Nonribosomal Peptide Synthetase Gene Research Articles

Orfamide production in Pseudomonas protegens CHA0T promotes rhizospheric colonization and influences assemblage of the bacterial community of wheat roots in soil.

Orfamides (Ofa) are a family of closely related cyclic lipopeptides (cLPs) produced by different soil-inhabiting and plant beneficial strains of the Pseudomonas protegens subgroup. Orfamides are required for swarming motility, they mediate toxicity on oomycetes, algae, and insects, and they can act as ISR elicitors. Ofa biosynthesis depends on a non-ribosomal peptide synthetase gene cluster whose expression is tightly controlled by the post-transcriptional regulatory cascade Gac-Rsm. Although the biological properties and physiological role of Ofa have been well characterized in vitro, the role of Ofa in the fitness of the producing bacterium in its niche has not been studied so far. Here, we used an Ofa biosynthesis-deficient mutant (ΔofaABC) derived from the biocontrol model strain P. protegens CHA0T to explore the relevance of this cLP in soil for the survival of strain CHA0T, its contribution to the bacterial competitiveness for colonization of wheat roots, and its impact on the corresponding rhizobacterial community structure. To facilitate CFU counts in non-sterile soil and root samples we used chromosomally tagged versions of CHA0T and its ΔofaABC mutant with mini-Tn7 constructs carrying antibiotic resistance markers. We confirmed that the tagged Ofa− mutant does not show drop-collapse activity neither it can swarm in semi-solid medium. We also confirmed that Ofa can be exploited in vitro as a social good by the Ofa− mutant to resume swarming in the presence of CHA0T cells. We found that Ofa production did not confer any advantage for bacterial survival along a period of 5 weeks after soil inoculation. However, we observed that Ofa production was associated with a higher competitiveness for colonization of the wheat rhizosphere. Expression of the ofa biosynthetic operon, by means of a transcriptional Pofa-gfp reporter fusion, was detected in the rhizoplane of colonized wheat roots, suggesting that Ofa is produced in situ on the surface of colonized wheat roots. Finally, we observed that seed inoculation with the wild type Ofa producer strain CHA0T resulted in a differential assemblage of the bacterial community of the wheat rhizosphere with respect to that of seeds bacterized with the Ofa deficient mutant. Overall, we provide evidence that Ofa is important for the competitiveness of P. protegens CHA0T cells to colonize the surface of wheat roots and that Ofa production in the rhizosphere can impact the structure of the assembled bacterial microbiome.

Isolation and identification of associated endophytic bacteria from barely seeds harbour non-ribosomal peptides and enhance tolerance to salinity stress

BackgroundBarely Hordeum vulgare L. is considered one of the most important cereal crops with economic and industrial importance in the world, but its productivity is affected by climate change and abiotic stresses. One of the most recent and important microbiological promising aspects is the use of associated microorganisms, especially the endophytic bacteria producers for non-ribosomal peptides which play an important role in promoting plant growth, productivity, and tolerance to biotic and abiotic stresses. This work aims to identify vertically transferred or inherited endophytic bacterial communities in barely seeds, detect the presence of non-ribosomal peptides from these isolated endophytic strains and study their effect on protein patterns as a response to salinity stress.ResultsFrom two different tolerant (Giza 126) and sensitive (Giza 123) barely seeds cultivars, six different endophytic bacterial strains were isolated and identified using 16S rRNA. Bacterial strains belonged to Bacillus, Paenibacillus, Staphylococcus and Acinetobacter genera. Three of them have been isolated from both sensitive and tolerant barely cultivar (Uncultured Staphylococcus, Acinetobacter and Priestia endophytica or Bacillus endophyticus), while the other three endophytes have been isolated uniquely from the tolerant barely cultivar (Paenibacillus glucanolyticus, Bacillus cereus and Bacillus sp.). Non-ribosomal peptide synthetases genes NRPs of two lipopeptide families; surfactins and kurstakins have been detected using both bioinformatic analysis and degenerate primers. On the other hand, fragments similar to NRPs genes might be considered new NRPS molecules in Paenibacillus glucanolyticus, Acinetobacter and Priestia endophytica which have been detected using degenerate primers and required whole genome sequencing. The effect of soaking barely seeds exposed to 2.5% NaCl using SDS-PAGE electrophoresis revealed the presence of 24 bands, 10 of them were monomorphic with 41.5%, and 14 were polymorphic with 58.5% polymorphism.ConclusionThe overnight soaking and co-cultivation of isolated endophytic strains with barely seeds before planting proved their capability in conferring salt stress tolerance to barely seedlings which appeared in protein patterns. We could consider these barely seeds endophytic among the PGPR strains promising to improve plant growth during abiotic stresses.

Decoding the chemical language of Suillus fungi: genome mining and untargeted metabolomics uncover terpene chemical diversity.

Ectomycorrhizal fungi establish mutually beneficial relationships with trees, trading nutrients for carbon. Suillus are ectomycorrhizal fungi that are critical to the health of boreal and temperate forest ecosystems. Comparative genomics has identified a high number of non-ribosomal peptide synthetase and terpene biosynthetic gene clusters (BGC) potentially involved in fungal competition and communication. However, the functionality of these BGCs is not known. This study employed co-culture techniques to activate BGC expression and then used metabolomics to investigate the diversity of metabolic products produced by three Suillus species (Suillus hirtellus EM16, Suillus decipiens EM49, and Suillus cothurnatus VC1858), core members of the pine microbiome. After 28 days of growth on solid media, liquid chromatography-tandem mass spectrometry identified a diverse range of extracellular metabolites (exometabolites) along the interaction zone between Suillus co-cultures. Prenol lipids were among the most abundant chemical classes. Out of the 62 unique terpene BGCs predicted by genome mining, 41 putative prenol lipids (includes 37 putative terpenes) were identified across the three Suillus species using metabolomics. Notably, some terpenes were significantly more abundant in co-culture conditions. For example, we identified a metabolite matching to isomers isopimaric acid, sandaracopimaric acid, and abietic acid, which can be found in pine resin and play important roles in host defense mechanisms and Suillus spore germination. This research highlights the importance of combining genomics and metabolomics to advance our understanding of the chemical diversity underpinning fungal signaling and communication.IMPORTANCEUsing a combination of genomics and metabolomics, this study's findings offer new insights into the chemical diversity of Suillus fungi, which serve a critical role in forest ecosystems.

Cyclo-diphenylalanine production in Aspergillus nidulans through stepwise metabolic engineering

Cyclo-diphenylalanine (cFF) is a symmetrical aromatic diketopiperazine (DKP) found wide-spread in microbes, plants, and resulting food products. As different bioactivities continue being discovered and relevant food and pharmaceutical applications gradually emerge for cFF, there is a growing need for establishing convenient and efficient methods to access this type of compound. Here, we present a robust cFF production system which entailed stepwise engineering of the filamentous fungal strain Aspergillus nidulans A1145 as a heterologous expression host. We first established a preliminary cFF producing strain by introducing the heterologous nonribosomal peptide synthetase (NRPS) gene penP1 to A. nidulans A1145. Key metabolic pathways involving shikimate and aromatic amino acid biosynthetic support were then engineered through a combination of gene deletions of competitive pathway steps, over-expressing feedback-insensitive enzymes in phenylalanine biosynthesis, and introducing a phosphoketolase-based pathway, which diverted glycolytic flux toward the formation of erythrose 4-phosphate (E4P). Through the stepwise engineering of A. nidulans A1145 outlined above, involving both heterologous pathway addition and native pathway metabolic engineering, we were able to produce cFF with titers reaching 611 mg/L in shake flask culture and 2.5 g/L in bench-scale fed-batch bioreactor culture. Our study establishes a production platform for cFF biosynthesis and successfully demonstrates engineering of phenylalanine derived diketopiperazines in a filamentous fungal host.

Complete Genome Sequence and Pan-Genome Analysis of Shewanella oncorhynchi Z-P2, a Siderophore Putrebactin-Producing Bacterium.

In this study, we reported the complete genome sequence of Shewanella oncorhynchi for the first time. S. oncorhynchi Z-P2 is a bacterium that produces the siderophore putrebactin. Its genome consists of a circular chromosome of 5,034,612 bp with a G + C content of 45.4%. A total of 4544 protein-coding genes, 109 tRNAs and 31 rRNAs were annotated by the RAST. Five non-ribosomal peptide synthetase (NRPS) and polyketide synthetase (PKS) gene clusters were identified by the antiSMASH analysis. The pan-genome analysis of Z-P2 and 10 Shewanella putrefaciens revealed 9228 pan-gene clusters and 2681 core gene clusters, with Z-P2 having 618 unique gene clusters. Additionally, the gene cluster involved in putrebactin biosynthesis in Z-P2 was annotated, and the mechanism of putrebactin biosynthesis was analyzed. The putrebactin produced by Z-P2 was detected using UPLC-MS analysis, with an [M + H]+ molecular ion at m/z 373.21. These findings provide valuable support for further research on the genetic engineering of putrebactin biosynthetic genes of Z-P2 and their potential applications.

A small step to discover candidate biological control agents from preexisting bioresources by using novel nonribosomal peptide synthetases hidden in activated sludge metagenomes.

Biological control agents (BCAs), beneficial organisms that reduce the incidence or severity of plant disease, have been expected to be alternatives to replace chemical pesticides worldwide. To date, BCAs have been screened by culture-dependent methods from various environments. However, previously unknown BCA candidates may be buried and overlooked because this approach preferentially selects only easy-to-culture microbial lineages. To overcome this limitation, as a small-scale test case, we attempted to explore novel BCA candidates by employing the shotgun metagenomic information of the activated sludge (AS) microbiome, which is thought to contain unutilized biological resources. We first performed genome-resolved metagenomics for AS taken from a municipal sewage treatment plant and obtained 97 nonribosomal peptide synthetase (NRPS)/polyketide synthase (PKS)-related gene sequences from 43 metagenomic assembled bins, most of which were assigned to the phyla Proteobacteria and Myxococcota. Furthermore, these NRPS/PKS-related genes are predicted to be novel because they were genetically dissimilar to known NRPS/PKS gene clusters. Of these, the condensation domain of the syringomycin-related NRPS gene cluster was detected in Rhodoferax- and Rhodocyclaceae-related bins, and its homolog was found in previously reported AS metagenomes as well as the genomes of three strains available from the microbial culture collections, implying their potential BCA ability. Then, we tested the antimicrobial activity of these strains against phytopathogenic fungi to investigate the potential ability of BCA by in vitro cultivation and successfully confirmed the actual antifungal activity of three strains harboring a possibly novel NRPS gene cluster. Our findings provide a possible strategy for discovering novel BCAs buried in the environment using genome-resolved metagenomics.

The Lrp transcriptional factor of an entomopathogenic bacterium, Xenorhabdus hominickii, activates non-ribosomal peptide synthetases to suppress insect immunity

Two bacterial genera, Xenorhabdus and Photorhabdus, are mutually symbiotic to the entomopathogenic nematodes, Steinernema and Heterorhabditis, respectively. The infective juveniles deliver the symbiotic bacteria to the hemocoel of target insects, in which the bacteria proliferate and help the development of the host nematode. The successful parasitism of the nematode-bacterial complex depends on host immunosuppression by the bacteria via their secondary metabolites. Leucine-responsive regulatory protein (Lrp) is a global bacterial transcriptional factor that plays a crucial role in parasitism. However, its regulatory targets to suppress insect immunity are not clearly understood. This study investigated the bacterial genes regulated by Lrp and the subsequent production of secondary metabolites in Xenorhabdus hominickii. Lrp expression occurred at the early infection stage of the bacteria in a target insect, Spodoptera exigua. A preliminary in silico screening indicated that 3.7% genes among 4075 predicted genes encoded in X. hominickii had the Lrp-response element on their promoters, including two non-ribosomal peptide synthetases (NRPSs). Eight NRPS (NRPS1-NRPS8) genes were predicted in the bacterial genome, in which six NRPS (NRPS3-NRPS8) expressions were positively correlated with Lrp expression in the infected larvae of S. exigua. Exchange of the Lrp promoter with an inducible promoter altered the production of the secondary metabolites and the NRPS expression levels. The immunosuppressive activities of X. hominickii were dependent on the Lrp expression level. The metabolites produced by Lrp expression included the eicosanoid-biosynthesis inhibitors and hemolytic factors. A cyclic dipeptide (=cPF) was produced by the bacteria at high Lrp expression and inhibited the phospholipase A2 activity of S. exigua in a competitive inhibitory manner. These results suggest that Lrp is a global transcriptional factor of X. hominickii and plays a crucial role in insect immunosuppression by modulating NRPS expression.

Profile of PKS and NRPS Gene Clusters in the Genome of Streptomyces cellostaticus NBRC 12849T

Polyketides and nonribosomal peptides are major secondary metabolites in members of the genus Streptomyces. Streptomyces cellostaticus is a validly recognized species and the type strain produces cellostatin. However, little is known about whether it has the potential to produce diverse polyketides and nonribosomal peptides. Here, we sequenced the whole genome of S. cellostaticus NBRC 12849T and surveyed polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) gene clusters in the genome. The genome encoded 12 PKS, one NRPS and eight hybrid PKS/NRPS gene clusters. Among the 21 gene clusters, products of 10 gene clusters were annotated to be an annimycin congener, fuelimycins, lankamycin, streptovaricin, spore pigment, flaviolin, foxicin, blasticidin, lankacidin and an incarnatapeptine congener via our bioinformatic analysis. Although the other clusters were orphan and their products were unknown, five of them were predicted to be compounds derived from two independent diketides, a tridecaketide, a triketide and a tetraketide with a cysteine residue, respectively. These results suggest that S. cellostaticus is a source of diverse polyketides and hybrid polyketide/nonribosomal peptides, including unknown and new secondary metabolites.

A Staphylococcus capitis strain with unusual bacteriocin production.

Staphylococcus capitis is a member of the human and mammal skin microbiomes and is considered less harmful than Staphylococcus aureus. S. capitis subsp. urealyticus BN2 was isolated from a cat and expressed strong antibacterial activity against a range of Gram-positive species, most notably including S. aureus strains with resistance to methicillin (MRSA) and strains with intermediate resistance to vancomycin (VISA). These latter strains are normally relatively resistant to bacteriocins, due to cell wall and cell membrane modifications. Genomic sequencing showed that the strain harboured at least two complete gene clusters for biosynthesis of antagonistic substances. The complete biosynthetic gene cluster of the well-known lantibiotic gallidermin was encoded on a large plasmid and the mature peptide was present in isopropanol cell extracts. In addition, a chromosomal island contained a novel non-ribosomal peptide synthetase (NRPS) gene cluster. Accidental deletion of two NRPS modules and partial purification of the anti-VISA activity showed that this novel bacteriocin represents a complex of differently decorated, non-ribosomal peptides. Additionally, a number of phenol-soluble modulins (PSMs) was detected by mass spectrometry of whole cells. Producing these compounds, the strain was able to outcompete several S. aureus strains, including MRSA and VISA, in tube cultures.

Global distribution and diversity of antimicrobial genes across subsurface bacterial and archaeal metagenome assembled genomes

Microorganisms have the capability to produce antimicrobial compounds through secondary metabolism, which are not essential within their natural environments, but have been found to have many effects on the ecosystem. Antimicrobial production genes have been identified in a wide range of microorganisms; however, research into natural ecosystems has historically been limited to continental soil environments. Antimicrobial production research has been limited in the deep continental subsurface and marine environments, especially deeply buried marine sediments. We analyzed 466 high-quality metagenome assembled genomes (MAGs) collected from continental and marine subsurface environments through the Deep Carbon Observatory’s Census of Deep Life. A total of 383 MAGs contained biosynthetic gene clusters, namely Type I and Type III polyketide synthase genes, non-ribosomal peptide synthetase genes, and other unspecified ribosomally synthesized and post-translationally modified peptide products. All of these genes were found across continental mines, subglacial lakes, hotsprings, and serpentinizing environments. These environments have previously not been investigated via metagenomics for antimicrobial gene diversity, which may be produced for competition or communication purposes. All other biosynthetic genes identified in this study were less than 50% similar to reference biosynthesis genes indicating the novelty of secondary metabolism in subsurface microorganisms. The majority of predicted antimicrobial products were found to be produced exergonically, which could indicate microbial populations use energy-conserving mechanisms to produce compounds that could offer a competitive advantage.

Isolation and Screening of Antibacterial Activity of Actinomycetota from the Medicinal Plant, Anthemis pseudocotula Boiss.

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, Actinomycetota (formerly, Actinobacteria) were isolated from Anthemis pseudocotula 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. Actinomycetota was identified using the polymerase chain reaction method targeting the 16S rRNA gene. To evaluate the antibacterial properties of the isolated Actinomycetota, the agar diffusion method was used. In parallel, the frequencies of biosynthetic gene clusters, including polyketide synthase (PKS-I and PKS-II) and nonribosomal peptide synthetase (NRPS) genes, were determined in the isolated Actinomycetota. Ninety bacteria were isolated from different parts of Anthemis flowers. Thirty-eight (42.2%) of these bacteria belonged to the phylum Actinomycetota, and out of these 38, 15 isolates (39.5%) had antibacterial properties. Of these, 11 isolates (73.3%) exhibited antibacterial effects against Staphylococcus aureus, 2 (13.3%) against Pseudomonas aeruginosa, 3 (20%) against Escherichia coli, and two isolates (13.3%) against Salmonella enterica sub-species of enterica serovar Typhimurium. The results of the molecular analysis of PKS-I, PKS-II, and NRPS genes showed that out of 38 isolated Actinomycetota strains, 23 isolates (60.5%) carried PKS-I gene, 6 (15.8%) harbored PKS-II gene, and 20 isolates (52.6%) had NRPS gene. This study indicates that Anthemis pseudocotula Boiss. has a number of active Actinomycetota that produce secondary metabolites with antibacterial properties.

Brevibacillus brevis HNCS-1: a biocontrol bacterium against tea plant diseases.

As a biocontrol bacteria, Brevibacillus has been the subject of extensive research for agricultural applications. Antibacterial peptides (AMPs) are the main antibacterial products of Brevibacillus. This study isolated a strain of Br. brevis HNCS-1 from tea garden soil, and the strain has an antagonistic effect against five types of pathogens of tea diseases, namely Gloeosporium theae-sinensis, Elsinoe leucospira, Phyllosticta theaefolia, Fusarium sp., and Cercospora theae. To determine the genetic characteristics implicated in the biocontrol mechanism, the genome sequence of the HNCS-1 strain was obtained and analyzed further, and the data are deposited in the GenBank repository (No. CP128411). Comparative genomics analyses revealed that the HNCS-1 strain and 17 public Br. brevis share a core genome composed of 3,742 genes. Interestingly, only one non-ribosomal peptide synthetase (NRPS) gene cluster annotated as edeine is present in the core genome. And UHPLC-MS/MS detection results showd that edeine B and edeine A were the principal antibacterial peptides in the HNCS-1 strain. This study proves that edeine is the main antibacterial peptide of Br. brevis, and provides a new strategy for the identification of antibacterial products from other biocontrol bacteria.

Nonribosomal Peptide Synthetases in Animals.

Nonribosomal peptide synthetases (NRPSs) are a class of cytosolic enzymes that synthesize a range of bio-active secondary metabolites including antibiotics and siderophores. They are widespread among both prokaryotes and eukaryotes but are considered rare among animals. Recently, several novel NRPS genes have been described in nematodes, schistosomes, and arthropods, which led us to investigate how prevalent NRPS genes are in the animal kingdom. We screened 1059 sequenced animal genomes and showed that NRPSs were present in 7 out of the 19 phyla analyzed. A phylogenetic analysis showed that the identified NRPSs form clades distinct from other adenylate-forming enzymes that contain similar domains such as fatty acid synthases. NRPSs show a remarkably scattered distribution over the animal kingdom. They are especially abundant in rotifers and nematodes. In rotifers, we found a large variety of domain architectures and predicted substrates. In the nematode Plectus sambesii, we identified the beta-lactam biosynthesis genes L-δ-(α-aminoadipoyl)-L-cysteinyl-D-valine synthetase, isopenicillin N synthase, and deacetoxycephalosporin C synthase that catalyze the formation of beta-lactam antibiotics in fungi and bacteria. These genes are also present in several species of Collembola, but not in other hexapods analyzed so far. In conclusion, our survey showed that NRPS genes are more abundant and widespread in animals than previously known.

Cyclic Peptides from the Opportunistic Pathogen Basidiobolus meristosporus.

Three new cyclic peptides, meristosporins A, B, and C (1-3), one of which features an unusual amino acid, were isolated from the opportunistic pathogen Basidiobolus meristosporus and identified by 1D, 2D NMR, MS/MS, and Marfey's analysis. The biosynthetic pathway of the hexapeptide meristosporin A (1) was deduced based on nonribosomal peptide synthetase gene clusters analysis. Compounds 1 and 2 showed cytotoxicity to RAW264.7 and 293T cells, respectively. These compounds may be involved in the fungal injury caused to human cells.

Detection of non-ribosomal and polyketide biosynthetic genes in bacteria from green mud crab Scylla serrata gut microbiome and their antagonistic activities

Multi-modular enzyme complexes known as non-ribosomal peptide synthetases (NRPSs) and polyketide synthetases (PKSs) have been widely reported in bacteria that produce secondary bioactive metabolites such as non-ribosomal peptides (NRPs) and polyketides (PKs), respectively. These NRPS/PKS pathways contribute to synthesizing several antibiotics, such as vancomycin, rifamycin, and bleomycin, which are vital in human medicine. The present study aimed to isolate gut-associated bacteria from mud crab Scylla serrata, and detect NRPS and PKS gene clusters associated with it. This study included 36 bacterial isolates from five mud crab gut samples. Biosynthetic gene clusters (NRPS and PKS), were detected by PCR using degenerative primers specific to these genes. Three isolates (FKP2–4, FKP4–1, and FKP2–16) were positive for NRPS and two for PKS (FKP2–4 and FKP4–1) genes. The isolates were subjected to 16S rRNA gene amplification and sequenced. In silico analysis of the sequences using the Basic Local Alignment Search Tool (BLAST) identified the isolates FKP2–4, FKP4–1, and FKP2–16 as Acinetobacter variabilis, Vagococcus fluvialis, and Staphylococcus arlettae, respectively, after comparing with the existing sequences available in the National Center for Biotechnology Information (NCBI) database. Compared to the control, it was observed that these isolates exhibited intriguing antagonistic activities against Escherichia coli and Staphylococcus aureus. However, these isolates failed to show significant activity against Candida albicans. Exopolysaccharide production by the isolated organisms was tested using Zobell marine agar (ZMA) with 5% sucrose, but none of the colonies were mucoid or slimy.

Trichoderma atroviride suppresses Fusarium graminearum by altering primary and secondary metabolite biosynthesis profiling

AbstractThe use of Trichoderma spp. offers an ecologically friendly tool for the struggle with mycotoxigenic Fusarium spp. Here, the alterations in transcriptome level were investigated in 6‐day‐old Fusarium graminearum cultures treated with Trichoderma atroviride and nontreated with T. atroviride (FGc), using whole transcriptome sequencing to better understand the associated biological processes. Transcriptome analysis indicated a total of 55 up‐regulated genes and 728 down‐regulated genes with padj < 0.05. Enrichment analysis revealed that the up‐regulated genes were related to fatty acid biosynthesis, AMP‐dependent biosynthesis, amino acid recognition/activation processes and secondary metabolite production, whereas down‐regulated genes were involved in amino acid synthesis, oxidation–reduction processes, metal ion‐binding and metabolic/catalytic activities. Among the down‐regulated genes, the expression of pigmentation‐related genes such as aurO, gip1 and aurR2 was remarkable. Similarly, the expression levels of key enzyme‐coding genes involved in deoxynivalenol mycotoxin production were significantly decreased in the range of −1.77 and −2.94. For up‐regulated genes, nonribosomal peptide synthetase and polyketide synthase genes were notably distinguished from the remaining down‐regulated genes as these genes can be involved in biosynthesis of common secondary metabolites. The results clearly emphasize that T. atroviride repressed the biosynthesis of primary metabolites in F. graminearum while simultaneously up‐regulating the expression of genes involved in the synthesis of secondary metabolites. This is the first report showing how T. atroviride leads to transcriptome alterations and the findings suggest that T. atroviride could serve as an effective fungus by employing a wide variety of strategies against phytopathogenic fungi.

Taxonogenomic Analysis of Marine-Derived Streptomyces sp. N11-50 and the Profile of NRPS and PKS Gene Clusters

Streptomyces sp. N11-50 was isolated from deep-sea water and found to produce diketopiperazine (DKP) compounds such as albonoursin and cyclo(Phe-Leu). This study aimed to reveal the potential to synthesize diverse nonribosomal peptide and polyketide compounds as the other secondary metabolites different from DKP after clarifying the taxonomic position. Strain N11-50 was identified as Streptomyces albus, as it showed 100% 16S rRNA gene sequence similarities and 95.5% DNA–DNA relatedness to S. albus NBRC 13014T. We annotated the nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) gene clusters in the genome. Consequently, five NRPS, one hybrid PKS/NRPS, five type-I PKS and one type-II PKS gene clusters were observed, of which we predicted the products through bioinformatic analysis. These gene clusters were well conserved in already whole-genome sequence (WGS)-published strains belonging to S. albus. On the other hand, our taxonogenomic analysis revealed that three WGS-published S. albus strains were not S. albus. Two of the three should be classified as Streptomyces albidoflavus, and the remaining one was likely a new genomospecies. After reclassifying these appropriately, we demonstrated species-specific profiles of the NRPS and PKS gene clusters with little strain-level diversities.

Genome-Guided Discovery of the Myxobacterial Thiolactone-Containing Sorangibactins.

In this study, an unprecedented myxobacterial siderophore termed sorangibactin was discovered by heterologous expression of a coelibactin-like nonribosomal peptide synthetase (NRPS) gene cluster from the Sorangiineae strain MSr11367 in the host Myxococcus xanthus DK1622. De novo structure elucidation uncovered a linear polycyclic structure consisting of an N-terminal phenol group, an oxazole, tandem N-methyl-thiazolidines, and an unusual C-terminal γ-thiolactone moiety. Except for the unprecedented oxazoline dehydrogenation to form an oxazole, which we show to be catalyzed by a cytochrome P450-dependent enzyme, other tailoring steps were found necessary for efficient downstream processing. The unusual thioesterase (TE) domain is proposed to select homocysteine or methionine for offloading involving an intramolecular γ-thiolactone formation. Its active site comprises a rare cysteine, which was found essential for product formation by point mutation to alanine or serine, which both abolished its activity. This unusual release mechanism and the resulting rare thiolactone structure can serve as a starting point for detailed biochemical investigations.

Bursts in biosynthetic gene cluster transcription are accompanied by surges of natural compound production in the myxobacterium Sorangium sp.

A better understanding of the genetic regulation of the biosynthesis of microbial compounds could accelerate the discovery of new biologically active molecules and facilitate their production. To this end, we have investigated the time course of genome-wide transcription in the myxobacterium Sorangium sp. So ce836 in relation to its production of natural compounds. Time-resolved RNA sequencing revealed that core biosynthesis genes from 48 biosynthetic gene clusters (BGCs; 92% of all BGCs encoded in the genome) were actively transcribed at specific time points in a batch culture. The majority (80%) of polyketide synthase and non-ribosomal peptide synthetase genes displayed distinct peaks of transcription during exponential bacterial growth. Strikingly, these bursts in BGC transcriptional activity were associated with surges in the net production rates of known natural compounds, indicating that their biosynthesis was critically regulated at the transcriptional level. In contrast, BGC read counts from single time points had limited predictive value about biosynthetic activity, since transcription levels varied >100-fold among BGCs with detected natural products. Taken together, our time-course data provide unique insights into the dynamics of natural compound biosynthesis and its regulation in a wild-type myxobacterium, challenging the commonly cited notion of preferential BGC expression under nutrient-limited conditions. The close association observed between BGC transcription and compound production warrants additional efforts to develop genetic engineering tools for boosting compound yields from myxobacterial producer strains.

In silico Analysis of Molecular Phylogeny of Genes Involved in the Synthesis of Bioactive Compounds in Cyanobacteria Strains Located in Tehran Cascade

Background: Cyanobacteria is an excellent candidate for discovering bioactive compounds with applications in pharmaceutical industries. Nowadays, the major bioactive metabolites isolated from cyanobacteria are polymerized, nonribosomal peptides (NRPS), or hybrids of both. Despite numerous studies on the gene distribution of bioactive compounds, no studies have been conducted on the cyanobacteria strains found in the Tehran cascade. Objectives: This monitoring study aimed to study the secondary structure of the protein and molecular phylogeny of polypeptides synthase genes and nonribosomal peptides along with the 16S rRNA gene. Methods: In this study, 20 strains of cyanobacteria found in the Tehran Cascade were identified based on a 16rRNA gene sequence. The PCR of the nonribosomal peptides (NRPS) and polyketide synthase (PKS) genes was performed using molecular techniques to analyze the phylogenetic genes responsible for secondary metabolite production. Bioinformatics software was used to predict the composition of peptides, activated amino acids, and signature sequences of the NRPS adenylation module. Finally, the strains studied in the Alborz Herbarium Microbial Culture Collection (CCC) were kept at the Azad Islamic University, Science and Research Unit. Results: The NRPS and PKS genes were found in some strains. Furthermore, the bioinformatics analyses revealed the type of natural compound, the signature sequences, and the predication of the activated amino acid. Clustering of NRPS and PKS protein sequences showed no clear phylogenetic correlation between adenylation domains and the type of activated amino acid, indicating wide diversity within adenylation domains. Conclusions: The biological analysis of polypeptide synthase and nonribosomal peptide synthetases genes may help estimate species that produce natural products and the possible role of these enzymatic complexes in the bio-synthesis of bioactive compounds in the pharmaceutical industry.