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Historically, small molecules biosynthesised by bacteria have been an excellent source for antibacterial drugs. Today, however, the rediscovery of known compounds is a significant hurdle to developing new antimicrobials. Here we use a genome mining and synthetic biology approach to discover the ambocidins: calcium-dependent lipodepsipeptides that are active against drug-resistant Gram-positive pathogens. By cloning a silent biosynthetic gene cluster (the amb cluster) from Streptomyces ambofaciens ATCC 2387 and integrating this into the chromosome of Streptomyces avermitilis we induce expression of ambocidin A and B: two new N ϵ -hydroxyarginine-containing cyclic lipodepsipeptides active against drug-resistant Gram-positive pathogens. Using a panel of Streptomyces host strains, we show that the choice of heterologous host is critical for producing the biologically active compounds, and that inappropriate host choice leads to aberrant production inactive derivatives. We show that N ϵ -hydroxyarginine is the product of a heme-dependent oxygenase and that it enhances biological activity. Ambocidin A inhibits cell wall biosynthesis by binding to Lipid II at a different site than vancomycin. Furthermore, unlike daptomycin, ambocidin A retains potent antimicrobial activity in the presence of lung surfactant, giving it the potential to treat bacterial pneumonia. Our work expands the family of calcium-dependent lipopeptide antibiotics with a new member exhibiting a distinct mechanism of action., (© 2024 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Carbapenem resistance among bacteria, especially Klebsiella pneumoniae and Acinetobacter baumannii, constitutes a dreadful threat to public health all over the world that requires developing new medications urgently. Carbapenem resistance emerges as a serious problem as this class is used as a last-line option to clear the multidrug-resistant bacteria. Arthrospira maxima (Spirulina) is a well-known cyanobacterium used as a food supplement as it is rich in protein, essential minerals and vitamins and previous studies showed it may have some antimicrobial activity against different organisms. Biosynthesized (green) zinc oxide nanoparticles have been investigated by several researchers as antibacterials because of their safety in health. In this article, previous studies were analyzed to get to a conclusion about their activity as antibacterials., Competing Interests: Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

The species Bacillus velezensis is known for its biosynthetic potential and metabolic versatility in producing several secondary metabolites and promoting plant growth. In this study, we isolated two B. velezensis strains, WGTg-8 and WGTm-299, from the termite gut and termitarium, which exhibited antimicrobial activity against multiple clinical and phytopathogens. The whole genomes of these strains were sequenced using the Illumina platform and annotated. The genome mining of the draft genome sequences revealed 48 biological gene clusters (BGCs) responsible for synthesizing various secondary metabolites. The construction of the similarity network of the BGCs and the comparative analysis with the genetically related organisms are aided in the identification of metabolites produced by these strains. We identified biosynthetic gene clusters (BGCs) coding for macrolactin H, bacilysin, bacillibactin, amylocyclin, comX4, and LCI, found in both strains with 100% similarity. The difficidin, bacillaene, thusin_alpha, and cericidin BGCs are exclusively found in strain WGTg-8, while the colicin BGC is exclusively present in the WGTm-299 strain. The fengycin and surfactin gene clusters are present in both strains with 80% similarity. Furthermore, 28 putative NRPS BGCs, NRPS-T1PKS hybrid clusters, a T1PKS, and a bacteriocin BGC were identified with very low similarity (≤ 25%) or no similarity with known antibiotics. In addition, we found several genes coding for plant growth-promoting properties, including nitrogen metabolism, hormone synthesis, sulfur metabolism, phosphate metabolism, and a few other properties., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Lipopeptides are an important family of natural products, some of which are clinically used as antibiotics to treat multidrug-resistant pathogens. Although the lipid moieties play a crucial role in balancing antibacterial activity and hemolytic toxicity, modifying the lipid moieties has been challenging due to the complexity of the lipidation process in lipopeptide biosynthesis. Here, we show that the lipid profile can be altered by engineering both secondary and primary metabolisms, using daptomycin as an example. First, swapping the fatty acyl AMP ligase (FAAL) gene dptF with foreign FAAL homologs improved the fatty acyl specificity of the lipidation process for decanoic acid. Then, the introduction of Mycobacterium type I fatty acid synthase operon (MvFAS-Ib/MvAcpS) and Cryptosporidium thioesterase (CpTEII) enriched the fatty acid pool with decanoic acid in Streptomyces roseosporus . The engineered fatty acid metabolism eliminates the need for external decanoic acid supplementation by enabling S. roseosporus to biosynthesize decanoic acid. By complete engineering of the lipidation process, we achieved, for the first time, high-purity, natural production of daptomycin. The lipidation engineering approach we demonstrate here lays the foundation for the lipidation control in lipopeptide biosynthesis.

Bacterial infections present a major global health threat, often displaying resistance to various antibiotics. Lipoteichoic acid (LTA) is a vital component of bacterial cell envelopes of Gram-positive bacteria, crucial for cell integrity, cell division, and host inflammation. Due to its essential role for bacteria, LTA and its biosynthesis are also attractive drug targets, however, there is only scant molecular knowledge on LTA and its precursor molecules in membranes. Here, we report the isolation and molecular characterization of diglucosyldiacylglycerol (Glc 2 -DAG), the glycolipid precursor molecule that anchors LTA in the bacterial plasma-membrane. Using a tailored growth medium and purification protocols, we isolated 13 C-isotope labelled Glc 2 -DAG from bacteria, which can then be used for high-resolution NMR studies. Using solution-state and solid-state NMR, we show an in-depth molecular characterization of Glc 2 -DAG, including in native-like membranes. Our approach may help to identify antibiotics that directly target LTA precursor molecules, and it offers a tool for future investigations into the role of Glc 2 -DAG in bacterial physiology., (© 2024 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

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.)

Daptomycin (DAP), a novel cyclic lipopeptide antibiotic produced by Streptomyces roseosporus, is clinically important for treatment of infections caused by multidrug-resistant Gram-positive pathogens, but the low yield hampers its large-scale industrial production. Here, we describe a combination metabolic engineering strategy for constructing a DAP high-yielding strain. Initially, we enhanced aspartate (Asp) precursor supply in S. roseosporus wild-type (WT) strain by separately inhibiting Asp degradation and competitive pathway genes using CRISPRi and overexpressing Asp synthetic pathway genes using strong promoter kasOp*. The resulting strains all showed increased DAP titre. Combined inhibition of acsA4, pta, pyrB, and pyrC increased DAP titre to 167.4 μg/mL (73.5% higher than WT value). Co-overexpression of aspC, gdhA, ppc, and ecaA led to DAP titre 168 μg/mL (75.7% higher than WT value). Concurrently, we constructed a chassis strain favourable for DAP production by abolishing by-product production (i.e., deleting a 21.1 kb region of the red pigment biosynthetic gene cluster (BGC)) and engineering the DAP BGC (i.e., replacing its native dptEp with kasOp*). Titre for the resulting chassis strain reached 185.8 μg/mL. Application of our Asp precursor supply strategies to the chassis strain further increased DAP titre to 302 μg/mL (2.1-fold higher than WT value). Subsequently, we cloned the engineered DAP BGC and duplicated it in the chassis strain, leading to DAP titre 274.6 μg/mL. The above strategies, in combination, resulted in maximal DAP titre 350.7 μg/mL (2.6-fold higher than WT value), representing the highest reported DAP titre in shake-flask fermentation. These findings provide an efficient combination strategy for increasing DAP production and can also be readily applied in the overproduction of other Asp-related antibiotics., (© 2024 The Author(s). Microbial Biotechnology published by John Wiley & Sons Ltd.)

The genus Serratia is a typical red bacterium involved in prodigiosin synthesis. Here, we report the genome sequence of Serratia rubidaea XU1, which was isolated from radiation-contaminated soil in Xinjiang, China. The genome of XU1 is composed of 4,972,898 base pairs with a GC content of 59.25%. The genome sequence contains 4707 genes and encodes 4573 proteins, 79 tRNAs, and 17 rRNAs. The prodigiosin biosynthesis gene cluster was identified and analyzed, showing a sequence similarity of 85.55-96.02% with Serratia rubidaea. After optimizing the biosynthesis process, XU1 was able to achieve a maximum titer of 574 units/cell of prodigiosin at a pH of 7.5 and a temperature of 25 °C for 36 h. Glycerol at 20 g/L and beef extract at 5 g/L were used as the carbon and nitrogen sources, respectively. Prodigiosin extracted from XU1 demonstrated inhibition of Escherichia coli, Staphylococcus aureus, Enterococcus faecalis and Pseudomonas aeruginosa. The availability of the sequenced genome of XU1 will be greatly beneficial and contribute to complementary studies on the biosynthetic mechanisms of prodigiosin., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Bacteriocin is a kind of natural substance that can effectively inhibit bacteria, but its production usually limited by environment. Co-culture is a strategy to stimulate bacteriocin production. Bifidocin A produced by Bifidobacterium animalis BB04, is a novel bacteriocin with a broad-spectrum antimicrobial active of foodborne bacteria. In order to enhance bifidocin A production, bacteriocin-inducing strains were screened firstly in co-cultivation. Then, the molecular mechanism of co-cultural induction was investigated by transcriptomic and proteomic analysis. Finally, the key inducing metabolites were identified by using targeted metabolomic technology. The results showed that Wickerhamomyces anomalus Y-5 in co-cultivation could significantly enhance bifidocin A production, with a 3.00-fold increase compared to mono-culture. The induction may not depend on direct contact with cells and may instead be attributed to be continuous exposure to inducing substances at specific concentration. In co-cultivation, W. anomalus Y-5 up-regulated Hxk2 and Tap42 to activate Glucose-cAMP and Tor and HOG-MAPK pathway, stimulated the expression of the retrograde gene, produced glutamine and glycerol to maintain activity. During this process, glutamine, inosine, guanosine, adenine, uracil, fumaric acid and pyruvic acid produced by W. anomalus Y-5 could induce the synthesis of bifidocin A. In conclusion, W. anomalus Y-5 in co-cultivation induced the synthesis of bifidocin A by regulating various signaling pathways to produce inducing substances. These findings establish a foundation for high-efficient synthesis of bifidocin A and provide a new perspective into the industrial production of bacteriocin., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Genome analysis of Kutzneria sp. CA-103260 revealed a putative lipopeptide-encoding biosynthetic gene cluster (BGC) that was cloned into a bacterial artificial chromosome (BAC) and heterologously expressed in Streptomyces coelicolor M1152. As a result, a novel cyclic lipo-tetrapeptide containing two diaminopropionic acid residues and an exotic N , N -acetonide ring, kutzneridine A ( 1 ), was isolated and structurally characterized. Evaluation of the extraction conditions and isotope-labeling chemical modifications showed that the acetonide ring originated from acetone during isolation. The BGC was analyzed in silico and a biosynthetic pathway to 1 was proposed. Kutzneridine A displayed remarkable antibacterial activity against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococci .

Lanthipeptides, a group of ribosomally synthesized and post-translationally modified peptides (RiPPs), exhibit diverse structures and bioactivities. Their biosynthetic enzymes serve as valuable tools for peptide bioengineering. Here, we report a class II lanthipeptide biosynthetic gene cluster in a Bacillus strain, driving the biosynthesis of a two-component lanthipeptide, termed rodencin, featured by the presence of two different d-amino acids, i.e., d-Ala and d-Abu. Rodencin displays synergistic antimicrobial activity against food-borne pathogens such as Bacillus cereus , Staphylococcus aureus , and Listeria monocytogenes . The α-peptide of rodencin contains one d-Ala and the β-peptide features both d-Ala and d-Abu. These are installed by dehydratases RodM1 and RodM2 and dehydrogenase RodJ A , the activities of which were successfully reconstituted using a dedicated E. coli expression system. To illustrate the unusual d-Abu incorporation potential of the enzymes, analogous to the d-amino acid-containing β peptide of lacticin 3147, was successfully produced with the rodencin heterologous expression system, by employing RodM2 and the dehydrogenase RodJ A .

Bacterial cellulose (BC) is a highly versatile biopolymer renowned for its exceptional mechanical strength, water retention, and biocompatibility. These properties make it a valuable material for various industrial and biomedical applications. In this study, Enterococcus faecalis synthesized extracellular BC, utilizing Phoenix dactylifera and Musa acuminata fruit extracts as sustainable carbon sources. LC-MS analysis identified glucose as the primary carbohydrate in these extracts, providing a suitable substrate for BC production. Scanning Electron Microscopy (SEM) revealed a network of BC nanofibers on Congo red agar plates. ATR-FTIR spectroscopy confirmed the presence of characteristic cellulose functional groups, further supporting BC synthesis. X-ray diffraction (XRD) analysis indicated a high crystallinity index of 71%, consistent with the cellulose I structure, as evidenced by peaks at 16.22°, 21.46°, 22.52°, and 34.70°. Whole-genome sequencing of E. faecalis identified vital genes involved in BC biosynthesis, including bcsA, bcsB, diguanylate cyclase (DGC), and 6-phosphofructokinase (pfkA). Antibiotic susceptibility tests revealed resistance to cefotaxime, ceftazidime, and ceftriaxone, while susceptibility to imipenem was observed. Quantitative assessment demonstrated that higher concentrations of fruit extracts (5.0-20 mg/mL) significantly enhanced BC production. Cytotoxicity testing via the MTT assay confirmed excellent biocompatibility with NIH/3T3 fibroblast cells, showing high cell viability (97-105%). Unlike commonly studied Gram-negative bacteria like Acetobacter xylinum for BC production, this research focuses on Gram-positive Enterococcus faecalis and utilizes Phoenix dactylifera and Musa acuminata fruit extracts as carbon sources. This approach offers a sustainable and promising avenue for BC production., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Since a transcriptional regulator has yet to be identified within the tunicamycin biosynthetic gene cluster in Streptomyces clavuligerus, we conducted a comprehensive investigation by focusing on the possible function of the pleiotropic regulator AdpA on tunicamycin. The genes encoding early steps of tunicamycin biosynthesis were significantly upregulated in S. clavuligerus ΔadpA. At the same time, they were downregulated in adpA overexpressed strain as shown by RNA-sequencing (RNA-seq) and reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) analysis. The tunicamycin gene cluster's co-transcription pattern was understood by reverse transcriptase polymerase chain reaction (RT-PCR). Our Electrophoretic Mobility Shift Assay (EMSA) data clearly showed AdpA's binding to the upstream sequence of the tunA gene, asserting its regulatory control. In addition to its direct negative regulation of tunicamycin biosynthesis, AdpA operates at a global level by orchestrating various regulatory genes in S. clavuligerus, such as wblA, whiB, bldM, arpA, brp, and adsA involved in morphological differentiation and secondary metabolite biosynthesis as depicted in RNA-seq data. This study represents a significant milestone by unveiling the AdpA regulator's pathway-specific and global regulatory effect in S. clavuligerus., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Background: Marine actinomycetes, especially Streptomyces, are recognized as excellent producers of diverse and bioactive secondary metabolites on account of the multiplicity of marine habitations and unique ecological conditions, which are yet to be explored in terms of taxonomy, ecology, and functional activity. Isolation, culture and genome analysis of novel species of Streptomyces to explore their potential for discovering bioactive compounds is an important approach in natural product research., Results: A marine actinobacteria, designated strain SCSIO 75703  T , was isolated, and the potential for bioactive natural product discovery was evaluated based on genome mining, compound detection, and antimicrobial activity assays. The phylogenetic, phenotypic and chemotaxonomic analyses indicate that strain SCSIO 75703  T represents a novel species in genus Streptomyces, for which the name Streptomyces sediminicola sp. nov. is proposed. Genome analysis revealed the presence of 25 secondary metabolite biosynthetic gene clusters. The screening for antibacterial activity reveals the potential to produce bioactive metabolites, highlighting its value for in-depth exploration of chemical constituents. Seven compounds (1-7) were separated from the fractions guided by antibacterial activities, including three indole alkaloids (1-3), three polyketide derivatives (4-6), and 4-(dimethylamino)benzoic acid (7). These primarily antibacterial components were identified as anthracimycin (4), 2-epi-anthracimycin (5) and β-rubromycin (6), presenting strong antibacterial activities against Gram-positive bacteria with the MIC value ranged from 0.125 to 16 μg/mL. Additionally,, monaprenylindole A (1) and 3-cyanomethyl-6-prenylindole (2) displayed moderate inhibitory activities against α-glucosidase with the IC 50 values of 83.27 and 86.21 μg/mL, respectively., Conclusion: Strain SCSIO 75703  T was isolated from marine sediment and identified as a novel species within the genus Streptomyces. Based on genomic analysis, compounds isolation and bioactivity studies, seven compounds were identified, with anthracimycin and β-rubromycin showing significant biological activity and promising potential for further applications., (© 2024. The Author(s).)

We report the identification of 3,6-dihydroxy-1,2-benzisoxazole (DHB) in a screen of Photorhabdus and Xenorhabdus , whose symbiotic relationship with eukaryotic nematodes favors secondary metabolites that meet several requirements matching those for clinically useful antibiotics. DHB is produced by Photorhabdus laumondii and is selective against the Gram-negative species Escherichia coli, Enterobacter cloacae, Serratia marcescens, Klebsiella pneumoniae, Proteus mirabilis, and Acinetobacter baumannii . It is inactive against anaerobic gut bacteria and nontoxic to human cells. Mutants resistant to DHB map to the ubiquinone biosynthesis pathway. DHB binds to 4-hydroxybenzoate octaprenyltransferase (UbiA) and prevents the formation of 4-hydroxy-3-octaprenylbenzoate. Remarkably, DHB itself is prenylated, forming an unusable chimeric product that likely contributes to the toxic effect of this antimicrobial. DHB appears to be both a competitive enzyme inhibitor and a prodrug; this dual mode of action is unusual for an antimicrobial compound., Importance: The spread of resistant pathogens has led to the antimicrobial resistance crisis, and the need for new compounds acting against Gram-negative pathogens is especially acute. From a screen of Photorhabdus symbionts of nematodes, we identified 3,6-dihydroxy-1,2-benzisoxazole (DHB) that acts against a range of Gram-negative bacteria, including Escherichia coli , Enterobacter cloacae , Klebsiella pneumoniae , and Acinetobacter baumannii . DHB had previously been isolated from other bacterial species, but its mechanism of action remained unknown. We show that DHB is unique among antimicrobials, with dual action as an inhibitor of an important enzyme, UbiA, in the biosynthesis pathway of ubiquinone and as a prodrug. DHB is a mimic of the natural substrate, and UbiA modifies it into a toxic product, contributing to the antimicrobial action of this unusual antibiotic. We also uncover the mechanism of DHB selectivity, which depends on a particular fold of the UbiA enzyme., Competing Interests: The authors declare no conflict of interest.

The genus Pseudomonas is a prolific source of specialized metabolites with significant biological activities, including siderophores, antibiotics, and plant hormones. These molecules play pivotal roles in environmental interactions, influencing pathogenicity, inhibiting microorganisms, responding to nutrient limitation and abiotic challenges, and regulating plant growth. These properties mean that pseudomonads are suitable candidates as biological control agents against plant pathogens. Multiple transposon-based screens have identified a Pseudomonas biosynthetic gene cluster (BGC) associated with potent antibacterial and antifungal activities, which produces 7-hydroxytropolone (7-HT). In this study, we show that this BGC also makes 3,7-dihydroxytropolone (3,7-dHT), which has strong antimicrobial activity toward Streptomyces scabies , a potato pathogen. Through metabolomics and reporter assays, we unveil the involvement of cluster-situated genes in generating phenylacetyl-coenzyme A, a key precursor for tropolone biosynthesis via the phenylacetic acid catabolon. The clustering of these phenylacetic acid genes within tropolone BGCs is unusual in other Gram-negative bacteria. Our findings support the interception of phenylacetic acid catabolism via an enoyl-CoA dehydratase encoded in the BGC, as well as highlighting an essential role for a conserved thioesterase in biosynthesis. Biochemical assays were used to show that this thioesterase functions after a dehydrogenation-epoxidation step catalyzed by a flavoprotein. We use this information to identify diverse uncharacterized BGCs that encode proteins with homology to flavoproteins and thioesterases involved in tropolone biosynthesis. This study provides insights into tropolone biosynthesis in Pseudomonas , laying the foundation for further investigations into the ecological role of tropolone production.IMPORTANCE Pseudomonas bacteria produce various potent chemicals that influence interactions in nature, such as metal-binding molecules, antibiotics, or plant hormones. This ability to synthesize bioactive molecules means that Pseudomonas bacteria may be useful as biological control agents to protect plants from agricultural pathogens, as well as a source of antibiotic candidates. We have identified a plant-associated Pseudomonas strain that can produce 3,7-dihydroxytropolone, which has broad biological activity and can inhibit the growth of Streptomyces scabies , a bacterium that causes potato scab. Following the identification of this molecule, we used a combination of genetic, chemical, and biochemical experiments to identify key steps in the production of tropolones in Pseudomonas species. Understanding this biosynthetic process led to the discovery of an array of diverse pathways that we predict will produce new tropolone-like molecules. This work should also help us shed light on the natural function of antibiotics in nature., Competing Interests: The authors declare no conflict of interest.

Starmerella bombicola is a native yeast strain producing sophorolipids as secondary metabolites. This study explores the production, characterization, and biological activities of sophorolipids and investigates the antimicrobial, anti-biofilm, and antifungal properties of sophorolipids produced from oil refinery wastes by the yeast Starmerella bombicola. The present work demonstrated that S. bombicola MTCC 1910 when grown in oil refinery wastes namely palm fatty acid distillates and soy fatty acid distillates enhanced the rate of sophorolipids production drastically in comparison to vegetable oil, sunflower oil used as hydrophobic feedstock. Sophorolipid yields were 18.14, 37.21, and 46.1 g/L with sunflower oil, palm, and soy fatty acid distillates respectively. The crude biosurfactants were characterized using TLC, FTIR, and HPLC revealing to be acetylated sophorolipids containing both the acidic and lactonic isomeric forms. The surface lowering and emulsifying properties of the sophorolipids from refinery wastes were significantly higher than the sunflower oil-derived sophorolipids. Also, all the sophorolipids exhibited strong antibacterial properties (minimum inhibitory concentrations were between 50 and 200 µg mL -1 ) against Salmonella typhimurium, Bacillus cereus, and Staphylococcus epidermidis and were validated with morphological analysis by Scanning electron microscopy. All the sophorolipids were potent biofilm inhibitors and eradicators (minimum biofilm inhibitory and eradication concentrations were between 12.5 to 1000 µg mL -1 ) for all the tested organisms. Furthermore, antifungal activities were also found to exhibit about 16-56% inhibition at 1 mg mL -1 for fungal mycelial growth. Therefore, this endeavour of sophorolipids production using palm and soy fatty acid distillates not only opens up a window for the bioconversion of industrial wastes into productive biosurfactants but also concludes that sophorolipids from oil refinery wastes are potent antimicrobial, anti-biofilm, and antifungal agents, highlighting their potential in biotechnological and medical applications., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

One of the major global health threats in the present era is antibiotic resistance. Biosynthesized iron oxide nanoparticles (FeNPs) can combat microbial infections and can be synthesized without harmful chemicals. In the present investigation, 16S rRNA gene sequencing was used to discover Streptomyces sp. SMGL39, an actinomycete isolate utilized to reduce ferrous sulfate heptahydrate (FeSO 4 .7H 2 O) to biosynthesize FeNPs, which were then characterized using UV-Vis, XRD, FTIR, and TEM analyses. Furthermore, in our current study, the biosynthesized FeNPs were tested for antimicrobial and antibiofilm characteristics against different Gram-negative, Gram-positive, and fungal strains. Additionally, our work examines the biosynthesized FeNPs' molecular docking and binding affinity to key enzymes, which contributed to bacterial infection cooperation via quorum sensing (QS) processes. A bright yellow to dark brown color shift indicated the production of FeNPs, which have polydispersed forms with particle sizes ranging from 80 to 180 nm and UV absorbance ranging from 220 to 280 nm. Biosynthesized FeNPs from actinobacteria significantly reduced the microbial growth of Fusarium oxysporum and L. monocytogenes , while they showed weak antimicrobial activity against P. aeruginosa and no activity against E. coli , MRSA , or Aspergillus niger . On the other hand, biosynthesized FeNPs showed strong antibiofilm activity against P. aeruginosa while showing mild and weak activity against B. subtilis and E. coli, respectively. The collaboration of biosynthesized FeNPs and key enzymes for bacterial infection exhibits hydrophobic and/or hydrogen bonding, according to this research. These results show that actinobacteria-biosynthesized FeNPs prevent biofilm development in bacteria.

The growth of material science and technology places high importance on creating better processes for synthesizing copper nanoparticles. Thus, an easy, ecological, and benign process for producing copper nanoparticles (CuNPs) has been developed using Priestia sp. bacteria utilizing a variety of low-cost agro-industrial wastes and byproducts. The biosynthesis of CuNPs was conducted using glucose medium and copper ions salt solution, then it was replaced by utilizing low-cost agro-industrial wastes. UV-visible spectroscopy, dynamic light scattering (DLS), X-ray diffraction (XRD), High-resolution transmission electron microscope (HR-TEM), Attenuated Total Reflectance and Fourier transform infrared (ATR-FTIR), and zeta potential were used to characterize the biosynthesized CuNPs. The cytotoxicity of CuNPs using Vero -CCL-81 cell lines, and antibacterial and antitumor properties using human colon epithelial colorectal adenocarcinoma Caco-2-HTB-37 cell lines were assessed. The UV-visible and DLS studies revealed CuNPs formation, with a maximum concentration of 6.19 ppm after 48 h, as indicated by a 0.58 Surface plasmon resonance (SPR) within 450 nm and 57.73 nm particle size. The 16S rRNA gene analysis revealed that Priestia sp. isolate is closely related to Priestia megaterium and has been deposited in the NCBI GenBank with accession number AMD 2024. The biosynthesis with various agro-industrial wastes indicated blackstrap sugar cane molasses being the most effective for reducing CuNPs size to 3.12 nm owing to various reducing and stabilizing active compounds. The CuNPs were free of contaminants, with a sphere-shaped structure and a cytotoxicity assessment with an IC 50 of 367.27 μg/mL. The antibacterial activity exhibited by the most susceptible bacteria were Bacillus cereus ATCC 11788 and Staphylococcus aureus ATCC 6538 with inhibition zones of 26.0 mm and 28.0 mm, respectively. The antitumor effect showed an IC 50 dose of 175.36 μg/mL. Based on the findings, the current work sought to lower product costs and provide a practical solution to the environmental contamination issues brought on by the buildup of agricultural wastes. In addition, the obtained CuNPs could be applied in many fields such as pharmaceuticals, water purification, and agricultural applications as future aspects., (© 2024. The Author(s).)

Modification of the N- and C-termini of peptides enhances their stability against degradation by exopeptidases. The biosynthetic pathways of many peptidic natural products feature enzymatic modification of their termini, and these enzymes may represent a valuable pool of biocatalysts. The lantibiotic cacaoidin carries an N , N -dimethylated N-terminal amine group. Its biosynthetic gene cluster encodes the putative methyltransferase Cao4. In this work, we present reconstitution of the activity of the enzyme, which we termed CaoS C following standardized lanthipeptide nomenclature, using a heterologously produced peptide as the model substrate. In vitro methylation of diverse lanthipeptides revealed the substrate requirements of CaoS C . The enzyme accepts peptides of varying lengths and C-terminal sequences but requires dehydroalanine or dehydrobutyrine at the second position. CaoS C -mediated dimethylation of natural lantibiotics resulted in modestly enhanced antimicrobial activity of the lantibiotic haloduracin compared to that of the native compound. Improved activity and/or metabolic stability as a result of methylation illustrates the potential future application of CaoS C in the bioengineering of therapeutic peptides.

Bacteriocins are antimicrobial compounds that have awakened interest across several industries due to their effectiveness. However, their large-scale production often becomes unfeasible on an industrial scale, primarily because of high process costs. Addressing this challenge, this work analyzes the potential of using low-cost whey permeate powder, without any supplementation, to produce bacteriocin-like inhibitory substances (BLIS) through the fermentation of Latilactobacillus sakei. For this purpose, different concentrations of whey permeate powder (55.15 gL -1 , 41.3 gL -1 and 27.5 gL -1 ) were used. The ability of L. sakei to produce BLIS was evaluated, as well as the potential of crude cell-free supernatant to act as a preservative. Raman spectroscopy and surface-enhanced Raman scattering (SERS) provided detailed insights into the composition and changes occurring during fermentation. SERS, in particular, enhanced peak definition significantly, allowing for the identification of key components, such as lactose, proteins, and phenylalanine, which are crucial in understanding the fermentation process and BLIS characteristics. The results revealed that the concentration of 55.15 gL -1 of whey permeate powder, in flasks without agitation and a culture temperature of 32.5 °C, presented the highest biological activity of BLIS, reaching 99% of inhibition of Escherichia coli and Staphylococcus aureus with minimum inhibitory concentration of 36-45%, respectively. BLIS production began within 60 h of cultivation and was associated with class II bacteriocins. The results demonstrate a promising approach for producing BLIS in an economical and environmentally sustainable manner, with potential implications for various industries., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Small pentacyclic peptides, represented by nisin, have been successfully utilized as preservatives in the food industry and have evolved into a paradigm for understanding the genetic structure, expression, and control of genes created by lantibiotics. Due to the ever-increasing antibiotic resistance, nisin-relevant antimicrobial peptides have received much attention, which calls for a summarization of their synthesis, modification and applications. In this review, we first provided a timeline of select highlights in nisin biosynthesis and engineering. Then, we outlined the current developments in nisin synthesis. We also provided an overview of the engineering, screening, and production of nisin-relevant antimicrobial peptides based on enzyme alteration, substrate modification, and sequence mining. Furthermore, an updated summary of applications of nisin-relevant antimicrobial peptides has been developed for food applications. Finally, this study offers insights into emerging technologies, limitations and the future development of nisin-relevant antimicrobial peptides for pathogen inhibition, food preservatives, and improved health.

The bacterial stringent response is a global regulatory process in which polyphosphate kinase (Ppk) and lon protease are important players. Previous studies have shown that overexpression of the lon gene and deletion of the ppk gene significantly increased actinorhodin production in Streptomyces coelicolor (SCO). In this study, a recombinant SCOΔppk-lon cell, expressing the extra lon gene in Δppk cells, was simulated using a modified in silico (computational) model, ecSco-GEM, and the negative effect of Ppk on actinorhodin production was confirmed. In addition, we identified key enzymes that play a positive role in actinorhodin production. Of these, NADH dehydrogenase/complex-I, beta-ketoacyl-[acyl-carrier-protein] synthase III, glycine cleavage system, and superoxide dismutase were identified as the most significant. By confirming these results with experiments, we have shown that GEMs can be a reliable starting point for in vitro (lab-based) studies of Streptomyces.., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Multidrug resistance in clinical pathogens is a significant challenge in healthcare, requiring the development of novel approaches to combat infections. In this study, we report the identification of novel antimicrobial biosynthetic gene clusters from Brevibacillus parabrevis WGTm-23, the bacterial strain isolated from a termitarium. This strain showed an antagonistic effect against drug-resistant clinical pathogens, such as Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella paratyphi, Streptococcus gordonii, and enteropathogenic Escherichia coli. The whole genome of this strain was sequenced using the Illumina platform. The genome mining revealed a total of 17 biosynthetic gene clusters (BGCs) responsible for the synthesis of secondary metabolites. The metabolites produced by this strain were predicted by constructing an identity network of the BGCs and performing a comparative analysis with genetically related strains. The genome contains multiple BGCs coding for ribosomally synthesized and post-translationally modified peptides (RiPPs). In the genome of Br. parabrevis WGTm-23, we identified BGCs that code for ulbactin F, ulbactin G, gramicidin, and bacillopaline with the highest identity. We also identified a few BGCs with less than 50% sequence identity to MC-LR/MC-LHty/MC-HphHty/MC-LHph/MC-HphHph, xenocoumacin 1/xenocoumacin II, and tyrocidine. In addition, we found fourteen BGCs that do not resemble or show identity to any entries within the antiSMASH database. Therefore, Br. parabrevis WGTm-23 has the potential to synthesize new classes of antimicrobial compounds., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Aminoglycosides are essential antibiotics used to treat severe infections caused mainly by Gram-negative bacteria. Gentamicin is an aminoglycoside and, despite its toxicity, is clinically used to treat several pulmonary and urinary infections. The commercial form of gentamicin is a mixture of five compounds with minor differences in the methylation of one of their aminosugars. In the case of two compounds, gentamicin C2 and C2a, the only difference is the stereochemistry of the methyl group attached to C-6'. GenB2 is the enzyme responsible for this epimerization and is one of the four PLP-dependent enzymes encoded by the gentamicin biosynthetic gene cluster. Herein, we have determined the structure of GenB2 in its holo form in complex with PMP and also in the ternary complex with gentamicin X2 and G418, two substrate analogues. Based on the structural analysis, we were able to identify the structural basis for the catalytic mechanism of this enzyme, which was also studied by site-directed mutagenesis. Unprecedently, GenB2 is a PLP-dependent enzyme from fold I, which is able to catalyze an epimerization but with a mechanism distinct from that of fold III PLP-dependent epimerases using a cysteine residue near the N-terminus. The substitution of this cysteine residue for serine or alanine completely abolished the epimerase function of the enzyme, confirming its involvement. This study not only contributes to the understanding of the enzymology of gentamicin biosynthesis but also provides valuable details for exploring the enzymatic production of new aminoglycoside derivatives.

Circular bacteriocins are known for their structural stability and effective antimicrobial properties, positioning them as potential natural food preservatives. However, their widespread application is impeded by restricted availability. This research developed a total biosynthesis platform for circular bacteriocins, with a focus on AS-48 by involving recombinant production of the linear precursor in Escherichia coli , followed by enzymatic cyclization of the precursor into cyclic AS-48 using the ligase butelase-1 in vitro . An important discovery is that, aside from fusion tags, the C-terminal motif LE and LEKKK also could affect the expression yield of the precursor. This biosynthesis platform is both versatile and high-yielding, achieving yields of 10-20 mg/L of AS-48. Importantly, the biosynthetic AS-48 exhibited a secondary structure and antimicrobial activities comparable to those of the native molecules. As such, this work proposes an effective synthetic approach for circular bacteriocins, facilitating their advancement and application in the food industry.

Many bacteria co-exist and produce antibiotics, yet we know little about how they cope and occupy the same niche. The purpose of the present study was to determine if and how two potent antibiotic-producing marine bacteria influence the secondary metabolome of each other. We established an agar- and broth-based system allowing co-existence of a Phaeobacter species and Pseudoalteromonas piscicida that, respectively, produce tropodithietic acid (TDA) and bromoalterochromides (BACs). Co-culturing of Phaeobacter sp. strain A36a-5a on Marine Agar with P. piscicida strain B39bio caused a reduction of TDA production in the Phaeobacter colony. We constructed a transcriptional gene reporter fusion in the tdaC gene in the TDA biosynthetic pathway in Phaeobacter and demonstrated that the reduction of TDA by P. piscicida was due to the suppression of the TDA biosynthesis. A stable liquid co-cultivation system was developed, and the expression of tdaC in Phaeobacter was reduced eightfold lower (per cell) in the co-culture compared to the monoculture. Mass spectrometry imaging of co-cultured colonies revealed a reduction of TDA and indicated that BACs diffused into the Phaeobacter colony. BACs were purified from Pseudoalteromonas ; however, when added as pure compounds or a mixture they did not influence TDA production. In co-culture, the metabolome was dominated by Pseudoalteromonas features indicating that production of other Phaeobacter compounds besides TDA was reduced. In conclusion, co-existence of two antibiotic-producing bacteria may be allowed by one causing reduction in the antagonistic potential of the other. The reduction (here of TDA) was not caused by degradation but by a yet uncharacterized mechanism allowing Pseudoalteromonas to reduce expression of the TDA biosynthetic pathway.IMPORTANCEThe drug potential of antimicrobial secondary metabolites has been the main driver of research into these compounds. However, in recent years, their natural role in microbial systems and microbiomes has become important to determine the assembly and development of microbiomes. Herein, we demonstrate that two potent antibiotic-producing bacteria can co-exist, and one mechanism allowing the co-existence is the specific reduction of antibiotic production in one bacterium by the other. Understanding the molecular mechanisms in complex interactions provides insights for applied uses, such as when developing TDA-producing bacteria for use as biocontrol in aquaculture., Competing Interests: The authors declare no conflict of interest.

Pseudomonas poae PMA22 produces safracins, a family of compounds with potent broad-spectrum anti-bacterial and anti-tumor activities. The safracins' biosynthetic gene cluster (BGC sac) consists of 11 ORFs organized in two divergent operons ( sacABCDEFGHK and sacIJ ) that are controlled by P a and P i promoters. Contiguous to the BGC sac, we have located a gene that encodes a putative global regulator of the LysR family annotated as MexT that was originally described as a transcriptional activator of the MexEF-OprN multidrug efflux pump in Pseudomonas . Through both in vitro and in vivo experiments, we have demonstrated the involvement of the dual regulatory system MexT-MexS on the BGC sac expression acting as an activator and a repressor, respectively. The MexEF-OprN transport system of PMA22, also controlled by MexT, was shown to play a fundamental role in the metabolism of safracin. The overexpression of mexEF-oprN in PMA22 resulted in fourfold higher production levels of safracin. These results illustrate how a pleiotropic regulatory system can be critical to optimizing the production of tailored secondary metabolites, not only through direct interaction with the BGC promoters, but also by controlling their transport.

In the face of escalating antibiotic resistance, the quest for novel antimicrobial compounds is critical. Actinobacteria is known for producing a substantial fraction of bioactive molecules from microorganisms, nonetheless there is the challenge of metabolic redundancy in bioprospecting. New sources of natural products are needed to overcome these current challenges. Our present work proposes an unexplored potential of Neotropical social wasp-associated microbes as reservoirs of novel bioactive compounds. Using social wasp-associated Tsukamurella sp. strains 8F and 8J, we aimed to determine their biosynthetic potential for producing novel antibiotics and evaluated phylogenetic and genomic traits related to environmental and ecological factors that might be associated with promising bioactivity and evolutionary specialization. These strains were isolated from the cuticle of social wasps and subjected to comprehensive genome sequencing. Our genome mining efforts, employing antiSMASH and ARTS, highlight the presence of BGCs with minimal similarity to known compounds, suggesting the novelty of the molecules they may produce. Previous, bioactivity assays of these strains against bacterial species which harbor known human pathogens, revealed inhibitory potential. Further, our study focuses into the phylogenetic and functional landscape of the Tsukamurella genus, employing a throughout phylogenetic analysis that situates strains 8F and 8J within a distinct evolutionary pathway, matching with the environmental and ecological context of the strains reported for this genus. Our findings emphasize the importance of bioprospecting in uncharted biological territories, such as insect-associated microbes as reservoirs of novel bioactive compounds. As such, we posit that Tsukamurella sp. strains 8F and 8J represent promising candidates for the development of new antimicrobials., (© 2024. The Author(s).)

We have created a novel synthetic biology expression system allowing easy refactoring of biosynthetic gene clusters (BGCs) as monocistronic transcriptional units. The system is based on a set of plasmids containing a strong kasOp* promoter, RBS and terminators. It allows the cloning of biosynthetic genes into transcriptional units kasOp*-gene(s)-terminator flanked by several rare restriction cloning sites that can be sequentially combined into the artificial BGC in three compatible Streptomyces integration vectors. They allow a simultaneous integration of these BGCs at three different attB sites in the Streptomyces chromosome. The system was validated with biosynthetic genes from two known BGCs for aromatic polyketides landomycin and mithramycin., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Jan Kormanec reports financial support was provided by Institute of Molecular Biology Slovak Academy of Sciences. Jan Kormanec reports financial support was provided by Slovak Research and Development Agency. Jan Kormanec reports financial support was provided by Slovak Academy of Sciences. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Istamycins (ISMs) are 2-deoxyfortamine-containing aminoglycoside antibiotics (AGAs) produced by Streptomyces tenjimariensis ATCC 31603 with broad-spectrum bactericidal activities against most of the clinically relevant pathogens. Therefore, this study aimed to statistically optimize the environmental conditions affecting ISMs production using the central composite design (CCD). Both the effect of culture media composition and incubation time and agitation rate were studied as one factor at the time (OFAT). The results showed that both the aminoglycoside production medium and the protoplast regeneration medium gave the highest specific productivity. Results also showed that 6 days incubation time and 200 rpm agitation were optimum for their production. A CCD quadratic model of 17 runs was employed to test three key variables: initial pH, incubation temperature, and concentration of calcium carbonate. A significant statistical model was obtained including, an initial pH of 6.38, incubation temperature of 30 ˚C, and 5.3% CaCO 3 concentration. This model was verified experimentally in the lab and resulted in a 31-fold increase as compared to the unoptimized conditions and a threefold increase to that generated by using the optimized culture media. To our knowledge, this is the first report about studying environmental conditions affecting ISM production as OFAT and through CCD design of the response surface methodology (RSM) employed for statistical optimization. In conclusion, the CCD design is an effective tool for optimizing ISMs at the shake flask level. However, the optimized conditions generated using the CCD model in this study should be scaled up in a fermenter for industrial production of ISMs by S. tenjimariensis ATCC 31603 considering the studied environmental conditions that significantly influence the production proces., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

The tricyclic-aromadendrene-type sesquiterpenes are widely distributed and exhibit a range of biological activities, including anti-inflammatory, analgesic, antioxidant, antibacterial, insecticidal and cytotoxic properties. Several key sesquiterpene synthases (STSs) of this type have been identified, of which, viridiflorol synthase has been engineered for efficiently biosynthesizing viridiflorol in an Escherichia coli strain. This paper comprehensively summarizes the distribution and biological activity of aromadendrene-type sesquiterpenes in plant essential oils and microorganisms. The progress in aromadendrene-type sesquiterpene biosynthesis research, including the modifications of key STSs and the optimization of synthetic pathways, is reviewed. Finally, the prospects and associated challenges for the application and biosynthesis of these natural products are also discussed.

Sundews ( Drosera sp.) are the source of biologically active secondary metabolites: phenolic acids, flavonoids, and 1,4-naphtoquinones. Because obtaining them from the natural environment is impossible (rare and endangered species), in this study modifications of traditional tissue cultures grown in solid medium (SM), such as agitated cultures (ACs) (cultures in liquid medium with rotary shaking) and temporary immersion bioreactors Plantform TM (TIB), were used for multiplication of four sundew species: Drosera peltata , Drosera indica , Drosera regia , and Drosera binata , with simultaneously effective synthesis of biologically active phenolic compounds. Each species cultivated on SM, AC, and TIB was tested for biomass accumulation, the content of total phenols and selected phenolic derivative concentrations (DAD-HPLC), the productivity on of phenolic compounds, as well as its antibacterial activity against two human pathogens: Staphylococcus aureus and Escherichia coli . The results showed that the type of culture should be selected for each species separately. Phytochemical analyses showed that the synthesis of secondary metabolites from the groups of phenolic acids, flavonoids, and 1,4-naphthoquinones can be increased by modifying the cultivation conditions. D. regia turned out to be the richest in phenolic compounds, including 1,4-naphtoquinones: plumbagin and ramentaceone. Extracts from D. indica and D. regia tissue showed strong antibacterial activity against both pathogens. It has also been shown that the growth conditions of sundews can modify the level of secondary metabolites, and thus, their biological activity.

The actinomycete genus Rhodococcus is known for its diverse biosynthetic enzymes, with potential in pollutant degradation, chemical biocatalysis, and natural product exploration. Comparative genomics have analyzed the distribution patterns of non-ribosomal peptide synthetases (NRPSs) in Rhodococcus . The diversity and specificity of its secondary metabolism offer valuable insights for exploring natural products, yet remain understudied. In the present study, we analyzed the distribution patterns of biosynthetic gene clusters (BGCs) in the most comprehensive Rhodococcus genome data to date. The results show that 86.5% of the gene cluster families (GCFs) are only distributed in a specific phylogenomic-clade of Rhodococcus , with the most predominant types of gene clusters being NRPS and ribosomally synthesized and post-translationally modified peptides (RiPPs). In-depth mining of RiPP gene clusters revealed that Rhodococcus encodes many clade-specific novel RiPPs, with thirteen core peptides showing antibacterial potential. High-throughput elicitor screening (HiTES) and non-targeted metabolomics revealed that a marine-derived Rhodococcus strain produces a large number of new aurachin-like compounds when exposed to specific elicitors. The present study highlights the diversity and specificity of secondary biosynthetic potential in Rhodococcus , and provides valuable information for the targeted exploration of novel natural products from Rhodococcus , especially for phylogenomic-clade-specific metabolites.

Pelgipeptins, tridecaptins, and elgicins are among the antimicrobials produced by Paenibacillus elgii. Growth in complex media is commonly applied to obtain lipopeptides from culture's supernatant, but it requires further purification. This study aimed to improve the yield of pelgipeptins and tridecaptins using chemically defined media. The kinetics of antimicrobial lipopeptide yield in chemically defined media were evaluated in P. elgii AC13. Pelgipeptins were detected in the supernatant and the culture pellet, but tridecaptins were mainly associated with cell debris or endospores. We investigated whether removing Ca2+ would impair P. elgii sporogenesis, consequently improving the yield of tridecaptin. The kinetics of both lipopeptides in the presence and absence of Ca2+ were quantitatively and qualitatively evaluated and further correlated with the cell cycle. The impairment of P. elgii AC13 sporogenesis had no effect on tridecaptin production, which remained undetected in the supernatant of the culture. On the other hand, the yield of pelgipeptin in a Ca2+-free medium increased. We showed for the first time that the removal of Ca2+ interrupted the sporogenesis in P. elgii and improved the yield of pelgipeptins. However, Ca2+ absence had no effect on tridecaptin yield, which is possibly degraded or associated with other cell debris components., (© The Author(s) 2024. Published by Oxford University Press on behalf of Applied Microbiology International.)

E50-52, a class IIa-peptidic bacteriocin produced by a strain of Enterococcus faecium , has broad-spectrum antimicrobial activity against various foodborne pathogens. However, effective utilization of the E50-52 has been limited by low production yields and challenges associated with separation and purification of this 39-amino acid antimicrobial peptide. In this study, we have successfully produced a biologically active recombinant form of E50-52 by fusing it with the 16-kDa catalytic domain of lysostaphin-class III bacteriocin (LssCAT), which resulted in high-yield production. Initially, the LssCAT-E50-52 chimeric protein was insoluble upon over-expression in Escherichia coli , but it became soluble using phosphate buffer (pH 7.4) supplemented with 8 M urea. Purification using immobilized-Ni 2+ affinity chromatography under urea denaturing conditions resulted in consistent production a homogenous products (LssCAT-E50-52) with >95% purity. The purified protein was refolded using an optimized stepwise dialysis process. The resulting refolded LssCAT-E50-52 protein exhibited dose-dependent inhibitory activity against Helicobacter pylori , a Gram-negative, flagellated, helical bacterium that is associated with gastric cancer. Overall, the optimized protocol described in this study effectively produced large quantities of high-purity recombinant LssCAT-E50-52 protein, yielding approximately 100 mg per liter of culture. To the best of our knowledge, this is the first report on the impact of LssCAT-E50-52 on H. pylori . This finding could pave the way for further research into bactericidal mechanism and potential applications of this bacteriocin in biomedical industry.

Pseudonocardia species comprise a genus of filamentous, sporulating bacteria belonging to the phylum Actinomycetota, formerly Actinobacteria. They are found in marine and freshwater sediments and soils and associated with marine animals, insects, and plants. To date, they have mostly been studied because of their mutually beneficial symbiosis with fungus-growing ants in the tribe Attini . They have also attracted interest due to their biosynthetic capabilities, including the production of variably glycosylated polyenes and other novel antifungal compounds, and for their capacity to grow on a variety of hydrocarbons. The majority of clinically used antibiotics are derived from the specialised metabolites of filamentous actinomycete bacteria and most of these come from the genus Streptomyces . However, in the quest for novel chemistry there is increasing interest in studying other filamentous actinomycete genera, including Pseudonocardia . Here we outline the biological properties, genome size and structure and key features of the genus Pseudonocardia , namely their specialised metabolites and ecological roles.

Colistin, also known as polymyxin E, is a lipopeptide antibiotic used to treat infections caused by multidrug-resistant gram-negative bacteria. It is considered a "last-line antibiotic", but its clinical development is hindered by low titer and impurities resulting from the presence of diverse homologs in microbial fermentation. To ensure consistent pharmaceutical activity and kinetics, it is crucial to have high-purity colistin active pharmaceutical ingredient (API) in the pharmaceutical industry. This study focused on the metabolic engineering of a natural colistin producer strain to produce colistin with a high titer and purity. Guided by genome mining, we identified Paenibacillus polymyxa ATCC 842 as a natural colistin producer capable of generating a high proportion of colistin A. By systematically inactivating seven non-essential biosynthetic gene clusters (BGCs) of peptide metabolites that might compete precursors with colistin or inhibit colistin production, we created an engineered strain, P14, which exhibited an 82% increase in colistin titer and effectively eliminated metabolite impurities such as tridecaptin, paenibacillin, and paenilan. Additionally, we engineered the L-2,4-diaminobutyric acid (L-2,4-DABA) pathway to further enhance colistin production, resulting in the engineered strain P19, which boosted a remarkable colistin titer of 649.3 mg/L - a 269% improvement compared to the original strain. By concurrently feeding L-isoleucine and L-leucine, we successfully produced high-purity colistin A, constituting 88% of the total colistin products. This study highlights the potential of metabolic engineering in improving the titer and purity of lipopeptide antibiotics in the non-model strain, making them more suitable for clinical use. These findings indicate that efficiently producing colistin API in high purity directly from fermentation can now be achieved in a straightforward manner., Competing Interests: Declaration of competing interest Yong-Xin Li and Nanzhu Chen have a financial interest in NeoPeptide Innovations., (Copyright © 2024 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Nanobiotechnology has gained significant attention due to its capacity to generate substantial benefits through the integration of microbial biotechnology and nanotechnology. Among microbial organisms, Actinomycetes, particularly the prominent genus Streptomycetes, have garnered attention for their prolific production of antibiotics. Streptomycetes have emerged as pivotal contributors to the discovery of a substantial number of antibiotics and play a dominant role in combating infectious diseases on a global scale. Despite the noteworthy progress achieved through the development and utilization of antibiotics to combat infectious pathogens, the prevalence of infectious diseases remains a prominent cause of mortality worldwide, particularly among the elderly and children. The emergence of antibiotic resistance among pathogens has diminished the efficacy of antibiotics in recent decades. Nevertheless, Streptomycetes continue to demonstrate their potential by producing bioactive metabolites for the synthesis of nanoparticles. Streptomycetes are instrumental in producing nanoparticles with diverse bioactive characteristics, including antiviral, antibacterial, antifungal, antioxidant, and antitumor properties. Biologically synthesized nanoparticles have exhibited a meaningful reduction in the impact of antibiotic resistance, providing resources for the development of new and effective drugs. This review succinctly outlines the significant applications of Streptomycetes as a crucial element in nanoparticle synthesis, showcasing their potential for diverse and enhanced beneficial applications., (© 2024 The Author(s). Journal of Basic Microbiology published by Wiley‐VCH GmbH.)

Lactococcus lactis, widely used in the manufacture of dairy products, encounters various environmental stresses both in natural habitats and during industrial processes. It has evolved intricate machinery of stress sensing and defense to survive harsh stress conditions. Here, we identified a novel TetR/AcrR family transcription regulator, designated AcrR1, to be a repressor for acid and antibiotic tolerance that was derepressed in the presence of vancomycin or under acid stress. The survival rates of acrR1 deletion strain ΔAcrR1 under acid and vancomycin stresses were about 28.7-fold (pH 3.0, HCl), 8.57-fold (pH 4.0, lactic acid) and 2.73-fold (300 ng/mL vancomycin) greater than that of original strain F44. We also demonstrated that ΔAcrR1 was better able to maintain intracellular pH homeostasis and had a lower affinity to vancomycin. No evident effects of AcrR1 deletion on the growth and morphology of strain F44 were observed. Subsequently, we characterized that the transcription level of genes associated with amino acids biosynthesis, carbohydrate transport and metabolism, multidrug resistance, and DNA repair proteins significantly upregulated in ΔAcrR1 using transcriptome analysis and quantitative reverse transcription-PCR assays. Additionally, AcrR1 could repress the transcription of the nisin post-translational modification gene, nisC, leading to a 16.3% increase in nisin yield after AcrR1 deletion. Our results not only refined the knowledge of the regulatory mechanism of TetR/AcrR family regulator in L. lactis, but presented a potential strategy to enhance industrial production of nisin., (The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Marine microorganisms offer a promising avenue for the eco-friendly synthesis of nanoparticles due to their unique biochemical capabilities and adaptability to various environments. This study focuses on exploring the potential of a marine bacterial species, Stenotrophomonas rhizophila BGNAK1, for the synthesis of biocompatible copper nanoparticles and their application for hindering biofilms formed by monomicrobial species. The study begins with the isolation of the novel marine S. rhizophila species from marine soil samples collected from the West coast region of Kerala, India. The isolated strain is identified through 16S rRNA gene sequencing and confirmed to be S. rhizophila species. Biosynthesis of copper nanoparticles using S. rhizophila results in the formation of nanoparticles with size of range 10-50 nm. The nanoparticles exhibit a face-centered cubic crystal structure of copper, as confirmed by X-Ray Diffraction analysis. Furthermore, the synthesized nanoparticles display significant antimicrobial activity against various pathogenic bacteria and yeast. The highest inhibitory activity was against Staphylococcus aureus with a zone of 27 ± 1.00 mm and the least activity was against Pseudomonas aeruginosa with a zone of 22 ± 0.50 mm. The zone of inhibition against Candida albicans was 16 ± 0.60 mm. The antibiofilm activity against biofilm-forming clinical pathogens was evidenced by the antibiofilm assay and SEM images. Additionally, the copper nanoparticles exhibit antioxidant activity, as evidenced by their scavenging ability against DPPH, hydroxyl, nitric oxide, and superoxide radicals, as well as their reducing power in the FRAP assay. The study highlights the potential of the marine bacterium S. rhizophila BGNAK1 for the eco-friendly biosynthesis of copper nanoparticles with diverse applications. Synthesized nanoparticles exhibit promising antibiofilm, antimicrobial, and antioxidant properties, suggesting their potential utility in various fields such as medicine, wastewater treatment, and environmental remediation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The fast spread of antibiotic resistance results in the requirement for a constant introduction of new candidates. Pentangular polyphenols, a growing family of actinomycetes-derived aromatic type II polyketides, have attracted considerable attention due to their intriguing polycyclic systems and potent antimicrobial activity. Among them, benastatins, anthrabenzoxocinones (ABXs), and fredericamycins, display unique variations in their polycyclic frameworks, yet concurrently share structural commonalities within their substitutions. The present review summarizes advances in the isolation, spectroscopic characteristics, biosynthesis, and biological activities of pentangular polyphenols benastatins (1-16), ABXs (17-39), and fredericamycins (40-42) from actinomycetes. The information presented here thus prompts researchers to further explore and discover additional congeners within these three small classes of pentangular polyphenols., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Silver nanoparticles can be produced by an array of procedures, such as chemical, physical, and biological processes. The process of biosynthesis is more economical and significantly more environmentally friendly. We describe an environmentally compatible method (biosynthesis) of producing silver nanoparticles (Ag: NPs) with the capping component Artocarpus heterophyllus in this research work. Powder-X-ray crystallography (P-XRD), Fourier Transform Infrared (FT-IR), UV-visible (UV-Vis), Photoluminescence (PL), Field emission scanning electron microscopy (FE-SEM), and an antimicrobial test were all used to examine the synthesized samples. The P-XRD analysis revealed that the produced NPs have an FCC form with a typical particle size of 23 nm. FT-IR spectra further demonstrate the availability of the functional groups in the synthesized nanoparticles. The absorbance and transmittance spectra of the UV-Vis study have shown substantial transparency and less absorbance of the Ag: NPs in the entire visible region. The bandgap of the Ag: NPs was found to be 3.25 eV using the Tauc relation. In the PL study, an emission peak at 472 nm was found, suggesting the fluorescence emission of Ag: NPs. The FE-SEM micrographs provide confirmation of the surface-wide aggregate of nanostructural homogeneities. The FE-SEM micrographs illustrate that Ag: NPs are homogeneous aggregates of very small spheres. Variations in particle size and surface area-to-volume ratios of synthesized NPs have been proven to be responsible for the antibacterial activities. According to the antibacterial study, Ag: NPs restrain the development of both normal and harmful bacteria and so have the potential to be utilized for coating surgical equipment for aseptic operators in the healthcare industry., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Seven previously undescribed compounds, including four diketomorpholine alkaloids (1‒4), one indole diketopiperazine alkaloid (9), one chromone (10), and one benzoic acid derivative (13), and nine known compounds (5-8, 11, 12, and 14-16) were isolated from two different fungal sources. Nine of these metabolites (1-9) were obtained from a seagrass-derived Aspergillus alabamensis SYSU-6778, while the others were obtained from a mixed culture of A. alabamensis SYSU-6778 and a co-isolated fungus A. fumigatiaffinis SYSU-6786. The chemical structures of the compounds were deduced via spectroscopic techniques (including HRESIMS, 1D and 2D NMR), chemical reactions, and ECD calculations. It is worth noting that compound 10 was identified as a defensive secondary metabolite of strain SYSU-6786, produced through the induction of compound 8 under co-culture conditions. Compounds 3 and 4 possessed a naturally rare isotryptophan core. Moreover, compounds 1 and 2 exhibited potent inhibitory activities against fish pathogenic bacterium Edwardsiella ictalurid, with minimum inhibitory concentration values of 10.0 μg/mL for both compounds., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. none., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Actinomycins are a class of cyclic lipopeptide antibiotics produced by Streptomyces, which have rich biological activities and demonstrate great potential value. Among them, actinomycin D is currently the effective drug for some malignant tumor diseases. Although the chemical properties, biological activities and biosynthesis of actinomycins have been extensively studied, the regulation of their biosynthesis remains poorly understood. Streptomyces antibioticus ZS isolated from deep-sea corals is a producer of actinomycin D and actinomycin V. Here, we reported the characterization of a cluster-situated regulator ActO in actinomycins biosynthetic gene cluster (act cluster) of S. antibioticus ZS, which belongs to LmbU family. Deletion of actO completely blocked the synthesis of actinomycins. Overexpression of actO increased the yields of actinomycin D and actinomycin V by 4.4 fold and 2.6 fold, respectively. The result of RT-qPCR showed that ActO activates the transcription of all genes in act cluster. However, no specific binding of His 6 -ActO to the promoters of target genes was observed after electrophoretic mobility shift assay (EMSA). These results proved that ActO serves as a positive regulator involved in the biosynthesis of actinomycins, affecting the transcription of all genes related to the synthesis of intermediates, skeleton modification and extracellular transportation of final products. Moreover, we demonstrated that overexpression of actO is a novel strategy to increase the yields of actinomycins., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Pseudoalteromonas is a genus of marine bacteria and a promising source of natural products with antibacterial, antifungal, and antifouling bioactivities. To accelerate the exploration of new compounds from this genus, we applied the gene-first approach to study 632 public Pseudoalteromonas genomes. We identified 3968 biosynthetic gene clusters (BGCs) involved in the biosynthesis of secondary metabolites and classified them into 995 gene cluster families (GCFs). Surprisingly, only 9 GCFs (0.9 %) included an experimentally identified reference biosynthetic gene cluster from the Minimum Information about a Biosynthetic Gene cluster database (MIBiG), suggesting a striking novelty of secondary metabolites in Pseudoalteromonas. Bioinformatic analysis of the biosynthetic diversity encoded in the identified BGCs uncovered six dominant species of this genus, P. citrea, P. flavipulchra, P. luteoviolacea, P. maricaloris, P. piscicida, and P. rubra, that encoded more than 17 BGCs on average. Moreover, each species exhibited a species-specific distribution of BGC. However, a deep analysis revealed two BGCs conserved across five of the six dominant species. These BGCS encoded an unknown lanthipeptide and the siderophore myxochelin B implying an essential role of antibiotics for Pseudoalteromonas. We chemically profiled 11 strains from the 6 dominant species and identified four new antibiotics, korormicins L-O (1-4), from P. citrea WJX-3. Our results highlight the unexplored biosynthetic potential for bioactive compounds in Pseudoalteromonas and provide an important guideline for targeting exploration., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

With the rise of antimicrobial resistance, there is high demand for novel antimicrobials to combat multi-drug resistant pathogens. The bacterial genus Pantoea produces a diversity of antimicrobial natural products effective against a wide range of bacterial and fungal targets. These antimicrobials are synthesized by specialized biosynthetic gene clusters that have unique distributions across Pantoea as well as several other genera outside of the Erwiniaceae. Phylogenetic and genomic evidence shows that these clusters can mobilize within and between species and potentially between genera. Pantoea antimicrobials belong to unique structural classes with diverse mechanisms of action, but despite their potential in antagonizing a wide variety of plant, human, and animal pathogens, little is known about many of these metabolites and how they function. This review will explore the known antimicrobials produced by Pantoea: agglomerins, andrimid, D-alanylgriseoluteic acid, dapdiamide, herbicolins, pantocins, and the various Pantoea Natural Products (PNPs). It will include information on the structure of each compound, their genetic basis, biosynthesis, mechanism of action, spectrum of activity, and distribution, highlighting the significance of Pantoea antimicrobials as potential therapeutics and for applications in biocontrol., Competing Interests: Declaration of Competing Interest The authors have no conflicts of interest to declare., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

The synthesis of magnesium hydroxide nanoparticles (Mg(OH) 2 NPs) using plant extracts are known to be a practical, economical, and an environmentally friendly approach. In this work, Mg(OH) 2 NPs were synthesized using aqueous leaf extract of Tinospora cordifolia, a medicinal plant commonly found in India. The synthesized Mg(OH) 2 NPs were characterized using various spectroscopic techniques. The ultraviolet-visible (UV-Vis) absorption peak of the Mg(OH) 2 NPs was detected at 289 nm, Fourier transform infrared (FTIR) analysis confirmed the presence of various functional groups, and X-ray diffraction (XRD) patterns revealed the well-crystallized structure of the Mg(OH) 2 NPs. High-resolution transmission electron microscopy (HR-TEM) and scanning electron microscopy (SEM) analyses depicted spherical morphology and an average particle size (PS) of 27.71 nm. The energy-dispersive X-ray (EDX) analysis confirmed the presence of C, O, and Mg elements, and the X-ray photoelectron spectroscopy (XPS) survey spectrum confirmed the elements for the Su 1 s peak at 280.2 eV. The dynamic light scattering (DLS) analysis displayed an average PS of 54.3 nm, and the Zeta potential (ZP) was of 9.89 mV. The fabricated Mg(OH) 2 NPs displayed notable antibacterial activity against S. epidermidis, E. coli, and S. aureus. In addition, these NPs exhibited strong antioxidant properties (> 75%) based on DPPH, ABTS, and hydrogen peroxide (H 2 O 2 ) assays. Further, the same NPs exerted a potent anti-inflammatory activity (> 65%) based on COX-1 and COX-2 evaluations. The anti-Alzheimer' disease (AD) potential of Mg(OH) 2 NPs was assessed through effective inhibition (> 70%) of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities. Molecular docking (MD) studies confirmed that caryophyllene has higher binding affinity with AChE (-5.3 kcal/mol) and BuChE (-6.4 kcal/mol) enzymes. This study emphasizes the green synthesis of Mg(OH) 2 NPs using T. cordifolia as a plant source and highlights their potential for biomedical applications., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Biosurfactants generated from lactic acid bacteria (LAB) offer an advantage over standard microbial surfactants due to their antifungal, antibacterial and antiviral capabilities. Many LAB strains have been related to the manufacture of biosurfactant, an essential chemical with uses in the treatment of a number of illnesses. Furthermore, their effectiveness as anti-adhesive agents against a diverse variety of pathogens proves their utility as anti-adhesive coating agents for medical insertional materials, reducing hospital infections without the need of synthetic drugs and chemicals. LAB produces both low and high molecular weight biosurfactants. Biosurfactants from L. pentosus , L. gasseri and L. jensenii have been reported to produce glycolipopeptides that comprise carbohydrates, proteins and lipids in the ratio of 1:3:6 with palmitic, stearic acid, and linoelaidic acid as the major fatty acid component, whereas L. plantarum has been reported to make surlactin due to the presence of non-ribosomal peptide synthetase genes (NRPS) genes. Antimicrobial activity of sophorolipids and rhamnolipids generated from LAB against B. subtilis , P. aeruginosa , S. epidermidis , Propionibacterium acnes and E. coli has been demonstrated. The safety of biosurfactants is being evaluated in compliance with a number of regulatory standards that emphasize the importance of safety in the pharmaceutical industry. This review attempts, for the first time, to provide a comprehensive evaluation of several approaches for the synthesis of biosurfactant-mediated molecular modulation in terms of their biological value. Future biosurfactant directions, as well as regulatory considerations that are crucial for the synthesis of biosurfactants from novel LAB, have also been explored.

Background: Several two-component systems of Streptomyces coelicolor, a model organism used for studying antibiotic production in Streptomyces, affect the expression of the bfr (SCO2113) gene that encodes a bacterioferritin, a protein involved in iron storage. In this work, we have studied the effect of the deletion mutant ∆bfr in S. coelicolor., Results: The ∆bfr mutant exhibits a delay in morphological differentiation and produces a lesser amount of the two pigmented antibiotics (actinorhodin and undecylprodigiosin) compared to the wild type on complex media. The effect of iron in minimal medium was tested in the wild type and ∆bfr mutant. Consequently, we also observed different levels of production of the two pigmented antibiotics between the two strains, depending on the iron concentration and the medium (solid or liquid) used. Contrary to expectations, no differences in intracellular iron concentration were detected between the wild type and ∆bfr mutant. However, a higher level of reactive oxygen species in the ∆bfr mutant and a higher tolerance to oxidative stress were observed. Proteomic analysis showed no variation in iron response proteins, but there was a lower abundance of proteins related to actinorhodin and ribosomal proteins, as well as others related to secondary metabolite production and differentiation. Additionally, a higher abundance of proteins related to various types of stress, such as respiration and hypoxia among others, was also revealed. Data are available via ProteomeXchange with identifier PXD050869., Conclusion: This bacterioferritin in S. coelicolor (Bfr) is a new element in the complex regulation of secondary metabolism in S. coelicolor and, additionally, iron acts as a signal to modulate the biosynthesis of active molecules. Our model proposes an interaction between Bfr and iron-containing regulatory proteins. Thus, identifying these interactions would provide new information for improving antibiotic production in Streptomyces., (© 2024. The Author(s).)