Search

In this study, the impact of culture media filtrate of QD3 actinobacterial isolate on two potato cultivars, Spunta and Diamond, infected with potato virus Y (PVY) was investigated. Various parameters, including infection percentage, PVY virus infectivity, disease severity scoring, PVY optical density, photosynthetic and defense-related biochemical markers, enzymatic profiling, phenolic compounds, proline content, salicylic acid levels, and growth and yield parameters, were assessed to elucidate the potential of the QD3 actinobacterial isolate culture filtrate in mitigating PVY-induced damage. The physiological and biochemical characteristics of the QD3 actinobacterial isolate, including its salinity tolerance, pH preferences, and metabolic traits, were investigated. Molecular identification via 16S rRNA gene sequencing confirmed its classification as Streptomyces fradiae QD3, and it was deposited in GenBank with the gene accession number MN160630. Distinct responses between Spunta and Diamond cultivars, with Spunta displaying greater resistance to PVY infection. Notably, pre-infection foliar application of the QD3 filtrate significantly reduced disease symptoms and virus infection in both cultivars. For post-PVY infection, the QD3 filtrate effectively mitigated disease severity and the PVY optical density. Furthermore, the QD3 filtrate positively influenced photosynthetic pigments, enzymatic antioxidant activities, and key biochemical components associated with plant defense mechanisms. Gas chromatography‒mass spectrometry (GC‒MS) analysis revealed palmitic acid (hexadecanoic acid, methyl ester) and oleic acid (9-octadecanoic acid, methyl ester) as the most prominent compounds, with retention times of 23.23 min and 26.41 min, representing 53.27% and 23.25%, respectively, of the total peak area as primary unsaturated fatty acids and demonstrating antiviral effects against plant viruses. Cytotoxicity assays on normal human skin fibroblasts (HSFs) revealed the safety of QD3 metabolites, with low discernible toxicity at high concentrations, reinforcing their potential as safe and effective interventions. The phytotoxicity results indicate that all the seeds presented high germination rates of approximately 95–98%, suggesting that the treatment conditions had no phytotoxic effect on the Brassica oleracea (broccoli) seeds, Lactuca sativa (lettuce) seeds, and Eruca sativa (arugula or rocket) seeds. Overall, the results of this study suggest that the S. fradiae filtrate has promising anti-PVY properties, influencing various physiological, biochemical, and molecular aspects in potato cultivars. These findings provide valuable insights into potential strategies for managing PVY infections in potato crops, emphasizing the importance of Streptomyces-derived interventions in enhancing plant health and crop protection. [ABSTRACT FROM AUTHOR]

Tylosin is an important macrolide antibiotic produced by Streptomyces fradiae. In the biosynthesis of tylosin, macrocin O-methyltransferase TylF catalyzes the conversion of the side-product tylosin C (macrocin) to the primary component tylosin A (C/A conversion). This conversion is the rate-limiting step in the biosynthesis of tylosin, and affects the quality of the end product. To find a high activity and environment-adapted TylF enzyme, a TylF variant pool has been constructed via protein evolution approach in our previous study (Fan et al., 2023 [41]). In this study, the TylF variants with higher C/A conversion rates were expressed in E. coli and purified. The variants TylFY139F, TylFQ138H, F232Y and TylFT36S, V54A were shown to have a higher C/A conversion rate at 30 °C than that of TylF at 38 °C. Moreover, they had a greater acid resistance and showed more adaptable to the pH change during fermentation. Further protein structural and substrate-binding affinity analyses revealed that the T36S, V54A, Q138H, Y139F, and F232Y mutations enlarged the volume of the substrate-binding pocket, thereby increasing the affinity of enzyme variants for their substrates of SAM and macrocin, and decreasing the inhibition of SAH. Three of the TylF variants were overexpressed in the industrial tylosin-producing S. fradiae strain, and the recombinant strains showed the highest C/A conversion at 30 °C without heating up to 38 °C during the last 24 h of fermentation. This is of great energy-saving significance for tylosin industrial production.

The lipid-producing gram-positive bacteria were isolated from soil and it was identified as Streptomyces fradiae JJ1 MK733985.1. Bacterial lipid synthesis was explored using rice washed water through the fermentation research. The exponential growth phase was reached on the fifth day of fermentation, according to the results. The ability of Streptomyces fradiae to produce microbial oil was compared to that of International Streptomyces Project-2 Medium (ISP2) medium. The results showed that RWW culture medium yielded 85.4% fatty acids and 60.3 g/L biomass, while ISP2 medium yielded 83.2% wt fatty acids and 59.5 g/L biomass. The microbial oil was transesterified into Fatty acid methyl esters (FAME), and its characteristics were studied using American Society for Testing Materials (ASTM) standards. The specific gravity was 0.864, the density was 0.863, the gross heating value was 42498 kj/kg, the cetane number was 37, and the acid value was 0.46. Streptomyces fradiae grew with an ISP2 density of 0.835, specific gravity of 0.845, gross heating value of 42326 kj/kg, and acid value of 0.43. According to the findings, Streptomyces fradiae isolated from soil can grow in RWW medium for the first time and provide the same yield as commercial media while being significantly less expensive.

To enrich 'epoxide hydrolase (EH) tool cabinet' for preparing enantiopure epoxides and 1,2-diols, a novel microbial EH, Sf EH2, was identified from Streptomyces fradiae SF-2. Bioinformatics analysis indicated that Sf EH2 has typical structural characteristics of α/β-hydrolase fold superfamily. Its coding gene, sfeh 2, was then codon-optimized and amplified. A recombinant (re) Sf EH2-expressing E. coli BL21(DE3) strain (E. coli / sfeh 2) was constructed through DNA manipulations. Substrate spectrum assay including fifteen common racemic (rac -) epoxides by E. coli / sfeh 2 showed that re Sf EH2 displayed the highest and best complementary regioselectivities (regioselectivity coefficients, α R = 89.7% and β S = 91.4%) towards rac - 14a , as well as the highest enantioselectivity (enantiomeric ratio, E = 15.3) in S -type towards rac - 15a. Molecular docking simulation revealed that Sf EH2 preferentially attacked C α of (R)- 14a and C β of (S)- 14a geometrically. In kinetic parameter analysis, the K m R (5.74 mM) of purified re Sf EH2 for (R)- 15a was 1.76-fold lower than K m S (10.09 mM) for (S)- 15a. At last, using 50 mg dry cell weight/mL E. coli / sfeh 2, the scale-up enantioconvergent hydrolysis of 100 mM rac - 14a at 30 °C for 6.0 h afforded (S)- 14b with 80.9% ee p and 90.1% yield, while the kinetic resolution of 150 mM rac - 15a retained (S)- 15a with >99.0% ee s and 42.2% yield. [Display omitted] • A novel epoxide hydrolase, Sf EH2, is identified and characterized. • Sf EH2 displays desired regioselectivity for racemic 4-methylphenyl glycidyl ether. • Sf EH2 displays desired enantioselectivity for racemic benzyl glycidyl ether phenyl. • Space-time yield of (S)-3-aryloxy-1,2-propanediol by Sf EH2 is the highest. • Space-time yield of (S)-benzyl glycidyl ether phenyl by Sf EH2 is the highest. [ABSTRACT FROM AUTHOR]

The lipid-producing gram-positive bacteria were isolated from soil and it was identified as Streptomyces fradiae JJ1 MK733985.1. Bacterial lipid synthesis was explored using rice washed water through the fermentation research. The exponential growth phase was reached on the fifth day of fermentation, according to the results. The ability of Streptomyces fradiae to produce microbial oil was compared to that of International Streptomyces Project-2 Medium (ISP2) medium. The results showed that RWW culture medium yielded 85.4% fatty acids and 60.3 g/L biomass, while ISP2 medium yielded 83.2% wt fatty acids and 59.5 g/L biomass. The microbial oil was transesterified into Fatty acid methyl esters (FAME), and its characteristics were studied using American Society for Testing Materials (ASTM) standards. The specific gravity was 0.864, the density was 0.863, the gross heating value was 42498 kj/kg, the cetane number was 37, and the acid value was 0.46. Streptomyces fradiae grew with an ISP2 density of 0.835, specific gravity of 0.845, gross heating value of 42326 kj/kg, and acid value of 0.43. According to the findings, Streptomyces fradiae isolated from soil can grow in RWW medium for the first time and provide the same yield as commercial media while being significantly less expensive. [ABSTRACT FROM AUTHOR]

The objective was to screen strains with excellent disease-controlling and plant growth-promoting properties from healthy pepper rhizosphere and apply them to bio-barrier soil, so as to provide probiotic resources for biocontrol of pepper root rot. The plate confrontation method was used to screen biocontrol strains to Fusarium solani, the causal agent of pepper root rot. The nitrogen fixation, phosphate solution, potassium solution, 3-indole acetic acid(IAA), siderophore, cellulase, and protease producing capacity and degradation of phenolic acids of the strains were measured. The actinomycetes were identified by morphological, physiological, biochemical, and 16S rRNA gene sequence analysis. In field experiments, plant growth-promoting effects were analyzed by measuring pepper yield and disease index of root rot. A total of six strains of biocontrol actinomycetes showed an antagonistic effect on F. solani. Strain G-1 was explored for the strongest inhibition rates to the five pathogens of the vegetable in greenhouse, and the superior characters of IAA, siderophore, cellulase, protease producing, organic-phosphorus dissociation and degradation of phenolic acids. Combined with colony morphological, physiological, biochemical, and 16S rRNA phylogenetic tree analysis, strain G-1 was identified as Streptomyces fradiae. In-situ field experiment showed that the strain G-1 had obvious disease prevention effect on pepper Fusarium root rot(P<0.05). It had certain growth-promoting effect on pepper plants, and G-1 increased the yield of pepper by 31.04%. S. fradiae(G-1) is considered a probiotic resource for pepper green agriculture, and we provide superior microbial inoculants that cater to the yield of pepper and the biocontrol of Fusarium root rot. [ABSTRACT FROM AUTHOR]

Macrolides are currently a class of extensively used antibiotics in human and animal medicine. Tylosin is not only one of the most important veterinary macrolides but also an indispensable material for the bio- and chemo-synthesis of new generations of macrolide antibiotics. Thus, improving its production yield is of great value. As the key rate-limiting enzyme catalyzing the terminal step of tylosin biosynthesis in Streptomyces fradiae (S. fradiae), TylF methyltransferase’s catalytic activity directly affects tylosin yield. In this study, a tylF mutant library of S. fradiae SF-3 was constructed based on error-prone PCR technology. After two steps of screening in 24-well plates and conical flask fermentation and enzyme activity assay, a mutant strain was identified with higher TylF activity and tylosin yield. The mutation of tyrosine to phenylalanine is localized at the 139th amino acid residue on TylF (TylFY139F), and protein structure simulations demonstrated that this mutation changed the protein structure of TylF. Compared with wild-type protein TylF, TylFY139F exhibited higher enzymatic activity and thermostability. More importantly, the Y139 residue in TylF is a previously unidentified position required for TylF activity and tylosin production in S. fradiae, indicating the further potential to engineer the enzyme. These findings provide helpful information for the directed molecular evolution of this important enzyme and the genetic modification of tylosin-producing bacteria.

Tylosin is an important macrolide antibiotic produced by Streptomyces fradiae . In the biosynthesis of tylosin, macrocin O -methyltransferase TylF catalyzes the conversion of the side-product tylosin C (macrocin) to the primary component tylosin A (C/A conversion). This conversion is the rate-limiting step in the biosynthesis of tylosin, and affects the quality of the end product. To find a high activity and environment-adapted TylF enzyme, a TylF variant pool has been constructed via protein evolution approach in our previous study (Fan et al., 2023 [41]). In this study, the TylF variants with higher C/A conversion rates were expressed in E. coli and purified. The variants TylF Y139F , TylF Q138H, F232Y and TylF T36S, V54A were shown to have a higher C/A conversion rate at 30 °C than that of TylF at 38 °C. Moreover, they had a greater acid resistance and showed more adaptable to the pH change during fermentation. Further protein structural and substrate-binding affinity analyses revealed that the T36S, V54A, Q138H, Y139F, and F232Y mutations enlarged the volume of the substrate-binding pocket, thereby increasing the affinity of enzyme variants for their substrates of SAM and macrocin, and decreasing the inhibition of SAH. Three of the TylF variants were overexpressed in the industrial tylosin-producing S. fradiae strain, and the recombinant strains showed the highest C/A conversion at 30 °C without heating up to 38 °C during the last 24 h of fermentation. This is of great energy-saving significance for tylosin industrial production., Competing Interests: 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., (© 2024 The Authors.)

To improve the screening efficiency of high-yield neomycin sulfate (NM) Streptomyces fradiae strains after mutagenesis, a high-throughput screening method using streptomycin resistance prescreening (8 μg/mL) and a 24-deep well plates/microplate reader (trypan blue spectrophotometry) rescreening strategy was developed. Using this approach, we identified a high-producing NM mutant strain, Sf6-2, via six rounds of atmospheric and room temperature plasma (ARTP) mutagenesis and screening. The mutant displayed a NM potency of 7780 ± 110 U/mL and remarkably stable genetic properties over six generations. Furthermore, the key components (soluble starch, peptone, and (NH4)2SO4) affecting NM potency in fermentation medium were selected using Plackett-Burman and optimized by Box-Behnken designs. Finally, the NM potency of Sf6-2 was increased to 10,849 ± 141 U/mL at the optimal concentration of each factor (73.98 g/L, 9.23 g/L, and 5.99 g/L, respectively), and it exhibited about a 40% and 100% enhancement when compared with before optimization conditions and the wild-type strain, respectively. In this study, we provide a new S. fradiae NM production strategy and generate valuable insights for the breeding and screening of other microorganisms.

Neomycin, an aminoglycoside antibiotic, is widely used in the livestock husbandry due to its higher antimicrobial activity and availability of feed additives in animals. However, its production yield is relatively low and cannot meet the needs of developing market and clinical application. Here, the entire natural neo cluster was cloned from Streptomyces fradiae CGMCC 4.576 by φBT1 integrase-mediated site-specific recombination. Then, the rational reconstruction of the neo cluster was performed by using λ-Red-mediated PCR targeting for improving neomycin production. In order to coordinate with this attempt, the supplementation of suitable precursors was carried out. The constructed recombinant strain Sf/pKCZ03 has multi-copy of the neo cluster modified by disrupting the negative regulatory gene neoI and replacing the native promoter of the neoE-D with PkasO*. Compared to the yield (1282 mg/L) of Streptomyces fradiae CGMCC 4.576, the engineered strain Sf/pKCZ03 had a 36% enhancement of neomycin production. Quantitative real-time PCR analysis revealed the increased transcription of structural genes (neoE, neoB, neoL, aacC8) and regulatory genes (neoR, neoH) in Sf/pKCZ03. Additionally, under the supplementation of 1 g/L N-acetyl-D-glucosamine and 5 g/L L-glutamine, the yield of engineered strain Sf/pKCZ03 showed 62% and 107% improvements compared to that of the wild-type strain in the original medium, respectively. These findings demonstrated that engineering the antibiotic gene cluster in combination with precursors feeding was an effective approach for strain improvement, and would be potentially extended to other Streptomyces for large-scale production of commercialized antibiotics. [ABSTRACT FROM AUTHOR]

We describe Streptomyces fradiae mechanisms of sensitivity to nitrone‐oligomycin A, a derivative of oligomycin A. We obtained S. fradiae‐nitR+bld, a nitrone‐oligomycin A resistant mutant with a «bald» phenotype. Comparative genomic analysis of the wild‐type S. fradiae ATCC19609 and S. fradiae‐nitR+bld revealed a mutation in padR – a gene encoding a multifunction transcription regulator, which resulted in the amino acid replacement in a highly conserved DNA‐binding domain. Bioinformatics genome analysis of S. fradiae ATCC19609 discovered a PadR binding site 13 bp upstream the start codon of the marR transcription factor gene. Induction of S. fradiaenitR+bld and w.t. strains with nitrone‐oligomycin A lead to a significant increase in expression level of the marR gene in the w.t. strain, but no change observed in mutant strain. We identified differences between DNA–protein interactions of the mutant and native PadR proteins with its putative binding site in S. fradiae ATCC19609. This allowed us to suggest that the padR gene, that harbored a single nucleotide mutation in the S. fradiaenitR+bld strain, might be involved in the mechanism of resistance to nitrone‐oligomycin A. We assume the participation of the transcriptional factorpadR in the formation of the bald phenotype. [ABSTRACT FROM AUTHOR]

The aim of the present study was to screen the antiviral activity of the secondary metabolite 9(10H)-Acridanone extracted from Streptomyces fradiae strain VITMK2 in Litopenaeus vannamei shrimp infected with white spot syndrome virus (WSSV). The strain was isolated from marine soil sediment sample collected from the mangrove forest region of Pichavaram, Tamil Nadu, India. Among the 31 isolates, the isolate VITMK-2 exhibited strong antiviral activity against the WSSV. The potential isolate was subjected to morphological, biochemical and molecular taxonomic characterisation. It was identified as Streptomyces species and designated as S . fradiae strain VITMK2. Purification of ethyl acetate (EA) extract by silica gel column chromatography yielded two compounds, C1 and C2. The shrimp infected with WSSV and treated with C1 compound (500 μg, 250 μg, and 125 μg) showed survival rates of 88.89%, 83.33% and 55.56% respectively. The survival of shrimp treated with the EA extract of S . fradiae strain VITMK2 (500 μg/animal) was 83.33%. The C1 compound (250 μg/animal) showed better antiviral activity when compared to EA extract. The chemical nature of the C1 compound was identified by FTIR, 1 H and 13 C NMR, and HRMS analysis. Based on the spectral data, the C1 compound was identified as 9(10H)-Acridanone with a molecular formula of C 13 H 9 NO and a molecular mass of 195.1048 Da. Docking of the lead compound with VP26 and VP28 of WSSV drug target proteins showed the least binding energy of −5.71 kcal/mol and −5.21 kcal/mol respectively predicting the strong interaction of the compound with VP26 and VP28. This is the first report on anti-WSSV activity of 9(10H)-Acridanone derived from marine S . fradiae strain VITMK2 and this compound can be probed further to be used as an effective antiviral agent for better control and management of WSSV infection in aquaculture farms. [ABSTRACT FROM AUTHOR]

Pretreated waste Streptomyces fradiae biomass was utilized as an eco-friendly sorbent for Congo Red (CR) and Methylene Blue (MB) removal from aqueous solutions. The biosorbent was characterized by Fourier transform infrared spectroscopy. Batch experiments were conducted to study the effect of pH, biosorbent dosage, initial concentration of adsorbates, contact time and temperature on the biosorption of the two dyes. The equilibrium adsorption data were analysed using Freundlich and Langmuir models. Both models fitted well to the experimental data. The maximum biosorption capacity of the pretreated Streptomyces fradiae biomass was 46.64 mg g-1 for CR and 59.63 mg g-1 for MB, at a pH = 6.0, a contact time of 120 min, a biosorbent dosage of 2 g dm-3 and a temperature of 298 K. Lagergren and Ho kinetic models were used to analyse the kinetic data obtained from different batch experiments. The biosorption of both dyes followed better the pseudo-second order kinetic model. The calculated values for ΔG°, ΔS°, and ΔH° indicated that the biosorption of CR and MB onto the waste pretreated biomass was feasible, spontaneous, and exothermic in the selected temperature range and conditions. [ABSTRACT FROM AUTHOR]

Penicillium sp. WC-29-5 was co-cultured with Streptomyces fradiae 007 to produce five natural products (1–3, 4a and 4b) that were isolated and characterized by spectroscopic analysis. Interestingly, these compounds were found to be different from those produced in discrete fungal and bacterial controls. Among these compounds, the absolute configurations of compounds 4a and 4b were determined for the first time by X-ray single crystal diffraction experiments and electronic circular dichroism (ECD) calculations. An evaluation of the cytotoxic activities of these compounds revealed that 4b was moderately cytotoxic towards HL-60 and H1975 tumor cells with IC50 values of 3.73 and 5.73 µM, respectively, whereas compound 4a was only moderately cytotoxic towards H1975 cells with an IC50 value of 3.97 µM.

Tetracycline repressors (TetRs) modulate multidrug efflux pathways in several pathogenic bacteria. In Streptomyces, they additionally regulate secondary metabolic pathways like antibiotic production. For instance, in the antibiotic producer Streptomyces fradiae, a layered network of TetRs regulates the levels of the commercially important antibiotic tylosin, with TylP occupying the top of this cascading network. TetRs exist in two functional states, the DNA-bound and the ligand-bound form, which are allosterically regulated. Here, to develop deeper insights into the factors that govern allostery, the crystal structure of TylP was solved to a resolution of 2.3 Å. The structure revealed that TylP possesses several unique features; notably, it harbors a unique C-terminal helix-loop extension that spans the entire length of the structure. This anchor connects the DNAbinding domain (DBD) with the ligand-binding domain (LBD) via a mix of positively charged and hydrogen-bonding interactions. Supporting EMSA studies with a series of ΔC truncated versions show that a systematic deletion of this region results in complete loss of DNA binding. The structure additionally revealed that TylP is markedly different in the orientation of its DBD and LBD architecture and the dimeric geometry from its hypothesized Streptomyces homologue CprB, which is a γ-butyrolactone regulator. Rather, TylP is closer in structural design to macrolide-binding TetRs found in pathogens. Supporting molecular dynamic studies suggested that TylP binds a macrolide intermediate in the tylosin pathway. Collectively, the structure along with corroborating biochemical studies provided insights into the novel mode of regulation of TetRs in antibiotic-producing organisms. [ABSTRACT FROM AUTHOR]

Data of draft genome sequencing of Streptomyces fradiae-АТСС19609-Olg4R strain, resistant to (33S)-33-deoxy-33-thiocyanatooligomycin A, are presented. Comparative analysis of the genomes of S. fradiae wild-type strain ATCC19609 and S. fradiae strain ATCC19609-Olg4R showed the single nucleotide substitution that led to A(600)T change in the conservative P-loop NTPase region of class IV helicase gene, which, probably, promoted the resistance of S. fradiae strain ATCC19609-Olg4R to (33S)-33-deoxy-33-thiocyanatooligomycin A. [ABSTRACT FROM AUTHOR]

TylP is one of five regulatory proteins involved in the regulation of antibiotic (tylosin) production, morphological and physiological differentiation in Streptomyces fradiae. Its function is similar to those of various γ-butyrolactone receptor proteins. In this report, N-terminally His-tagged recombinant TylP protein (rTylP) was overproduced in Escherichia coli and purified to homogeneity. The rTylP protein was crystallized from a reservoir solution comprising 34%( v/ v) ethylene glycol and 5%( v/ v) glycerol. The protein crystals diffracted X-rays to 3.05 Å resolution and belonged to the trigonal space group P3121, with unit-cell parameters a = b = 126.62, c = 95.63 Å. [ABSTRACT FROM AUTHOR]

In order to excavate microbial epoxide hydrolases (EHs) with desired catalytic properties, a novel EH, Sf EH1, was identified based on the genome annotation of Streptomyces fradiae and sequence alignment analysis with local protein library. The Sf EH1-encoding gene, sfeh 1, was then cloned and over-expressed in soluble form in Escherichia coli /BL21(DE3). The optimal temperature and pH of recombinant Sf EH1 (re Sf EH1) and re Sf EH1-expressing E. coli (E. coli / sfeh 1) were both determined as 30 ℃ and 7.0, also indicating that the influences of temperature and pH on re Sf EH1′s activities were more obvious than those of E. coli / sfeh 1 whole cells. Subsequently, using E. coli / sfeh 1 as catalyst, its catalytic properties towards thirteen common mono-substituted epoxides were tested, in which E. coli / sfeh 1 had the highest activity of 28.5 U/g dry cells for rac -1,2-epoxyoctane (rac - 6a), and (R)-1,2-pentanediol ((R)- 3b) (or (R)-1,2-hexanediol ((R)- 4b)) with up to 92.5% (or 94.1%) ee p was obtained at almost 100% conversion ratio. Regioselectivity coefficients (α S and β R) displayed in the enantioconvergent hydrolysis of rac - 3a (or rac - 4a) were calculated to be 98.7% and 93.8% (or 95.2% and 98.9%). Finally, the reason of the high and complementary regioselectivity was confirmed by both kinetic parameter analysis and molecular docking simulations. • An epoxide hydrolase from S. fradiae , Sf EH1, was characterized for the first time. • By codon optimization, recombinant Sf EH1 was overexpressed mainly in soluble form. • Sf EH1 had high and complementary regioselectivity for rac - 3a (or 4a). • Hydrolysis of rac - 3a (or 4a) generated (R)- 3b (or 4b) with 92.5% (or 94.1%) ee p. [ABSTRACT FROM AUTHOR]

Tylosin is a potent veterinary macrolide antibiotic produced by the fermentation of Streptomyces fradiae ; however, it is necessary to modify S. fradiae strains to improve tylosin production. In this study, we established a high-throughput, 24-well plate screening method for identifying S. fradiae strains that produce increased yields of tylosin. Additionally, we constructed mutant libraries of S. fradiae via ultraviolet (UV) irradiation and/or sodium nitrite mutagenesis. A primary screening of the libraries in 24-well plates and UV spectrophotometry identified S. fradiae mutants producing increased yields of tylosin. Mutants with tylosin yield 10% higher than the wild-type strain were inoculated into shake flasks, and the tylosin concentrations produced were determined by high-performance liquid chromatography (HPLC). Joint (UV irradiation and sodium nitrite) mutagenesis resulted in higher yields of mutants with enhanced tylosin production. Finally, 10 mutants showing higher tylosin yield were re-screened in shake flasks. The yield of tylosin A by strains UN-C183 (6767.64 ± 82.43 μg/ml) and UN-C137 (6889.72 ± 70.25 μg/ml) was significantly higher than that of the wild-type strain (6617.99 ± 22.67 μg/ml). These mutant strains will form the basis for further strain breeding in tylosin production.

Macrolides are currently a class of extensively used antibiotics in human and animal medicine. Tylosin is not only one of the most important veterinary macrolides but also an indispensable material for the bio- and chemo-synthesis of new generations of macrolide antibiotics. Thus, improving its production yield is of great value. As the key rate-limiting enzyme catalyzing the terminal step of tylosin biosynthesis in Streptomyces fradiae ( S. fradiae ), TylF methyltransferase's catalytic activity directly affects tylosin yield. In this study, a tylF mutant library of S . fradiae SF-3 was constructed based on error-prone PCR technology. After two steps of screening in 24-well plates and conical flask fermentation and enzyme activity assay, a mutant strain was identified with higher TylF activity and tylosin yield. The mutation of tyrosine to phenylalanine is localized at the 139th amino acid residue on TylF (TylF Y139F ), and protein structure simulations demonstrated that this mutation changed the protein structure of TylF. Compared with wild-type protein TylF, TylF Y139F exhibited higher enzymatic activity and thermostability. More importantly, the Y139 residue in TylF is a previously unidentified position required for TylF activity and tylosin production in S . fradiae , indicating the further potential to engineer the enzyme. These findings provide helpful information for the directed molecular evolution of this important enzyme and the genetic modification of tylosin-producing bacteria., Competing Interests: 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., (© 2023 The Authors.)

An attempt has been made to isolate, purify and characterize antifouling compound from Streptomyces fradiae PE7 isolated from Vellar estuarine sediment, Parangipettai, South India. The microbial identification was done at species level based on its phenotypic, cell wall and molecular characteristics. Strain PE7 produced high quantity of antifouling compounds in agar surface fermentation when compared to submerged fermentation. In fermentation optimization, wide range of sugars, amino acids, minerals, pH, temperature and NaCl concentration was found to influence the antifouling compound production from the strain PE7. Antifouling compound PE7-C was purified from the crude extract by preparative TLC, and its activity against biofouling bacteria was confirmed by bioautography. Based on the physico-chemical characteristics, the chemical structure of the antifouling compound PE7-C was identified as quercetin (CHO), a flavonoid class of compound with the molecular weight 302.23 g/mol. The purified quercetin was active against 18 biofouling bacteria with MIC range between 1.6 and 25 μg/ml, algal spore germination and mollusc foot adherence found at 100 μg/ml and 306 ± 19.6 μg ml respectively. The present study, for the first time, reported quercetin from marine-derived Streptomyces sp. PE7 with antifouling activity. This also leads to the repurposing of quercetin for the development of antifouling agent. [ABSTRACT FROM AUTHOR]

Was studied the antimicrobial activity (antibacterial, antifungal) of the strain Streptomyces fradiae 19 isolated from chernozem soil of the Central Part of the Republic of Moldova. Study of the properties was carried out by using the test-cultures of opportunistic pathogens (E. coli, P. aeruginosa, E. faecalis, S. aureus, C. albicans) microorganisms and plant pathogenic bacteria (A. tumefaciens, C. michiganensis, X. campestris) and fungi (A. alternata, A. niger, B. cinerea, F. oxysporum, F. solani). Metabolites of studied strain showed the highest activity against the following test-microorganisms: S. aureus, C. albicans, growth inhibition zones ranged from 18.0 mm till complete inhibition in dependence of growing medium, but against phytopathogenic bacterium X. campestris, the diameter of the growth inhibition zones were from 24.5 till 32.0 mm. [ABSTRACT FROM AUTHOR]

The paper provides the annotation and data on sequencing the antibiotic resistance genes in Strep tomyces fradiae strain ATCC19609, highly sensitive to different antibiotics. Genome analysis revealed four groups of genes that determined the resistome of the tested strain. These included classical antibiotic resistance genes (nine aminoglycoside phosphotransferase genes, two beta-lactamase genes, and the genes of puromycin N-acetyltransferase, phosphinothricin N-acetyltransferase, and aminoglycoside acetyltransferase); the genes of ATP-dependent ABC transporters, involved in the efflux of antibiotics from the cell (MacB-2, BcrA, two-subunit MDR1); the genes of positive and negative regulation of transcription ( whiB and padR families); and the genes of post-translational modification (serine-threonine protein kinases). A comparative characteristic of aminoglycoside phosphotransferase genes in S. fradiae ATCC19609, S. liv idans TK24, and S. albus J1074, the causative agent of actinomycosis, is provided. The possibility of using the S. fradiae strain ATCC19609 as the test system for selection of the macrolide antibiotic oligomycin A derivatives with different levels of activity is demonstrated. Analysis of more than 20 semisynthetic oligomycin A derivatives made it possible to divide them into three groups according to the level of activity: inactive (>1 nmol/disk), 10 substances; with medium activity level (0.05-1 nmol/disk), 12 substances; and more active (0.01-0.05 nmol/disk), 2 substances. Important for the activity of semisynthetic derivatives is the change in the position of the 33rd carbon atom in the oligomycin A molecule. [ABSTRACT FROM AUTHOR]

Waste Streptomyces fradiae biomass from pharmaceutical industry was successfully used for Zn(II) removal from aqueous solutions. In the present study process parameters (initial pH of the solution, amount of biomass, initial metal concentration, stirring speed and contact time) and their influence on Zn(II) biosorption were investigated and optimized. Langmuir and Freundlich adsorption isotherms were used to describe sorption behavior between the waste biomass and the Zn ions. The biosorption process was better described by the Langmuir adsorption isotherm with 61.09 mg g-1 maximum adsorption capacity. Lagergren and Ho models were used to analyze the kinetic data. Ho model fitted better to the experimental results. [ABSTRACT FROM AUTHOR]

Abstract: In the present work, we analyze the mutant strains of Streptomyces fradiae ATCC 19609 resistant to (33S)-azido-33-deoxyoligomycin A, obtained at a frequency of 10-8 to 2.5 × 10-9. Analysis of the obtained mutant strains allows them to be divided into two groups based on the level of resistance to oligomycin A. One mutant out of 15 obtained is resistant to oligomycin A at MIC of 0.1 nm/mL, and 14 mutant strains are resistant at MIC of 0.01 nm/mL of oligomycin A (MIC for the initial strain: 0.001 nm/mL). Using PCR analysis and subsequent sequencing by the Sanger method, three candidate genes the products of which are related to resistance to oligomycin A are analyzed: helicase IV, the C subunit and the A subunit of FoF1-ATP synthase. It is found that, in the genomes of all 15 mutants, there is a single nucleotide substitution (SNP) in the helicase IV gene, while in one strain most resistant to oligomycin A there is an additional SNP in the gene of the A subunit of FoF1-ATP synthase. This mutant strain will be used to obtain the mutants of resistance to oligomycin A with the aim of further sequencing of genomes and detection of biotarget of oligomycin A. [ABSTRACT FROM AUTHOR]

The patterns of protein phosphorylation in inverted membrane vesicles from the strain Streptomyces fradiae ATCC 19609 were investigated to elucidate the mechanisms of regulation of bacterial membrane bound FF-ATP synthase. We found for the first time by two-dimensional gel electrophoresis and mass spectrometry that the β- and b-subunits of the FF-ATP synthase complex undergo phosphorylation; 20 proteins with known functions were identified. All eight subunits of FF-ATP synthase, i.e. α, β, γ, δ, ɛ, a, b, and c, were cloned into Escherichia coli and expressed as recombinant proteins. Using a crude preparation of serine/threonine protein kinases, we demonstrated the phosphorylation of recombinant γ-, β-, α- and ɛ-subunits. The β-subunit was phosphorylated both as a recombinant protein and in vesicles. Differential phosphorylation of membrane-bound and recombinant proteins can be attributed to different pools of protein kinases in each preparation; in addition, certain steps of FF-ATP synthase assembly and function might be accompanied by individual phosphorylation patterns. The structure of the operon containing all subunits and regulatory protein I was identified. The phylogenetic similarity of FF-ATP synthase from Streptomyces fradiae ATCC 19609 with the respective proteins in saprophytic and pathogenic (including Mycobacterium tuberculosis) bacteria was investigated. Thus, bacterial serine/threonine protein kinases are important for the regulation of FF-ATP synthase. From the practical standpoint, our results provide a basis for designing targeted antibacterial drugs. [ABSTRACT FROM AUTHOR]

The feasibility of an electro-Fenton process to treat tylosin (TYL), a non-biodegradable antibiotic, was examined in a discontinuous electrochemical cell with divided cathodic and anodic compartments. Only 15 min electrolysis was needed for total tylosin degradation using a carbon felt cathode and a platinum anode; while 6 h electrolysis was needed to achieve high oxidation and mineralization yields, 96 and 88 % respectively. Biodegradability improvement was shown since BOD/COD increased from 0 initially to 0.6 after 6 h electrolysis (for 100 mg L initial TYL). With the aim of combining electro-Fenton with a biological treatment, an oxidation time in the range 2 to 4 h has been however considered. Results of AOS (average oxidation state) and COD/TOC suggested that the pretreatment could be stopped after 2 h rather than 4 h; while in the same time, the increase of biodegradability between 2 and 4 h suggested that this latter duration seemed more appropriate. In order to conclude, biological cultures have been therefore carried out for various electrolysis times. TYL solutions electrolyzed during 2 and 4 h were then treated with activated sludge during 25 days, showing 57 and 67 % total organic carbon (TOC) removal, respectively, namely 77 and 88 % overall TOC removal if both processes were considered. Activated sludge cultures appeared, therefore, in agreement with the assessment made from the analysis of physico-chemical parameters (AOS and COD/TOC), since the gain in terms of mineralization expected from increasing electrolysis duration appeared too low to balance the additional energy consumption. [ABSTRACT FROM AUTHOR]

Tylosin is a veterinary antibiotic and is commercially produced using Streptomyces fradiae. Previously, we developed a mutant γ-1 of S. fradiae NRRL-2702 with a 6.87-fold increase in tylosin yield as compared with the wild-type strain through irradiation mutagenesis. The present studies were conducted to explore mutational changes in regulatory genes ( TylQ, TylP, TylS, TylR, and TylT) of Tyl cluster that may lead to an enhanced expression of tylosin. Expression analysis by RT-PCR revealed that TylQ was switched off earlier in mutant γ-1 while no change in expression pattern of TylP was observed between the wild-type and mutant γ-1 strains. However, a point mutation with a substitution of T to A was recorded at position 214 in the 420-bp product of TylP from mutant γ-1 that resulted in a change of one amino acid (serine to threonine) at position 72. Moreover, no mutation in the nucleotide sequence of TylS, TylR, and TylT genes was detected. [ABSTRACT FROM AUTHOR]

A study was conducted to evaluate the biocontrol potential of the optimized culture filtrates of Streptomyces fradiae against root-knot nematode Meloidogyne incognita in tomato under field conditions. The fermentation medium of S. fradiae optimized with pH 6 at 35oC with starch and yeast extract as carbon and nitrogen sources respectively maximized the colonization rate of S. fradiae. The culture filtrate of the optimized medium of S. fradiae caused in higher degree of inhibition in egg hatching, and mortality of juveniles of M. incognita. The effectiveness caused medium of S. fradiae against M .incognita was related to higher production of secondary metabolites subsequent to maximization of colonization. The S. fradiae experimented for the management of M. incognita under field conditions revealed its effectiveness for the suppression of nematode population and reduced the severity of nematode disease in terms of gall index. The suppression in M. incognita population / incidence following application of S. fradiae resulted in significant improvement in yield attributes including fruit yield of tomato. [ABSTRACT FROM AUTHOR]

The stereospecificity of an enzymatic reaction depends on the way in which a substrate and its enantiomer bind to the active site. These binding modes cannot be easily predicted. We have studied the stereospecificity and stereoselectivity of the ketoreductase domain Tyl-KR1 of the tylactone polyketide synthase from Streptomyces fradiae by analysing the stereochemical outcome of the reduction of five different keto ester substrates. The absolute configuration of the Tyl-KR1 reduction products was determined by using vibrational circular dichroism (VCD) spectroscopy combined with quantum chemical calculations. The conversion of only one of the tested substrates, 2-methyl-3-oxovaleric acid N-acetylcysteamine thioester, afforded the expected anti-(2 R,3 R) configuration of the α-methyl-β-hydroxyl ester product, representing the stereochemistry observed for the physiological polyketide product tylactone. For all other substrates, which were modified with respect to the type of ester and/or the chain length (C4 instead of C5), the opposite configuration ( anti-(2 S,3 S)) was obtained with significant enantio- and diastereoselectivity. Inversion of both stereocentres suggests completely different binding modes invoked by only minor modifications of the substrate structure. [ABSTRACT FROM AUTHOR]