerging shortage in DHA supply has increased interest in microbial cell factories that can provide the compound de novo. In this regard, the present work aimed to improve DHA production in the oleaginous yeast strain Y. lipolytica Af4, which synthetized the PUFA via a heterologous myxobacterial polyketide synthase (PKS)-like gene cluster. As starting point, we used transcriptomics, metabolomics, and C-based metabolic pathway profiling to study the cellular dynamics of Y. lipolytica Af4. The shift from the growth to the stationary DHA-production phase was associated with fundamental changes in carbon core metabolism, including a strong upregulation of the PUFA gene cluster, as well as an increase in citrate and fatty acid degradation. At the same time, the intracellular levels of the two DHA precursors acetyl-CoA and malonyl-CoA dropped by up to 98% into the picomolar range. Interestingly, the degradation pathways for the ketogenic amino acids l-lysine, l-leucine, and l-isoleucine were transcriptionally activated, presumably to provide extra acetyl-CoA. Supplementation with small amounts of these amino acids at the beginning of the DHA production phase beneficially increased the intracellular CoA-ester pools and boosted the DHA titer by almost 40%. Isotopic C-tracer studies revealed that the supplements were efficiently directed toward intracellular CoA-esters and DHA. Hereby, l-lysine was found to be most efficient, as it enabled long-term activation, due to storage within the vacuole and continuous breakdown. The novel strategy enabled DHA production in Y. lipolytica at the gram scale for the first time. DHA was produced at a high selectivity (27% of total fatty acids) and free of the structurally similar PUFA DPA, which facilitates purification for high-value medical applications that require API-grade DHA. The assembled multi-omics picture of the central metabolism of Y. lipolytica provides valuable insights into this important yeast. Beyond our work, the enhanced catabolism of ketogenic amino acids seems promising for the overproduction of other compounds in Y. lipolytica, whose synthesis is limited by the availability of CoA ester precursors.
Topics: Yarrowia; Polyketide Synthases; Acetyl Coenzyme A; Lysine; Multiomics; Esters; Polyketides; Metabolic Engineering
PubMed: 37683719
DOI: 10.1016/j.ymben.2023.09.003 -
G3 (Bethesda, Md.) Dec 2023Several species of sacoglossan sea slugs possess the incredible ability to sequester chloroplasts from the algae they consume. These "photosynthetic animals" incorporate...
Several species of sacoglossan sea slugs possess the incredible ability to sequester chloroplasts from the algae they consume. These "photosynthetic animals" incorporate stolen chloroplasts, called kleptoplasts, into the epithelial cells of tubules that extend from their digestive tracts throughout their bodies. The mechanism by which these slugs maintain functioning kleptoplasts in the absence of an algal nuclear genome is unknown. Here, we report a draft genome of the sacoglossan slug Elysia crispata morphotype clarki, a morphotype native to the Florida Keys that can retain photosynthetically active kleptoplasts for several months without feeding. We used a combination of Oxford Nanopore Technologies long reads and Illumina short reads to produce a 786-Mb assembly (N50 = 0.459 Mb) containing 68,514 predicted protein-coding genes. A phylogenetic analysis found no evidence of horizontal acquisition of genes from algae. We performed gene family and gene expression analyses to identify E. crispata genes unique to kleptoplast-containing slugs that were more highly expressed in fed versus unfed developmental life stages. Consistent with analyses in other kleptoplastic slugs, our investigation suggests that genes encoding lectin carbohydrate-binding proteins and those involved in regulation of reactive oxygen species and immunity may play a role in kleptoplast retention. Lastly, we identified four polyketide synthase genes that could potentially encode proteins producing UV- and oxidation-blocking compounds in slug cell membranes. The genome of E. crispata is a quality resource that provides potential targets for functional analyses and enables further investigation into the evolution and mechanisms of kleptoplasty in animals.
Topics: Animals; Phylogeny; Photosynthesis; Chloroplasts; Gastropoda; Genome
PubMed: 37816307
DOI: 10.1093/g3journal/jkad234 -
Nature Communications Jan 2024Animals synthesize simple lipids using a distinct fatty acid synthase (FAS) related to the type I polyketide synthase (PKS) enzymes that produce complex specialized...
Animals synthesize simple lipids using a distinct fatty acid synthase (FAS) related to the type I polyketide synthase (PKS) enzymes that produce complex specialized metabolites. The evolutionary origin of the animal FAS and its relationship to the diversity of PKSs remain unclear despite the critical role of lipid synthesis in cellular metabolism. Recently, an animal FAS-like PKS (AFPK) was identified in sacoglossan molluscs. Here, we explore the phylogenetic distribution of AFPKs and other PKS and FAS enzymes across the tree of life. We found AFPKs widely distributed in arthropods and molluscs (>6300 newly described AFPK sequences). The AFPKs form a clade with the animal FAS, providing an evolutionary link bridging the type I PKSs and the animal FAS. We found molluscan AFPK diversification correlated with shell loss, suggesting AFPKs provide a chemical defense. Arthropods have few or no PKSs, but our results indicate AFPKs contributed to their ecological and evolutionary success by facilitating branched hydrocarbon and pheromone biosynthesis. Although animal metabolism is well studied, surprising new metabolic enzyme classes such as AFPKs await discovery.
Topics: Animals; Polyketides; Fatty Acids; Lipid Metabolism; Phylogeny; Polyketide Synthases; Fatty Acid Synthases
PubMed: 38172109
DOI: 10.1038/s41467-023-44497-0 -
Marine Drugs Aug 2023With the increase in antimicrobial resistance and the subsequent demand for novel therapeutics, the deep-sea fish microbiome can be a relatively untapped source of...
With the increase in antimicrobial resistance and the subsequent demand for novel therapeutics, the deep-sea fish microbiome can be a relatively untapped source of antimicrobials, including bacteriocins. Previously, bacterial isolates were recovered from the gut of deep-sea fish sampled from the Atlantic Ocean.In this study, we used in vitro methods to screen a subset of these isolates for antimicrobial activity, and subsequently mined genomic DNA from isolates of interest for bacteriocin and other antimicrobial metabolite genes. We observed antimicrobial activity against foodborne pathogens, including , , and . In total, 147 candidate biosynthetic gene clusters were identified in the genomic sequences, including 35 bacteriocin/RiPP-like clusters. Other bioactive metabolite genes detected included non-ribosomal peptide synthases (NRPS), polyketide synthases (PKS; Types 1 and 3), beta-lactones and terpenes. Moreover, four unique bacteriocin gene clusters were annotated and shown to encode novel peptides: a class IIc bacteriocin, two class IId bacteriocins and a class I lanthipeptide (LanM subgroup). Our dual in vitro and in silico approach allowed for a more comprehensive understanding of the bacteriocinogenic potential of these deep-sea isolates and an insight into the antimicrobial molecules that they may produce.
Topics: Animals; Genomics; Anti-Infective Agents; Atlantic Ocean; Bacteriocins; Fishes; Microbiota
PubMed: 37623725
DOI: 10.3390/md21080444 -
Scientific Reports Mar 2024Actinobacteria are one of the predominant groups that successfully colonize and survive in various aquatic, terrestrial and rhizhospheric ecosystems. Among...
Actinobacteria are one of the predominant groups that successfully colonize and survive in various aquatic, terrestrial and rhizhospheric ecosystems. Among actinobacteria, Nocardia is one of the most important agricultural and industrial bacteria. Screening and isolation of Nocardia related bacteria from extreme habitats such as endolithic environments are beneficial for practical applications in agricultural and environmental biotechnology. In this work, bioinformatics analysis revealed that a novel strain Nocardia mangyaensis NH1 has the capacity to produce structurally varied bioactive compounds, which encoded by non-ribosomal peptide synthases (NRPS), polyketide synthase (PKS), and post-translationally modified peptides (RiPPs). Among NRPS, five gene clusters have a sequence homology with clusters encoding for siderophore synthesis. We also show that N. mangyaensis NH1 accumulates both catechol- and hydroxamate-type siderophores simultaneously under iron-deficient conditions. Untargeted LC-MS/MS analysis revealed a variety of metabolites, including siderophores, lipopeptides, cyclic peptides, and indole-3-acetic acid (IAA) in the culture medium of N. mangyaensis NH1 grown under iron deficiency. We demonstrate that four CAS (chrome azurol S)-positive fractions display variable affinity to metals, with a high Fe chelating capability. Additionally, three of these fractions exhibit antioxidant activity. A combination of iron scavenging metabolites produced by N. mangyaensis NH1 showed antifungal activity against several plant pathogenic fungi. We have shown that the pure culture of N. mangyaensis NH1 and its metabolites have no adverse impact on Arabidopsis seedlings. The ability of N. mangyaensis NH1 to produce siderophores with antifungal, metal-chelating, and antioxidant properties, when supplemented with phytohormones, has the potential to improve the release of macro- and micronutrients, increase soil fertility, promote plant growth and development, and enable the production of biofertilizers across diverse soil systems.
Topics: Nocardia; Siderophores; Ecosystem; Antifungal Agents; Chromatography, Liquid; Tandem Mass Spectrometry; Actinobacteria; Iron; Bacteria; Genomics; Metabolome; Soil
PubMed: 38453942
DOI: 10.1038/s41598-024-54095-9 -
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 -
ACS Omega May 2024is a fungal pathogen capable of producing two mycotoxins of concern, ochratoxin A (OTA) and citrinin (CIT). The production profile of these two mycotoxins is not well...
is a fungal pathogen capable of producing two mycotoxins of concern, ochratoxin A (OTA) and citrinin (CIT). The production profile of these two mycotoxins is not well understood but could help mitigate co-contamination in the food supply. As such, the production of OTA and CIT from DAOMC 242724 was investigated under different growing conditions in liquid culture. We found that among the different liquid media chosen, liquid YES (yeast extract sucrose) medium induced the highest production of both OTA and CIT, when DAOMC 242724 was cultured in stationary mode. Shake culture significantly reduced the amounts of OTA and CIT produced. Among all culture conditions tested, far greater amounts of CIT were produced compared to OTA. Consequently, upon transcriptomic data analysis, a statistically significant increase in the expression of CIT biosynthetic genes was easier to detect than the expression of OTA biosynthetic genes. Our study also revealed that the putative biosynthetic gene clusters of OTA and CIT in DAOMC 242724 are likely distinct from each other. It appears that despite sharing a highly similar structure, the isocoumarin rings of OTA and CIT are each assembled by a specialized polyketide synthase enzyme. Our data identified a putative nonreducing polyketide synthase responsible for assembling the carbo-skeleton of CIT. In contrast, a highly reducing polyketide synthase appears to be involved in the biosynthesis of OTA.
PubMed: 38737015
DOI: 10.1021/acsomega.4c00874