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Streptomyces are ubiquitous soil-dwelling bacteria with large, linear genomes that are of special importance as a source of metabolites used in human and veterinary medicine, agronomy, and industry. Conjugative elements (actinomycetes integrative and conjugative elements, AICEs) are the main drivers of Streptomyces Horizontal Gene Transfer. AICE transfer has long been known to be accompanied by mobilization of chromosomal DNA. However, the magnitude of DNA transfer, or the localization of acquired DNA across their linear chromosome, has remained undetermined. We here show that conjugative crossings in sympatric strains of Streptomyces result in the large-scale, genome-wide distributed replacement of up to one-third of the recipient chromosome, a phenomenon for which we propose the name "Streptomyces Chromosomal Transfer" (SCT). Such chromosome blending results in the acquisition, loss, and hybridization of Specialized Metabolite Biosynthetic Gene Clusters, leading to a novel metabolic arsenal in exconjugant offspring. Harnessing conjugation-mediated specialized metabolite biosynthesis gene cluster diversification holds great promise in the discovery of new bioactive compounds including antibiotics., Competing Interests: Conflict of Interest The authors declare that there are no conflicts of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Microorganisms can produce a vast array of bioactive secondary metabolites, including DNA-intercalating agents like actinomycin D, doxorubicin, which hold great potential for cancer chemotherapy. However, discovering novel DNA-intercalating compounds remains challenging due to the limited sensitivity and specificity of conventional activity assays, which require large-scale fermentation and purification. Here, we introduced the single-molecule stretching assay (SMSA) directly to microbial cultures or extracts for discovering DNA-intercalating agents, even in trace amounts of microbial cultures (5 μl). We showed that the unique changes of dsDNA in contour length and overstretching transition enable the specific detection of intercalators from complex samples without the need for extensive purification. Applying force to dsDNA also enhanced the sensitivity by increasing both the binding affinity Ka and the quantity of ligands intercalation, thus allowing the detection of weak intercalators, which are often overlooked using traditional methods. We demonstrated the effectiveness of SMSA, identified two DNA intercalator-producing strains: Streptomyces tanashiensis and Talaromyces funiculosus, and isolated three DNA intercalators: medermycin, kalafungin and ligustrone B. Interestingly, both medermycin and kalafungin, classified as weak DNA intercalators (Ka ∼103 M-1), exhibited potent anti-cancer activity against HCT-116 cancer cells, with IC50 values of 52 ± 6 and 70 ± 7 nM, respectively., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Aim: Fire blight, attributed to the bacterium Erwinia amylovora, significantly damages economically important crops, such as apples and pears. Conventional methods for managing fire blight involve the application of chemical pesticides, such as streptomycin and oxytetracycline. Nevertheless, apprehensions are increasing regarding developing antibiotic and pesticide-resistant strains, compounded by documented instances of plant toxicity. Here, we present that Streptomyces recifensis SN1E1 has exhibited remarkable efficacy in suppressing apple fire blight disease. This study aims to unravel the molecular-level antimicrobial mechanisms employed by the SN1E1 strain., Methods and Results: We identified four antimicrobial-associated biosynthetic gene clusters within the genomics of S. recifensis SN1E1. To validate antimicrobial activity against E. amylovora, knock-out mutants of biosynthetic genes linked to antimicrobial activity were generated using the CRISPR/Cas9 mutagenesis system. Notably, the whiE4 and phzB deficient mutants displayed statistically reduced antibacterial activity against E. amylovora., Conclusion: This research establishes a foundation for environmental and biological control studies. The potential utilization of environmentally friendly microbial agents derived from the SN1E1 strain holds promise for the biological control of fire blight disease., (© The Author(s) 2024. Published by Oxford University Press on behalf of Applied Microbiology International.)

This study reports the isolation and characterization of a Streptomyces sp. from soil, capable of producing bioactive secondary metabolites active against a variety of bacterial human pathogens. We targeted the antimicrobial activity against Escherichia coli ATCC-BAA 2469, a clinically relevant strain of bacteria harbouring resistance genes for carbapenems, extended spectrum beta-lactams, tetracyclines, fluoroquinones, etc. Preliminary screening using the spot inoculation technique identified Streptomyces sp. NP73 as the potent strain among the 74 isolated Actinomycetia strain. 16S rRNA gene and whole genome sequencing (WGS) confirmed its taxonomical identity and helped in the construction of the phylogenetic tree. WGS revealed the predicted pathways and biosynthetic gene clusters responsible for producing various types of antibiotics including the isolated compound. Bioactivity guided fractionation and chemical characterization of the active fraction, carried out using liquid chromatography, gas chromatography-mass spectrometry, infra-red spectroscopy, and nuclear magnetic resonance spectroscopy, led to the tentative identification of the active compound as Pyrrolo[1,2-a] pyrazine-1,4-dione, hexahydro-, a diketopiperazine molecule. This compound exhibited excellent antimicrobial and anti-biofilm properties against E. coli ATCC-BAA 2469 with an MIC value of 15.64 µg ml-1, and the low cytotoxicity of the compound identified in this study provides hope for future drug development., (© The Author(s) 2024. Published by Oxford University Press on behalf of Applied Microbiology International.)

Filamentous Actinobacteria, recently renamed Actinomycetia, are the most prolific source of microbial bioactive natural products. Studies on biosynthetic gene clusters benefit from or require chromosome-level assemblies. Here, we provide DNA sequences from >1000 isolates: 881 complete genomes and 153 near-complete genomes, representing 28 genera and 389 species, including 244 likely novel species. All genomes are from filamentous isolates of the class Actinomycetia from the NBC culture collection. The largest genus is Streptomyces with 886 genomes including 742 complete assemblies. We use this data to show that analysis of complete genomes can bring biological understanding not previously derived from more fragmented sequences or less systematic datasets. We document the central and structured location of core genes and distal location of specialized metabolite biosynthetic gene clusters and duplicate core genes on the linear Streptomyces chromosome, and analyze the content and length of the terminal inverted repeats which are characteristic for Streptomyces. We then analyze the diversity of trans-AT polyketide synthase biosynthetic gene clusters, which encodes the machinery of a biotechnologically highly interesting compound class. These insights have both ecological and biotechnological implications in understanding the importance of high quality genomic resources and the complex role synteny plays in Actinomycetia biology., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Streptomyces are soil bacteria with complex life cycle. During sporulation Streptomyces linear chromosomes become highly compacted so that the genetic material fits within limited spore volume. The key players in this process are nucleoid-associated proteins (NAPs). Among them, HU (heat unstable) proteins are the most abundant NAPs in the cell and the most conserved in bacteria. HupS, one of the two HU homologues encoded by the Streptomyces genome, is the best-studied spore-associated NAP. In contrast to other HU homologues, HupS contains a long, C-terminal domain that is extremely rich in lysine repeats (LR domain) similar to eukaryotic histone H2B and mycobacterial HupB protein. Here, we have investigated, whether lysine residues in HupS are posttranslationally modified by reversible lysine acetylation. We have confirmed that Streptomyces venezuelae HupS is acetylated in vivo. We showed that HupS binding to DNA in vitro is controlled by the acetylation. Moreover, we identified that CobB1, one of two Sir2 homologues in Streptomyces, controls HupS acetylation levels in vivo. We demonstrate that the elimination of CobB1 increases HupS mobility, reduces chromosome compaction in spores, and affects spores maturation. Thus, our studies indicate that HupS acetylation affects its function by diminishing DNA binding and disturbing chromosome organization., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Aims: Developing energy-saving and ecofriendly strategies for treating harvested Microcystis biomass., Methods and Results: Streptomyces amritsarensis HG-16 was first reported to effectively kill various morphotypes of natural Microcystis colonies at very high cell densities. Concurrently, HG-16 grown on lysed Microcystis maintained its antagonistic activity against plant pathogenic fungus Fusarium graminearum. It could completely inhibit spore germination and destroy mycelial structure of F. graminearum. Transcriptomic analysis revealed that HG-16 attacked F. graminearum in a comprehensive way: interfering with replication, transcription, and translation processes, inhibiting primary metabolisms, hindering energy production and simultaneously destroying stress-resistant systems of F. graminearum., Conclusions: The findings of this study provide a sustainable and economical option for resource reclamation from Microcystis biomass: utilizing Microcystis slurry to propagate HG-16, which can subsequently be employed as a biocontrol agent for managing F. graminearum., (© The Author(s) 2024. Published by Oxford University Press on behalf of Applied Microbiology International.)

Streptomyces sp. SN-593, a reveromycin producer, was previously thought to belong to the genus Streptomyces based on its morphological and chemotaxonomic characteristics. In this paper, we re-considered its taxonomic position according to the current criteria. Phylogenetic analysis using 16S rRNA gene sequences showed that the strain belongs to the genus Actinacidiphila. In multilocus sequence and phylogenomic analyses, the strain SN-593 represented a distinct evolutionary lineage within this genus, and its closest neighbor was A. yanglinensis. Digital DNA-DNA hybridization indicated that the strain shares less than 32% DNA-DNA relatedness with the type strains of closely related species, confirming this strain is a novel genomospecies. According to its phenotypic distinctiveness from the closest neighbor, we propose Actinacidiphila reveromycinica sp. nov. for the strain SN-593T. Additionally, as Streptomyces acidipaludis belonged to the genus Actinacidiphila in these analyses, it should be transferred to the genus, for which Actinacidiphila acidipaludis comb. nov. is proposed., (© The Author(s) 2024. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

The linear chromosome of Streptomyces exhibits a highly compartmentalized structure with a conserved central region flanked by variable arms. As double strand break (DSB) repair mechanisms play a crucial role in shaping the genome plasticity of Streptomyces, we investigated the role of EndoMS/NucS, a recently characterized endonuclease involved in a non-canonical mismatch repair (MMR) mechanism in archaea and actinobacteria, that singularly corrects mismatches by creating a DSB. We showed that Streptomyces mutants lacking NucS display a marked colonial phenotype and a drastic increase in spontaneous mutation rate. In vitro biochemical assays revealed that NucS cooperates with the replication clamp to efficiently cleave G/T, G/G and T/T mismatched DNA by producing DSBs. These findings are consistent with the transition-shifted mutational spectrum observed in the mutant strains and reveal that NucS-dependent MMR specific task is to eliminate G/T mismatches generated by the DNA polymerase during replication. Interestingly, our data unveil a crescent-shaped distribution of the transition frequency from the replication origin towards the chromosomal ends, shedding light on a possible link between NucS-mediated DSBs and Streptomyces genome evolution., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The increasing global concern of antimicrobial resistance and shortage of new antimicrobials necessitates exploring untapped terrestrial environments for new bioactive microbiome diversity. The low-temperature and oligotrophic North Western Himalaya (NWH) region has a vast diversity of Streptomyces with potential antimicrobial properties that remain largely unexplored. This study evaluates the diversity of culturable Streptomyces from high-altitude NWH and their potential as a source of new antimicrobials through genus-specific isolation and identification. The results demonstrate a distinct phylogenetic clustering of Streptomyces from different sampling regions of NWH, site-specific variation in culturable β-diversity and species commonness with varying intersite bioactivity among different sites. Further, the study optimized the media selection for large-scale culture cultivation in antibiotic production processes and demonstrated the antimicrobial efficacy of Streptomyces against a range of pathogens through in vitro bioassays using minimum inhibitory concentration determination and antibiofilm activity. Untargeted label-free proteomic profiling also revealed variable expression of stress-response proteins and antibiotic regulators as a competitive survival strategy for selective antagonistic Streptomyces. The findings highlight the potential of NWH in augmenting antimicrobial discovery and combating antimicrobial resistance through the isolation and study of novel bioactive Streptomyces., (© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.)

The demand for discovering novel microbial secondary metabolites is growing to address the limitations in bioactivities such as antibacterial, antifungal, anticancer, anthelmintic, and immunosuppressive functions. Among microbes, the genus Streptomyces holds particular significance for secondary metabolite discovery. Each Streptomyces species typically encodes approximately 30 secondary metabolite biosynthetic gene clusters (smBGCs) within its genome, which are mostly uncharacterized in terms of their products and bioactivities. The development of next-generation sequencing has enabled the identification of a large number of potent smBGCs for novel secondary metabolites that are imbalanced in number compared with discovered secondary metabolites. The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system has revolutionized the translation of enormous genomic potential into the discovery of secondary metabolites as the most efficient genetic engineering tool for Streptomyces. In this review, the current status of CRISPR/Cas applications in Streptomyces is summarized, with particular focus on the identification of secondary metabolite biosynthesis gene clusters and their potential applications.This review summarizes the broad range of CRISPR/Cas applications in Streptomyces for natural product discovery and production., One-Sentence Summary: This review summarizes the broad range of CRISPR/Cas applications in Streptomyces for natural product discovery and production., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society of Industrial Microbiology and Biotechnology.)

Narrow-spectrum antibiotics are of great interest given their ability to spare the microbiome and decrease widespread antibiotic resistance compared to broad-spectrum antibiotics. Herein, we screened an in-house library of Actinobacteria strains for selective activity against Acinetobacter baumannii and successfully identified Streptomyces sp. CS-62 as a producer of a natural product with this valuable activity. Analysis of the cultures via high-resolution mass spectrometry and tandem mass spectrometry, followed by comparison with molecules in the Natural Product Atlas and the Global Natural Products Social Molecular Networking platform, suggested a novel natural product. Genome mining analysis initially supported the production of a novel kirromycin derivative. Isolation and structure elucidation via mass spectrometry and Nuclear Magnetic Resonance (NMR) analyses revealed that the active natural product was the known natural product factumycin, exposing omissions and errors in the consulted databases. While public databases are generally very useful for avoiding rediscovery of known molecules, rediscovery remains a problem due to public databases either being incomplete or having errors that result in failed dereplication. Overall, the work describes the ongoing problem of dereplication and the continued need for public database curation., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society of Industrial Microbiology and Biotechnology.)

Aim: The present study aimed to investigate a novel antifungal compound produced by Streptomyces blastmyceticus S108 strain. Its effectiveness against clinical isolates of Candida species and its synergistic effect with conventional antifungal drugs were assessed, and its molecular mechanism of action was further studied against Candida albicans., Methods and Results: A newly isolated strain from Tunisian soil, S. blastmyceticus S108, showed significant antifungal activity against Candida species by well diffusion method. The butanolic extract of S108 strain supernatant exhibited the best anti-Candida activity with a minimal inhibitory concentration (MIC) value of 250 μg ml-1, determined by the microdilution method. The bio-guided purification steps of the butanolic extract were performed by chromatographic techniques. Among the fractions obtained, F13 demonstrated the highest level of activity, displaying a MIC of 31.25 μg ml-1. Gas chromatography-mass spectrometry and electrospray ionization mass spectrometry analyses of this fraction (F13) revealed the glycolipidic nature of the active molecule with a molecular weight of 685.6 m/z. This antifungal metabolite remained stable to physicochemical changes and did not show hemolytic activity even at 4MIC corresponding to 125 µg ml-1 toward human erythrocytes. Besides, the glycolipid compound was combined with 5-flucytosine and showed a high synergistic effect with a fractional inhibitory concentration index value 0.14 against C. albicans ATCC 10231. This combination resulted in a decrease of MIC values of 5-flucytosine and the glycolipid-like compound by 8- and 64-fold, respectively. The examination of gene expression in treated C. albicans cells by quantitative polymerase chain reaction (qPCR) revealed that the active compound tested alone or in combination with 5-flucytosine blocks the ergosterol biosynthesis pathway by downregulating the expression of ERG1, ERG3, ERG5, ERG11, and ERG25 genes., Conclusion and Impact of the Study: The new glycolipid-like compound, produced by Streptomyces S108 isolate, could be a promising drug for medical use against pathogenic Candida isolates., (© The Author(s) 2023. Published by Oxford University Press on behalf of Applied Microbiology International.)

Streptomyces lividans is an efficient host for extracellular overproduction of recombinant proteins. To enhance the overexpression strength of S. lividans, we designed several kinds of expression plasmids with different positioning of repeat promoters. The effect of repeat promoters was evaluated by measuring the accumulated amounts of a stable transglutaminase or an unstable carboxypeptidase that was secreted into the medium. Successive tandem positions of repeat promoters upstream of the normal promoter did not enhance the expression of transglutaminase. Discrete positions of repeat promoters both upstream and downstream of the normal promoter enhanced the expression of transglutaminase to 2-fold, and the downstream ones also enhanced the expression of carboxypeptidase to 1.7-fold. On the other hand, there were still some constructs of plasmids with discrete repeat promoters that did not promote the expression of the target enzymes, indicating the complexity of the mechanisms of repeat promoters working on gene expression., (© The Author(s) 2023. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Grisemycin, salinipeptin, and cypemycin belong to the linaridin class of ribosomally synthesized and posttranslationally modified peptides that contain multiple dehydrobutyrine and D-amino acid residues. The biosynthetic gene clusters of these linaridins lack obvious candidate genes for the dehydratase and epimerase required to introduce dehydrobutyrine and D-amino acid residues, respectively. However, we previously demonstrated that the grisemycin (grm) cluster contained cryptic dehydratase and epimerase genes by heterologous expression of this biosynthetic gene cluster in Streptomyces lividans and proposed that two genes (grmH and grmL) with unknown functions catalyze dehydration and epimerization reactions. In this study, we confirmed that both GrmH and GrmL, which were shown to constitute a protein complex by a co-purification experiment, were required to catalyze the dehydration, epimerization, and proteolytic cleavage of a precursor peptide GrmA by in vivo experiments. Furthermore, we demonstrated that GrmH/GrmL complex accepted salinipeptin and cypemycin precursor peptides, which possess three additional amino acids., (© The Author(s) 2023. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Aims: We evaluated whether the randomness of mutation breeding can be regulated through a double-reporter system. We hope that by establishing a new precursor feeding strategy, the production capacity of industrial microorganisms after pilot scale-up can be further improved., Methods and Results: In this study, the industrial strain Streptomyces roseosporus L2796 was used as the starter strain for daptomycin production, and a double-reporter system with the kanamycin resistance gene Neo and the chromogenic gene gusA was constructed to screen for high-yield strain L2201 through atmospheric and room temperature plasma (ARTP). Furthermore, the composition of the culture medium and the parameters of precursor replenishment were optimized, resulting in a significant enhancement of the daptomycin yield of the mutant strain L2201(752.67 mg/l)., Conclusions: This study successfully screened a high-yield strain of daptomycin through a double-reporter system combined with ARTP mutation. The expression level of two reporter genes can evaluate the strength of dptEp promoter, which can stimulate the expression level of dptE in the biosynthesis of daptomycin, thus producing more daptomycin. The developed multi-stage feeding rate strategy provides a novel way to increase daptomycin in industrial fermentation., (Published by Oxford University Press on behalf of Applied Microbiology International 2023.)

Due to the rise in bacterial resistance towards various therapeutic agents, interest is now developing towards fatty acid based antimicrobials because of their non-specific mode of action. A strain SORS 24 isolated from Sonchus oleraceus (Sow thistle) showed significant activity against Escherichia coli ATCC 25922 (25 mm), Chlorella vulgaris (20 mm), Bacillus subtilis DSM 10 (ATCC 6051) and Pseudomonas sp. (15 mm). It displayed an LC50 value of 10 µg/ml against Artemia salina (Brine shrimp) nauplii and an EC50 value of 0.8 µg/ml in the (DPPH) diphenylpicrylhydrazyl antioxidant assay. The strain also displayed genotoxicity against a PolA deficient strain, E. coli K-12 AB 3027 (15 mm). Mass spectrometry (HPLC-MS) showed that the strain produced oleamide (9-Octadecenamide) and erucamide (13-Docosenamide). Both of the purified fatty acid amides showed prominent activity against B. subtilis DSM 10 (ATCC 6051) (20 mm) and E. coli ATCC 25922 (15 mm). Significant genotoxicity was observed against E. coli K-12 AB 3027 (15 mm). The 16S gene sequencing revealed that the strain belonged to species, Streptomyces tanashiensis. As far as our understanding, this is the first report of this species producing these fatty acid based antimicrobials., (© The Author(s) 2023. Published by Oxford University Press on behalf of Applied Microbiology International.)

AtrA belongs to the TetR family and has been well characterized for its roles in antibiotic biosynthesis regulation. Here, we identified an AtrA homolog (AtrA-lin) in Streptomyces lincolnensis. Disruption of atrA-lin resulted in reduced lincomycin production, whereas the complement restored the lincomycin production level to that of the wild-type. In addition, atrA-lin disruption did not affect cell growth and morphological differentiation. Furthermore, atrA-lin disruption hindered the transcription of regulatory gene lmbU, structural genes lmbA and lmbW inside the lincomycin biosynthesis gene cluster, and 2 other regulatory genes, adpA and bldA. Completement of atrA-lin restored the transcription of these genes to varying degrees. Notably, we found that AtrA-lin directly binds to the promoter region of lmbU. Collectively, AtrA-lin positively modulated lincomycin production via both pathway-specific and global regulators. This study offers further insights into the functional diversity of AtrA homologs and the mechanism of lincomycin biosynthesis regulation., (© The Author(s) 2023. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Streptomyces lividans is a potent host for the extracellular overproduction of heterologous proteins. To further improve the usability and productivity of S. lividans, a dual gene expression vector of "pTSKr duet" containing two strong constitutive promoters, scmpPc and kasOp*, was constructed. The success in the overproduction of two secretory enzymes simultaneously without interference with each other indicated that the "pTSKr duet" vector can realize the coexpression of two genes simultaneously and independently. Further, using the two-gene coexpression vector, we screened the effects of the overexpression of five factors that possibly promote secretion on the extracellular overproduction of heterologous secretory proteins. Interestingly, the coexpression of a quality control regulator (CssR) promoted the overproduction level to 1.3-fold for a stable heterologous protein of SMTG (transglutaminase from S. mobaraensis), while other four factors limited the overproduction of SMTG at different degrees., (© The Author(s) 2022. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Kusaya, a traditional Japanese fermented fish product, is known for its high preservability, as it contains natural antibiotics derived from microorganisms, and therefore molds and yeasts do not colonize it easily. In this study, the Streptomyces diastaticus strain TUA-NKU25 was isolated from Kusaya, and its growth as well as the production of antibiotics were investigated. Strain TUA-NKU25 showed advantageous growth characteristics in the presence, but not in the absence, of sodium chloride (NaCl). Antimicrobial assay, high-performance liquid chromatography, and electrospray ionization-mass spectrometry analysis showed that this strain produced surugamide A and uncharacterized antimicrobial compound(s) during growth in the presence of NaCl, suggesting that the biosynthesis of these compounds was upregulated by NaCl. Draft genomic analysis revealed that strain TUA-NKU25 possesses a surugamide biosynthetic gene cluster (sur BGC), although it is incomplete, lacking surB/surC. Phylogenetic analysis of strain TUA-NKU25 and surugamide-producing Streptomyces showed that sur BGC formed a clade distinct from other known groups., (© The Author(s) 2022. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Streptomyces are prolific producers of specialized metabolites with applications in medicine and agriculture. These bacteria possess a large linear chromosome genetically compartmentalized: core genes are grouped in the central part, while terminal regions are populated by poorly conserved genes. In exponentially growing cells, chromosome conformation capture unveiled sharp boundaries formed by ribosomal RNA (rrn) operons that segment the chromosome into multiple domains. Here we further explore the link between the genetic distribution of rrn operons and Streptomyces genetic compartmentalization. A large panel of genomes of species representative of the genus diversity revealed that rrn operons and core genes form a central skeleton, the former being identifiable from their core gene environment. We implemented a new nomenclature for Streptomyces genomes and trace their rrn-based evolutionary history. Remarkably, rrn operons are close to pericentric inversions. Moreover, the central compartment delimited by rrn operons has a very dense, nearly invariant core gene content. Finally, this compartment harbors genes with the highest expression levels, regardless of gene persistence and distance to the origin of replication. Our results highlight that rrn operons are structural boundaries of a central functional compartment prone to transcription in Streptomyces., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Aims: Sequencing and genome analysis of two co-isolated streptomycetes, named BV410-1 and BV410-10, and the effect of their co-cultivation on the staurosporine production., Methods and Results: Identification of two strains through genome sequencing and their separation using different growth media was conducted. Sequence analysis revealed that the genome of BV410-1 was 9.5 Mb, whilst that of BV410-10 was 7.1 Mb. AntiSMASH analysis identified 28 biosynthetic gene clusters (BGCs) from BV410-1, including that responsible for staurosporine biosynthesis, whilst 20 BGCs were identified from BV410-10. The addition of cell-free supernatant from BV410-10 monoculture to BV410-1 fermentations improved the staurosporine yield from 8.35 mg L -1 up to 15.85 mg L -1 , whilst BV410-10 monoculture ethyl acetate extract did not have the same effect. Also, there was no improvement in staurosporine production when artificial mixed cultures were created using three different BV410-1 and BV410-10 spore ratios., Conclusions: The growth of BV410-10 was inhibited when the two strains were grown together on agar plates. Culture supernatants of BV410-10 showed potential to stimulate staurosporine production in BV410-1, but overall co-cultivation attempts did not restore the previously reported yield of staurosporine produced by the original mixed isolate., Significance and Impact of Study: This work confirmed complex relations between streptomycetes in soil that are difficult to recreate under the laboratory conditions. Also, mining of streptomycetes genomes that mainly produce known bioactive compounds could still be the fruitful approach in search for novel bioactive molecules., (© 2022 Society for Applied Microbiology.)

Aims: Assessing the role of ramR sl , a gene absent in a lincomycin over-producing strain, in the regulation of morphological development and lincomycin biosynthesis in Streptomyces lincolnensis., Methods and Results: The gene ramR sl was deleted from the wild-type strain NRRL 2936 and the ΔramR mutant strain was characterized by a slower growth rate and a delayed morphological differentiation compared to the original strain NRRL 2936. Furthermore, the ΔramR produced 2.6-fold more lincomycin than the original strain, and consistently the level of expression of all lincomycin cluster located genes was enhanced at 48 and 96 h in the ΔramR. Complementation of ΔramR with an intact copy of ramR sl restored all wild-type features, whereas the over-expression of ramR sl led to a reduction of 33% of the lincomycin yield. Furthermore, the level of expression of glnR, bldA and SLCG_2919, three of known lincomycin biosynthesis regulators, was lower in the ΔramR than in the original strain at the early stage of fermentation and we demonstrated, using electrophoretic mobility shift assay and XylE reporter assay, that glnR is a novel direct target of RamR., Conclusions: Altogether, these results indicated that, beyond promoting the morphological development, RamR regulates negatively lincomycin biosynthesis and positively the expression of the nitrogen regulator GlnR., Significance and Impact of the Study: We demonstrated that RamR plays a negative role in the regulation of lincomycin biosynthesis in S. lincolnensis. Interestingly, the deletion of this gene in other antibiotic-producing Streptomyces strains might also increase their antibiotic-producing abilities., (© 2022 Society for Applied Microbiology.)

A structurally unique aminoglycoside produced in Streptoalloteichus tenebrarius, Apramycin is used in veterinary medicine or the treatment of Salmonella, Escherichia coli, and Pasteurella multocida infections. Although apramycin was discovered nearly 50 years ago, many biosynthetic steps of apramycin remain unknown. In this study, we identified a HemK family methyltransferase, AprI, to be the 7'-N-methyltransferase in apramycin biosynthetic pathway. Biochemical experiments showed that AprI converted demethyl-aprosamine to aprosamine. Through gene disruption of aprI, we identified a new aminoglycoside antibiotic demethyl-apramycin as the main product in aprI disruption strain. The demethyl-apramycin is an impurity in apramycin product. In addition to demethyl-apramycin, carbamyltobramycin is another major impurity. However, unlike demethyl-apramycin, tobramycin is biosynthesized by an independent biosynthetic pathway in S. tenebrarius. The titer and rate of apramycin were improved by overexpression of the aprI and disruption of the tobM2, which is a crucial gene for tobramycin biosynthesis. The titer of apramycin increased from 2227 ± 320 mg/L to 2331 ± 210 mg/L, while the titer of product impurity demethyl-apramycin decreased from 196 ± 36 mg/L to 51 ± 9 mg/L. Moreover, the carbamyltobramycin titer of the wild-type strain was 607 ± 111 mg/L and that of the engineering strain was null. The rate of apramycin increased from 68% to 87% and that of demethyl-apramycin decreased from 1.17% to 0.34%., (© The Author(s) 2022. Published by Oxford University Press on behalf of Society of Industrial Microbiology and Biotechnology.)

The rise of antibiotic-resistant bacteria represents a major threat to global health, creating an urgent need to discover new antibiotics. Natural products derived from the genus Streptomyces represent a rich and diverse repertoire of chemical molecules from which new antibiotics are likely to be found. However, a major challenge is that the biosynthetic gene clusters (BGCs) responsible for natural product synthesis are often poorly expressed under laboratory culturing conditions, thus preventing the isolation and screening of novel chemicals. To address this, we describe a novel approach to activate silent BGCs through rewiring endogenous regulation using synthetic gene regulators based upon CRISPR-Cas. First, we refine CRISPR interference (CRISPRi) and create CRISPR activation (CRISPRa) systems that allow for highly programmable and effective gene repression and activation in Streptomyces. We then harness these tools to activate a silent BGC by perturbing its endogenous regulatory network. Together, this work advances the synthetic regulatory toolbox for Streptomyces and facilitates the programmable activation of silent BGCs for novel chemical discovery., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

We report the discovery and functional characterization of a new bacterial tRNA species. The tRNA-Asp-AUC, from a fast-growing desert streptomycete, decodes GAU codons. In the absence of queuosine tRNA anticodon modification in streptomycetes, the new tRNA circumvents inefficient wobble base-pairing during translation. The tRNA, which is constitutively expressed, greatly enhances synthesis of 4 different antibiotics in the model mesophilic species Streptomyces coelicolor, including the product of a so-called cryptic pathway, and increases yields of medically-important antibiotics in other species. This can be rationalised due to increased expression of both pleiotropic and pathway-specific transcriptional activators of antibiotic biosynthesis whose genes generally possess one or more GAT codons; the frequency of this codon in these gene sets is significantly higher than the average for streptomycete genes. In addition, the tRNA enhances production of cobalamin, a precursor of S-adenosyl methionine, itself an essential cofactor for synthesis of many antibiotics. The results establish a new paradigm of inefficient wobble base-pairing involving GAU codons as an evolved strategy to regulate gene expression and, in particular, antibiotic biosynthesis. Circumventing this by expression of the new cognate tRNA offers a generic strategy to increase antibiotic yields and to expand the repertoire of much-needed new bioactive metabolites produced by these valuable bacteria., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Direct cloning of biosynthetic gene clusters (BGCs) from microbial genomes facilitates natural product-based drug discovery. Here, by combining Cas12a and the advanced features of bacterial artificial chromosome library construction, we developed a fast yet efficient in vitro platform for directly capturing large BGCs, named CAT-FISHING (CRISPR/Cas12a-mediated fast direct biosynthetic gene cluster cloning). As demonstrations, several large BGCs from different actinomycetal genomic DNA samples were efficiently captured by CAT-FISHING, the largest of which was 145 kb with 75% GC content. Furthermore, the directly cloned, 110 kb long, cryptic polyketide encoding BGC from Micromonospora sp. 181 was then heterologously expressed in a Streptomyces chassis. It turned out to be a new macrolactam compound, marinolactam A, which showed promising anticancer activity. Our results indicate that CAT-FISHING is a powerful method for complicated BGC cloning, and we believe that it would be an important asset to the entire community of natural product-based drug discovery., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Aims: The current study aimed to evaluate the occurrence of actinomycetes in the Coast of Bejaia City using selective isolation, as well as their bioactivity and phylogenitic diversity., Methods and Results: Different selective media and methods were used, leading to the isolation of 103 actinomycete strains. The number of strains was influenced by isolation procedures and their interactions based on a three-way ANOVA and a post hoc Tukey test, which revealed that using M2 medium, dilution of samples followed by moderate heat treatment, and sampling at 10-20 m yielded the highest numbers of actinomycetes. The isolates were screened for their antimicrobial activity against human pathogenic microorganisms using agar and well diffusion methods. Of all the isolates, ten displayed activity against at least one Gram-positive bacterium, of which P21 showed the highest activity against Staphylococcus aureus, Methicillin-resistant S. aureus and Bacillus subtilis, with a diameter of 32, 28 and 25 mm respectively. Subsequently, active isolates were assigned to Streptomyces spp. and Nocardiopsis spp. based on 16S rRNA gene sequencing, including a putative new Streptomyces species (S3). The phenotypic characteristics of the P21 strain were determined, and interesting enzymatic capacities were shown., Conclusion: The recovery of actinomycetes along the Coast of Bejaia City was influenced by the isolation procedure. Ten strains displayed interesting antibacterial activity against Gram-positive bacteria, of which the P21 strain was selected as the most active strain., Significance and Impact of the Study: This work provides a new insight into the occurrence of actinobacteria in the Coast of Bejaia. It suggests also that polluted environments such as Bejaia Bay could provide access to interesting actinomycetes as sources of antibiotic leads., (© 2021 The Society for Applied Microbiology.)

Extensively produced by members of the genus Streptomyces, piericidins are a large family of microbial metabolites, which consist of main skeleton of 4-pyridinol with methylated polyketide side chain. Nonetheless, these metabolites show differences in their bioactive potentials against micro-organisms, insects and tumour cells. Due to its close structural similarity with coenzyme Q, piericidins also possess an inhibitory activity against NADH dehydrogenase as well as Photosystem II. This review studied the latest research progress of piericidins, covering the chemical structure and physical properties of newly identified members, bioactivities, biosynthetic pathway with gene clusters and future prospect. With the increasing incidence of drug-resistant human pathogen strains and cancers, this review aimed to provide clues for the development of either new potential antibiotics or anti-tumour agents., (© 2021 The Society for Applied Microbiology.)

Streptomyces antibiotic regulatory proteins (SARPs) are well characterized as transcriptional activators for secondary metabolites in Streptomyces species. Streptomyces rochei 7434AN4 harbors 15 SARP genes, among which 3 were located on a giant linear plasmid pSLA2-L and others were on the chromosome. Some SARP genes were cloned into an integrative thiostrepton-inducible vector pIJ8600, and their recombinants were cultivated. The recombinant of SARP gene, SRO_3163, accumulated a UV-active compound YM3163-A, which was not detected in the parent strain and other SARP recombinants. Its molecular formula was established to be C8H11NO. Extensive NMR analysis revealed that YM3163-A is a novel enamide, 2-(cyclohex-2-en-1-ylidene)acetamide, and its structure was confirmed by chemical synthesis including Horner-Wadsworth-Emmons reaction and ammonolysis., (© The Author(s) 2021. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

For over a decade, Streptomyces venezuelae has been used to study the molecular mechanisms that control morphological development in streptomycetes and is now a well-established model strain. Its rapid growth and ability to sporulate in a near-synchronised manner in liquid culture, unusual among streptomycetes, greatly facilitates the application of modern molecular techniques such as ChIP-seq and RNA-seq, as well as time-lapse fluorescence imaging of the complete Streptomyces life cycle. Here we describe a high-quality genome sequence of our isolate of the strain (Northern Regional Research Laboratory [NRRL] B-65442) consisting of an 8.2 Mb chromosome and a 158 kb plasmid, pSVJI1, which had not been reported previously. Surprisingly, while NRRL B-65442 yields green spores on MYM agar, the American Type Culture Collection (ATCC) type strain 10712 (from which NRRL B-65442 was derived) produces grey spores. While comparison of the genome sequences of the two isolates revealed almost total identity, it did reveal a single nucleotide substitution in a gene, vnz_33525, involved in spore pigment biosynthesis. Replacement of the vnz_33525 allele of ATCC 10712 with that of NRRL B-65442 resulted in green spores, explaining the discrepancy in spore pigmentation. We also applied CRISPR-Cas9 to delete the essential parB of pSVJI1 to cure the plasmid from the strain without obvious phenotypic consequences., (© The Author(s) 2021. Published by Oxford University Press on behalf of Society of Industrial Microbiology and Biotechnology.)

The Streptomyces clavuligerus genome consists in a linear chromosome of about 6.7 Mb and four plasmids (pSCL1 to pSCL4), the latter one of 1.8 Mb. Deletion of pSCL4, results in viable mutants with high instability in the chromosome arms, which may lead to chromosome circularisation. Transcriptomic and proteomic studies comparing different mutants with the wild-type strain improved our knowledge on the biosynthesis and regulation of clavulanic acid, cephamycin C and holomycin. Additional knowledge has been obtained on the SARP-type CcaR activator and the network of connections with other regulators (Brp, AreB, AdpA, BldG, RelA) controlling ccaR expression. The transcriptional pattern of the cephamycin and clavulanic acid clusters is supported by the binding of CcaR to different promoters and confirmed that ClaR is a CcaR-dependent activator that controls the late steps of clavulanic biosynthesis. Metabolomic studies allowed the detection of new metabolites produced by S. clavuligerus such as naringenin, desferroxamines, several N-acyl tunicamycins, the terpenes carveol and cuminyl alcohol or bafilomycin J. Heterologous expression of S. clavuligerus terpene synthases resulted in the formation of no less than 15 different terpenes, although none of them was detected in S. clavuligerus culture broth. In summary, application of the Omic tools results in a better understanding of the molecular biology of S. clavuligerus, that allows the use of this strain as an industrial actinobacterial platform and helps to improve CA production., (© The Author(s) 2021. Published by Oxford University Press on behalf of Society of Industrial Microbiology and Biotechnology.)

The iron-dependent regulator IdeR is the main transcriptional regulator controlling iron homeostasis genes in Actinobacteria, including species from the Corynebacterium, Mycobacterium and Streptomyces genera, as well as the erythromycin-producing bacterium Saccharopolyspora erythraea. Despite being a well-studied transcription factor since the identification of the Diphtheria toxin repressor DtxR three decades ago, the details of how IdeR proteins recognize their highly conserved 19-bp DNA target remain to be elucidated. IdeR makes few direct contacts with DNA bases in its target sequence, and we show here that these contacts are not required for target recognition. The results of our structural and mutational studies support a model wherein IdeR mainly uses an indirect readout mechanism, identifying its targets via the sequence-dependent DNA backbone structure rather than through specific contacts with the DNA bases. Furthermore, we show that IdeR efficiently recognizes a shorter palindromic sequence corresponding to a half binding site as compared to the full 19-bp target previously reported, expanding the number of potential target genes controlled by IdeR proteins., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Aim: Chemically disparate toxic organic and/or inorganic molecules produced by anthropogenic activities often hinder the bioremediation process. This research was conducted to understand the capacity of Streptomyces sp. MC1 to remove chemically disparate toxics such as Cr(VI) or phenanthrene., Methods and Results: Genomic, metabolic modeling and proteomic approaches were used in this study. Our results demonstrated that Streptomyces sp. MC1 has the genetic determinants to remove Cr(VI) or degrade phenanthrene. Proteomics showed that these genetic determinants were expressed. Metabolic versatility of the strain was confirmed by two metabolic models in complex and minimal media. Interestingly, our results also suggested a connection between the degradation of phenanthrene and synthesis of specialized metabolites., Conclusions: Streptomyces sp. MC1 has the genetic and physiological potential to remove Cr(VI) or degrade phenanthrene SIGNIFICANCE AND IMPACT OF STUDY: The probability of a microorganism to survive in the presence of different contaminants depends on its genetic potential and the ability to express it. The genetic and proteomic profiles obtained for Streptomyces sp. MC1 can be recommended as model and predict if other Streptomyces strains can be used in bioremediation processes. Our work also hypothesized that intermediates of the phenanthrene degradation serve as precursors for the specialized metabolism., (© 2021 The Society for Applied Microbiology.)

Uncialamycin (UCM) belongs to the anthraquinone-fused subfamily of 10-membered enediyne natural products that exhibits an extraordinary cytotoxicity against a wide spectrum of human cancer cell lines. Antibody-drug conjugates, utilizing synthetic analogues of UCM as payloads, are in preclinical development. UCM is exclusively produced by Streptomyces uncialis DCA2648 on solid agar medium with low titers (∼0.019 mg/l), limiting its supply by microbial fermentation and hampering its biosynthetic and engineering studies by in vivo pathway manipulation. Here, we report cultivation conditions that enable genetic manipulation of UCM biosynthesis in vivo and allow UCM production, with improved titers, by submerged fermentation of the engineered S. uncialis strains. Specifically, the titer of UCM was improved nearly 58-fold to ∼1.1 mg/l through the combination of deletion of biosynthetic gene clusters encoding unrelated metabolites from the S. uncialis wild-type, chemical mutagenesis and manipulation of pathway-specific regulators to generate the engineered S. uncialis strains, and finally medium optimization of the latter for UCM production. Genetic manipulation of UCM biosynthesis was demonstrated by inactivating selected genes in the engineered S. uncialis strains, one of which afforded a mutant strain accumulating tiancimycin B, a common biosynthetic intermediate known for the anthraquinone-fused subfamily of enediyne natural products. These findings highlight a biotechnology platform for UCM biosynthesis, engineering, and production that should facilitate both its fundamental studies and translational applications., (© The Author(s) 2021. Published by Oxford University Press on behalf of Society of Industrial Microbiology and Biotechnology.)

The cyclic lipopeptide antibiotics structurally related to daptomycin were first reported in the 1950s. Several have common lipopeptide initiation, elongation, and termination mechanisms. Initiation requires the use of a fatty acyl-AMP ligase (FAAL), a free-standing acyl carrier protein (ACP), and a specialized condensation (CIII) domain on the first NRPS elongation module to couple the long chain fatty acid to the first amino acid. Termination is carried out by a dimodular NRPS that contains a terminal thioesterase (Te) domain (CAT-CATTe). Lipopeptide BGCs also encode ABC transporters, apparently for export and resistance. The use of this mechanism of initiation, elongation, and termination, coupled with molecular target-agnostic resistance, has provided a unique basis for robust natural and experimental combinatorial biosynthesis to generate a large variety of structurally related compounds, some with altered or different antibacterial mechanisms of action. The FAAL, ACP, and dimodular NRPS genes were used as molecular beacons to identify phylogenetically related BGCs by BLASTp analysis of finished and draft genome sequences. These and other molecular beacons have identified: (i) known, but previously unsequenced lipopeptide BGCs in draft genomes; (ii) a new daptomycin family BGC in a draft genome of Streptomyces sedi; and (iii) novel lipopeptide BGCs in the finished genome of Streptomyces ambofaciens and the draft genome of Streptomyces zhaozhouensis., (© The Author(s) 2021. Published by Oxford University Press on behalf of Society of Industrial Microbiology and Biotechnology.)

Clavulanic acid (CA) produced by Streptomyces clavuligerus is a clinically important β-lactamase inhibitor. It is known that glycerol utilization can significantly improve cell growth and CA production of S. clavuligerus. We found that the industrial CA-producing S. clavuligerus strain OR generated by random mutagenesis consumes less glycerol than the wild-type strain; we then developed a mutant strain in which the glycerol utilization operon is overexpressed, as compared to the parent OR strain, through iterative random mutagenesis and reporter-guided selection. The CA production of the resulting S. clavuligerus ORUN strain was increased by approximately 31.3% (5.21 ± 0.26 g/l) in a flask culture and 17.4% (6.11 ± 0.36 g/l) in a fermenter culture, as compared to that of the starting OR strain. These results confirmed the important role of glycerol utilization in CA production and demonstrated that reporter-guided mutant selection is an efficient method for further improvement of randomly mutagenized industrial strains., (© The Author(s) 2021. Published by Oxford University Press on behalf of Society of Industrial Microbiology and Biotechnology.)

The ammosamides (AMMs) are a family of pyrroloquinoline alkaloids that exhibits a wide variety of bioactivities. A biosynthetic gene cluster (BGC) that is highly homologous in both gene content and genetic organization to the amm BGC was identified by mining the Streptomyces uncialis DCA2648 genome, leading to the discovery of a sub-family of new AMM congeners, named ammosesters (AMEs). The AMEs feature a C-4a methyl ester, differing from the C-4a amide functional group characteristic to AMMs, and exhibit modest cytotoxicity against a broad spectrum of human cancer cell lines, expanding the structure-activity relationship for the pyrroloquinoline family of natural products. Comparative analysis of the ame and amm BGCs supports the use of a scaffold peptide as an emerging paradigm for the biosynthesis of the pyrroloquinoline family of natural products. AME and AMM biosynthesis diverges from a common intermediate by evolving the pathway-specific Ame24 O-methyltransferase and Amm20 amide synthetase, respectively. These findings will surely inspire future efforts to mimic Nature's combinatorial biosynthetic strategies for natural product structural diversity., (© The Author(s) 2021. Published by Oxford University Press on behalf of Society of Industrial Microbiology and Biotechnology.)

Streptomyces species are soil-dwelling bacteria that produce vast numbers of pharmaceutically valuable secondary metabolites (SMs), such as antibiotics, immunosuppressants, antiviral, and anticancer drugs. On the other hand, the biosynthesis of most SMs remains very low due to tightly controlled regulatory networks. Both global and pathway-specific regulators are involved in the regulation of a specific SM biosynthesis in various Streptomyces species. Over the past few decades, many of these regulators have been identified and new ones are still being discovered. Among them, a global regulator of SM biosynthesis named WblA was identified in several Streptomyces species. The identification and understanding of the WblAs have greatly contributed to increasing the productivity of several Streptomyces SMs. This review summarizes the characteristics and applications on WblAs reported to date, which were found in various Streptomyces species and other actinobacteria., (© The Author(s) 2021. Published by Oxford University Press on behalf of Society of Industrial Microbiology and Biotechnology.)

Streptomyces are efficient chemists with a capacity to generate diverse and potent chemical scaffolds. The secondary metabolism of these soil-dwelling prokaryotes is stimulated upon interaction with other microbes in their complex ecosystem. We observed such an interaction when a Streptomyces isolate was cultivated in a media supplemented with dead yeast cells. Whole-genome analysis revealed that Streptomyces sp. MBK6 harbors the red cluster that is cryptic under normal environmental conditions. An interactive culture of MBK6 with dead yeast triggered the production of the red pigments metacycloprodigiosin and undecylprodigiosin. Streptomyces sp. MBK6 scavenges dead-yeast cells and preferentially grows in aggregates of sequestered yeasts within its mycelial network. We identified that the activation depends on the cluster-situated regulator, mbkZ, which may act as a cross-regulator. Cloning of this master regulator mbkZ in S. coelicolor with a constitutive promoter and promoter-deprived conditions generated different production levels of the red pigments. These surprising results were further validated by DNA-protein binding assays. The presence of the red cluster in Streptomyces sp. MBK6 provides a vivid example of horizontal gene transfer of an entire metabolic pathway followed by differential adaptation to a new environment through mutations in the receiver domain of the key regulatory protein MbkZ., (© The Author(s) 2021. Published by Oxford University Press on behalf of FEMS.)

Streptomyces incarnatus NRRL8089 produces the antiviral, antifungal, antiprotozoal nucleoside antibiotic sinefungin. To enhance sinefungin production, multiple mutations were introduced to the rpoB gene encoding RNA polymerase (RNAP) β-subunit at the target residues, D447, S453, H457, and R460. Sparse regression analysis using elastic-net lasso-ridge penalties on previously reported H457X mutations identified a numeric parameter set, which suggested that H457R/Y/F may cause production enhancement. H457R/R460C mutation successfully enhanced the sinefungin production by 3-fold, while other groups of mutations, such as D447G/R460C or D447G/H457Y, made moderate or even negative effects. To identify why the rif cluster residues have diverse effects on sinefungin production, an RNAP/DNA/mRNA complex model was constructed by homology modeling and molecular dynamics simulation. The 4 residues were located near the mRNA strand. Density functional theory-based calculation suggested that D447, H457, and R460 are in direct contact with ribonucleotide, and partially positive charges are induced by negatively charged chain of mRNA., (© The Author(s) 2021. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

A novel methymycin analog, 12-ketomethymycin N-oxide, was produced by the heterologous expression of the pikromycin/methymycin biosynthetic gene cluster of Streptomyces sp. AM4900 together with 12-ketomethymycin, which was only isolated by the biotransformation of the synthetic intermediate before. Their structures were determined by the spectroscopic data and the chemical derivatization. 12-Ketomethymycin showed a weak cytotoxicity against SKOV-3 and Jurkat cells, although its N-oxide analog did not show any activity. Both showed no antibacterial activities against Escherichia coli and Micrococcus luteus., (© The Author(s) 2020. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

In this study, strain Streptomyces sp. Act4Zk was isolated based on a method developed for the isolation of myxobacteria. Due to the low efficiency of the majority of conventional DNA extraction techniques, for molecular identification of the strain Streptomyces sp. Act4Zk, a new technique for DNA extraction of Actinobacteria was developed. In order to explore potential bioactivities of the strain, extracts of the fermented broth culture were prepared by an organic solvent (i.e. ethyl acetate) extraction method using. These ethyl acetate extracts were subjected to HPLC fractionation against standard micro-organisms, followed by LC/MS analysis. Based on morphological, physiological, biochemical and 16S rRNA gene sequence data, strain Streptomyces sp. Act4Zk is likely to be a new species of Streptomyces, close to Streptomyces genecies and Streptomyces roseolilacinus. Antimicrobial assay indicated high antifungal activity as well as antibacterial activity against Mycobacterium smegmatis and Gram-positive bacteria for the new strain. HPLC and LC/MS analyses of the extracts led to the identification of three different compounds and confirmed our hypothesis that the interesting species of the genus Streptomyces being a good producer of staurosporine and some derivatives., (© 2020 The Society for Applied Microbiology.)

Computational analysis of biosynthetic gene clusters (BGCs) has revolutionized natural product discovery by enabling the rapid investigation of secondary metabolic potential within microbial genome sequences. Grouping homologous BGCs into Gene Cluster Families (GCFs) facilitates mapping their architectural and taxonomic diversity and provides insights into the novelty of putative BGCs, through dereplication with BGCs of known function. While multiple databases exist for exploring BGCs from publicly available data, no public resources exist that focus on GCF relationships. Here, we present BiG-FAM, a database of 29,955 GCFs capturing the global diversity of 1,225,071 BGCs predicted from 209,206 publicly available microbial genomes and metagenome-assembled genomes (MAGs). The database offers rich functionalities, such as multi-criterion GCF searches, direct links to BGC databases such as antiSMASH-DB, and rapid GCF annotation of user-supplied BGCs from antiSMASH results. BiG-FAM can be accessed online at https://bigfam.bioinformatics.nl., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Increasing evidence supports the notion that different regions of a genome have unique rates of molecular change. This variation is particularly evident in bacterial genomes where previous studies have reported gene expression and essentiality tend to decrease, whereas substitution rates usually increase with increasing distance from the origin of replication. Genomic reorganization such as rearrangements occur frequently in bacteria and allow for the introduction and restructuring of genetic content, creating gradients of molecular traits along genomes. Here, we explore the interplay of these phenomena by mapping substitutions to the genomes of Escherichia coli, Bacillus subtilis, Streptomyces, and Sinorhizobium meliloti, quantifying how many substitutions have occurred at each position in the genome. Preceding work indicates that substitution rate significantly increases with distance from the origin. Using a larger sample size and accounting for genome rearrangements through ancestral reconstruction, our analysis demonstrates that the correlation between the number of substitutions and the distance from the origin of replication is significant but small and inconsistent in direction. Some replicons had a significantly decreasing trend (E. coli and the chromosome of S. meliloti), whereas others showed the opposite significant trend (B. subtilis, Streptomyces, pSymA and pSymB in S. meliloti). dN, dS, and ω were examined across all genes and there was no significant correlation between those values and distance from the origin. This study highlights the impact that genomic rearrangements and location have on molecular trends in some bacteria, illustrating the importance of considering spatial trends in molecular evolutionary analysis. Assuming that molecular trends are exclusively in one direction can be problematic., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Chromosomes are dynamic entities, whose organization and structure depend on the concerted activity of DNA-binding proteins and DNA-processing enzymes. In bacteria, chromosome replication, segregation, compaction and transcription are all occurring simultaneously, and to ensure that these processes are appropriately coordinated, all bacteria employ a mix of well-conserved and species-specific proteins. Unusually, Streptomyces bacteria have large, linear chromosomes and life cycle stages that include multigenomic filamentous hyphae and unigenomic spores. Moreover, their prolific secondary metabolism yields a wealth of bioactive natural products. These different life cycle stages are associated with profound changes in nucleoid structure and chromosome compaction, and require distinct repertoires of architectural-and regulatory-proteins. To date, chromosome organization is best understood during Streptomyces sporulation, when chromosome segregation and condensation are most evident, and these processes are coordinated with synchronous rounds of cell division. Advances are, however, now being made in understanding how chromosome organization is achieved in multigenomic hyphal compartments, in defining the functional and regulatory interplay between different architectural elements, and in appreciating the transcriptional control exerted by these 'structural' proteins., (© FEMS 2020.)

The gram-positive bacterium, Streptomyces, is noticed for its ability to produce a wide array of pharmaceutically active compounds through secondary metabolism. To discover novel bioactive secondary metabolites and increase the production, Streptomyces species have been extensively studied for the past decades. Among the cellular components, RNA molecules play important roles as the messengers for gene expression and diverse regulations taking place at the RNA level. Thus, the analysis of RNA-level regulation is critical to understanding the regulation of Streptomyces' metabolism and secondary metabolite production. A dramatic advance in Streptomyces research was made recently, by exploiting high-throughput technology to systematically understand RNA levels. In this review, we describe the current status of the system-wide investigation of Streptomyces in terms of RNA, toward expansion of its genetic potential for secondary metabolite synthesis.

Aim: The aim of the present work was to investigate the overexpression of the wysR gene in Streptomyces albulus var. wuyiensis strain CK-15 based on the ΔwysR3 mutant strain including the effect on morphological development, wuyiencin production and antibacterial activity. At the same time, we report a new rapid method for producing genetically engineered strains for industrial production of wuyiencin., Methods and Results: We developed a method to create a wysR overexpression strain based on the ΔwysR3 mutant strain by direct transformation. In this method, the desired gene fragment to be overexpressed was amplified by polymerase chain reaction (PCR) using Phusion High Fidelity DNA polymerase and fused with the linearized pSETC integrative plasmid by Gibson assembly. The resulting recombinant plasmid was transformed into ΔwysR3 mutant strain by the intergeneric conjugation method. The plasmid was then integrated into the chromosome and the resulting apramycin-resistant overexpression strain was confirmed by PCR using the Apra-F and Apra-R primers. Finally, we successfully screened the genetically engineered strain with overexpression of wysR gene in ΔwysR3 mutant., Conclusion: We can conclude that overexpression of wysR gene in ΔwysR3 mutant strain proved to be an effective strategy for significantly increasing wuyiencin production together with faster morphological development. Quantitative real-time RT-PCR analysis showed that wysR regulated wuyiencin biosynthesis by modulating other putative regulatory genes and bld, whi, chp, rdl and ram family genes are crucial for the morphological development., Significance and Impact of the Study: Overexpression of wysR gene in the ΔwysR3 mutant strain named OoWysR strain may increase the efficiency in the industrial fermentation processes for wuyiencin production. The mechanism by which wysR overexpression promotes rapid sporulation and a high yield of wuyiencin production is likely related to modulation of other putative regulatory genes., (© 2020 The Society for Applied Microbiology.)

Quorum-sensing (QS) mediated dynamic regulation has emerged as an effective strategy for optimizing product titers in microbes. However, these QS-based circuits are often created on heterologous systems and require careful tuning via a tedious testing/optimization process. This hampers their application in industrial microbes. Here, we design a novel QS circuit by directly integrating an endogenous QS system with CRISPRi (named EQCi) in the industrial rapamycin-producing strain Streptomyces rapamycinicus. EQCi combines the advantages of both the QS system and CRISPRi to enable tunable, autonomous, and dynamic regulation of multiple targets simultaneously. Using EQCi, we separately downregulate three key nodes in essential pathways to divert metabolic flux towards rapamycin biosynthesis and significantly increase its titers. Further application of EQCi to simultaneously regulate these three key nodes with fine-tuned repression strength boosts the rapamycin titer by ∼660%, achieving the highest reported titer (1836 ± 191 mg/l). Notably, compared to static engineering strategies, which result in growth arrest and suboptimal rapamycin titers, EQCi-based regulation substantially promotes rapamycin titers without affecting cell growth, indicating that it can achieve a trade-off between essential pathways and product synthesis. Collectively, this study provides a convenient and effective strategy for strain improvement and shows potential for application in other industrial microorganisms., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Aim: To evaluate the antimicrobial potential of Juglomycins isolated from Streptomyces achromogenes E91CS4, an endophyte of Crocus sativus Linn., Methods and Results: The extract from E91CS4 displayed significant antimicrobial activity against several pathogens. The endophyte was identified as S. achromogenes on by 16S ribosomal gene analysis. Chemical investigation of the extract led to the isolation of two naphthoquinone antibiotics, Juglomycin A and B. Juglomycin A inhibited several pathogens, with an MIC value of 13·7µg ml -1 , whereas it was most potent against Escherichia coli, Bacillus thuringiensis and Xanthobacter flavus with MIC values of 6·8, 3·4 and 6·8 µg ml -1 respectively. It was found to reduce the biofilm formation in E. coli through inhibition of swimming and swarming motilities and downregulation of fimH gene. The α-haemolysin-related gene (hlyA) was also downregulated indicating that the compound is also reducing the virulence in E. coli. In vitro time kill kinetics showed efficient bactericidal activity of this compound. Furthermore, Juglomycin A inhibited bacterial transcription/translation in vitro, while also inducing postantibiotic effect in E. coli., Conclusions: Juglomycin A is a potential antimicrobial compound against several bacterial pathogens, particularly, E. coli., Significance and Impact of the Study: This study showed the promising potential of Juglomycin A as an antimicrobial agent. Efforts should be made to scale up the production of this compound and conduct further studies to explore its efficacy as an antibiotic, using in vivo models., (© 2019 The Society for Applied Microbiology.)