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Scientific Reports Nov 2023The discovery of novel bioactive compounds produced by microorganisms holds significant potential for the development of therapeutics and agrochemicals. In this study,...
The discovery of novel bioactive compounds produced by microorganisms holds significant potential for the development of therapeutics and agrochemicals. In this study, we conducted genome mining to explore the biosynthetic potential of entomopathogenic bacteria belonging to the genera Xenorhabdus and Photorhabdus. By utilizing next-generation sequencing and bioinformatics tools, we identified novel biosynthetic gene clusters (BGCs) in the genomes of the bacteria, specifically plu00736 and plu00747. These clusters were identified as unidentified non-ribosomal peptide synthetase (NRPS) and unidentified type I polyketide synthase (T1PKS) clusters. These BGCs exhibited unique genetic architecture and encoded several putative enzymes and regulatory elements, suggesting its involvement in the synthesis of bioactive secondary metabolites. Furthermore, comparative genome analysis revealed that these BGCs were distinct from previously characterized gene clusters, indicating the potential for the production of novel compounds. Our findings highlighted the importance of genome mining as a powerful approach for the discovery of biosynthetic gene clusters and the identification of novel bioactive compounds. Further investigations involving expression studies and functional characterization of the identified BGCs will provide valuable insights into the biosynthesis and potential applications of these bioactive compounds.
Topics: Genome, Bacterial; Bacteria; Computational Biology; Multigene Family; Biosynthetic Pathways
PubMed: 38007490
DOI: 10.1038/s41598-023-47121-9 -
Marine Drugs Nov 2023The hadal biosphere is the most mysterious ecosystem on the planet, located in a unique and extreme environment on Earth. To adapt to extreme environmental conditions,...
The hadal biosphere is the most mysterious ecosystem on the planet, located in a unique and extreme environment on Earth. To adapt to extreme environmental conditions, hadal microorganisms evolve special strategies and metabolisms to survive and reproduce. However, the secondary metabolites of the hadal microorganisms are poorly understood. In this study, we focused on the isolation and characterization of hadal fungi, screening the potential strains with bioactive natural products. The isolates obtained were detected further for the polyketide synthase (PKS) genes. Two isolates of were picked up as the representatives, which had the potential to synthesize active natural products. The epigenetic modifiers were used for the two isolates to stimulate functional gene expression in hadal fungi under laboratory conditions. The results showed that the chemical epigenetic modifier, 5-Azacytidine (5-Aza), affected the phenotype, PKS gene expression, production of secondary metabolites, and antimicrobial activity of the hadal fungus . The influence of epigenetic modification on natural products was strongest when the concentration of 5-Aza was 50 μM. Furthermore, the modification of epigenetic agents on hadal fungi under high hydrostatic pressure (HHP) of 40 MPa displayed significant effects on PKS gene expression, and also activated the production of new compounds. Our study demonstrates the high biosynthetic potential of cultivable hadal fungi, but also provides evidence for the utility of chemical epigenetic modifiers on active natural products from hadal fungi, providing new ideas for the development and exploitation of microbial resources in extreme environments.
Topics: Ecosystem; Polyketide Synthases; Hydrostatic Pressure; Epigenesis, Genetic; Biological Products
PubMed: 37999409
DOI: 10.3390/md21110585 -
Microbiology Spectrum Jun 2024The cyanosiphophage Mic1 specifically infects the bloom-forming FACHB 1339 from Lake Chaohu, China. Previous genomic analysis showed that its 92,627 bp double-stranded...
UNLABELLED
The cyanosiphophage Mic1 specifically infects the bloom-forming FACHB 1339 from Lake Chaohu, China. Previous genomic analysis showed that its 92,627 bp double-stranded DNA genome consists of 98 putative open reading frames, 63% of which are of unknown function. Here, we investigated the transcriptome dynamics of Mic1 and its host using RNA sequencing. In the early, middle, and late phases of the 10 h lytic cycle, the Mic1 genes are sequentially expressed and could be further temporally grouped into two distinct clusters in each phase. Notably, six early genes, including that encodes a TnpB-like transposase, immediately reach the highest transcriptional level in half an hour, representing a pioneer cluster that rapidly regulates and redirects host metabolism toward the phage. An in-depth analysis of the host transcriptomic profile in response to Mic1 infection revealed significant upregulation of a polyketide synthase pathway and a type III-B CRISPR system, accompanied by moderate downregulation of the photosynthesis and key metabolism pathways. The constant increase of phage transcripts and relatively low replacement rate over the host transcripts indicated that Mic1 utilizes a unique strategy to gradually take over a small portion of host metabolism pathways after infection. In addition, genomic analysis of a less-infective Mic1 and a Mic1-resistant host strain further confirmed their dynamic interplay and coevolution via the frequent horizontal gene transfer. These findings provide insights into the mutual benefit and symbiosis of the highly polymorphic cyanobacteria and cyanophages.
IMPORTANCE
The highly polymorphic is one of the predominant bloom-forming cyanobacteria in eutrophic freshwater bodies and is infected by diverse and abundant cyanophages. The presence of a large number of defense systems in genome suggests a dynamic interplay and coevolution with the cyanophages. In this study, we investigated the temporal gene expression pattern of Mic1 after infection and the corresponding transcriptional responses of its host. Moreover, the identification of a less-infective Mic1 and a Mic1-resistant host strain provided the evolved genes in the phage-host coevolution during the multiple-generation cultivation in the laboratory. Our findings enrich the knowledge on the interplay and coevolution of and its cyanophages and lay the foundation for the future application of cyanophage as a potential eco-friendly and bio-safe agent in controlling the succession of harmful cyanobacterial blooms.
Topics: Microcystis; Bacteriophages; China; Transcriptome; Lakes; Genome, Viral; Evolution, Molecular
PubMed: 38695606
DOI: 10.1128/spectrum.00298-24 -
Research Square Jul 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 habits,...
BACKGROUND
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 habits, and how microbes survive, proliferate and spread in space conditions, is coming more and 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-positive bacteria, including two spore-forming and three non-spore-forming species, were isolated from the environmental surfaces of the International Space Station (ISS).
RESULTS
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 (WGS) 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 ucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values, when compared to any known bacterial species, were less than <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.
CONCLUSIONS
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: IIF3SC-B10 (=NRRL B-65660), , F6_8S_P_1A (=NRRL B-65661), , F6_8S_P_1B (=NRRL B- 65662 and DSMZ 115932), Paenibacillus vandeheii, F6_3S_P_1C(=NRRL B-65663 and DSMZ 115940), and F6_3S_P_2 T(=NRRL B-65664 and DSMZ 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.
PubMed: 37461605
DOI: 10.21203/rs.3.rs-3126314/v1 -
Plant Biotechnology Journal Jan 2024Lipid biosynthesis and transport are essential for plant male reproduction. Compared with Arabidopsis and rice, relatively fewer maize lipid metabolic genic...
Lipid biosynthesis and transport are essential for plant male reproduction. Compared with Arabidopsis and rice, relatively fewer maize lipid metabolic genic male-sterility (GMS) genes have been identified, and the sporopollenin metabolon in maize anther remains unknown. Here, we identified two maize GMS genes, ZmTKPR1-1 and ZmTKPR1-2, by CRISPR/Cas9 mutagenesis of 14 lipid metabolic genes with anther stage-specific expression patterns. Among them, tkpr1-1/-2 double mutants displayed complete male sterility with delayed tapetum degradation and abortive pollen. ZmTKPR1-1 and ZmTKPR1-2 encode tetraketide α-pyrone reductases and have catalytic activities in reducing tetraketide α-pyrone produced by ZmPKSB (polyketide synthase B). Several conserved catalytic sites (S128/130, Y164/166 and K168/170 in ZmTKPR1-1/-2) are essential for their enzymatic activities. Both ZmTKPR1-1 and ZmTKPR1-2 are directly activated by ZmMYB84, and their encoded proteins are localized in both the endoplasmic reticulum and nuclei. Based on protein structure prediction, molecular docking, site-directed mutagenesis and biochemical assays, the sporopollenin biosynthetic metabolon ZmPKSB-ZmTKPR1-1/-2 was identified to control pollen exine formation in maize anther. Although ZmTKPR1-1/-2 and ZmPKSB formed a protein complex, their mutants showed different, even opposite, defective phenotypes of anther cuticle and pollen exine. Our findings discover new maize GMS genes that can contribute to male-sterility line-assisted maize breeding and also provide new insights into the metabolon-regulated sporopollenin biosynthesis in maize anther.
Topics: Zea mays; Gene Editing; CRISPR-Cas Systems; Molecular Docking Simulation; Pyrones; Plant Breeding; Arabidopsis; Lipids; Pollen; Infertility; Gene Expression Regulation, Plant; Plant Proteins
PubMed: 37792967
DOI: 10.1111/pbi.14181 -
AMB Express Aug 2023Streptomyces lavendulae subsp. lavendulae CCM 3239 (formerly Streptomyces aureofaciens CCM 3239) contains a type II polyketide synthase (PKS) biosynthetic gene cluster...
Streptomyces lavendulae subsp. lavendulae CCM 3239 (formerly Streptomyces aureofaciens CCM 3239) contains a type II polyketide synthase (PKS) biosynthetic gene cluster (BGC) aur1 whose genes were highly similar to angucycline BGCs. However, its product auricin is structurally different from all known angucyclines. It contains a spiroketal pyranonaphthoquinone aglycone similar to griseusins and is modified with D-forosamine. Here, we describe the characterization of the initial steps in auricin biosynthesis using a synthetic-biology-based approach. We have created a plasmid system based on the strong kasOp* promoter, RBS and phage PhiBT1-based integration vector, where each gene in the artificial operon can be easily replaced by another gene using unique restriction sites surrounding each gene in the operon. The system was validated with the initial landomycin biosynthetic genes lanABCFDLE, leading to the production of rabelomycin after its integration into Streptomyces coelicolor M1146. However, the aur1DEFCGHA homologous genes from the auricin aur1 BGC failed to produce rabelomycin in this system. The cause of this failure was inactive aur1DE genes encoding ketosynthases α and β (KSα, KSβ). Their replacement with homologous aur2AB genes from the adjacent aur2 BGC resulted in rabelomycin production that was even higher after the insertion of two genes from the aur1 BGC, aur1L encoding 4-phosphopantetheinyl transferase (PPTase) and aur1M encoding malonyl-CoA:ACP transacylase (MCAT), suggesting that Aur1L PPTase is essential for the activation of the acyl carrier protein Aur1F. These results suggest an interesting communication of two BGCs, aur1 and aur2, in the biosynthesis of the initial structure of auricin aglycone.
PubMed: 37552435
DOI: 10.1186/s13568-023-01591-2 -
ACS Synthetic Biology Nov 2023Type I polyketide synthases (T1PKSs) hold enormous potential as a rational production platform for the biosynthesis of specialty chemicals. However, despite great...
Type I polyketide synthases (T1PKSs) hold enormous potential as a rational production platform for the biosynthesis of specialty chemicals. However, despite great progress in this field, the heterologous expression of PKSs remains a major challenge. One of the first measures to improve heterologous gene expression can be codon optimization. Although controversial, choosing the wrong codon optimization strategy can have detrimental effects on the protein and product levels. In this study, we analyzed 11 different codon variants of an engineered T1PKS and investigated in a systematic approach their influence on heterologous expression in , , and . Our best performing codon variants exhibited a minimum 50-fold increase in PKS protein levels, which also enabled the production of an unnatural polyketide in each of these hosts. Furthermore, we developed a free online tool (https://basebuddy.lbl.gov) that offers transparent and highly customizable codon optimization with up-to-date codon usage tables. In this work, we not only highlight the significance of codon optimization but also establish the groundwork for the high-throughput assembly and characterization of PKS pathways in alternative hosts.
Topics: Polyketide Synthases; Codon; Polyketides
PubMed: 37851920
DOI: 10.1021/acssynbio.3c00367 -
BioRxiv : the Preprint Server For... Dec 2023A subset of nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) are encoded in their biosynthetic gene clusters (BGCs) with enzymes annotated as...
A subset of nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) are encoded in their biosynthetic gene clusters (BGCs) with enzymes annotated as lantibiotic dehydratases. The functions of these putative lantibiotic dehydratases remain unknown. Here, we characterize an NRPS-PKS BGC with a putative lantibiotic dehydratase from the bacterium (). Heterologous expression revealed several metabolites produced by the BGC, and the omission of selected biosynthetic enzymes revealed the biosynthetic sequence towards these compounds. The putative lantibiotic dehydratase catalyzes peptide bond formation that extends the peptide scaffold opposite to the NRPS and PKS biosynthetic direction. The condensation domain of the NRPS catalyzes the formation of a ureido group, and bioinformatics analysis revealed distinct active site residues of ureido-generating condensation (UreaC) domains. This work demonstrates that the annotated lantibiotic dehydratase serves as a separate amide bond-forming machinery in addition to the NRPS, and that the lantibiotic dehydratase enzyme family possesses diverse catalytic activities in the biosynthesis of both ribosomal and non-ribosomal natural products.
PubMed: 38187666
DOI: 10.1101/2023.12.23.573212 -
Mycobiology 2023Hispidin is an important styrylpyrone produced by . To analyze hispidin biosynthesis in , the transcriptomes of hispidin-producing and non-producing were determined by...
Hispidin is an important styrylpyrone produced by . To analyze hispidin biosynthesis in , the transcriptomes of hispidin-producing and non-producing were determined by Illumina sequencing. Five PKSs were identified using genome annotation. Comparative analysis with the reference transcriptome showed that two PKSs ( and ) had low expression levels in four types of media. The gene expression pattern of only was consistent with the yield variation of hispidin. The combined analyses of gene expression with qPCR and hispidin detection by liquid chromatography-mass spectrometry coupled with ion-trap and time-of-flight technologies (LCMS-IT-TOF) showed that was involved in hispidin biosynthesis in is a partially reducing gene with extra AMP and ACP domains before the KS domain. The domain architecture of ShPKS1 was AMP-ACP-KS-AT-DH-KR-ACP-ACP. Phylogenetic analysis shows that ShPKS1 and other PKS genes from Hymenochaetaceae form a unique monophyletic clade closely related to the clade containing Agaricales hispidin synthase. Taken together, our data indicate that ShPKS1 is a novel PKS of involved in hispidin biosynthesis.
PubMed: 37929012
DOI: 10.1080/12298093.2023.2257999 -
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