Soil microorganisms with diverse bioactive compounds such as Streptomyces are appreciated as valuable resources for the discovery of eco-friendly fungicides. This study isolated a novel Streptomyces from soil samples collected in the organic green tea fields in South Korea. The isolation process involved antifungal activity screening around 2400 culture extracts, revealing a strain designated as S. collinus Inha504 with remarkable antifungal activity against diverse phytopathogenic fungi. S. collinus Inha504 not only inhibited seven phytopathogenic fungi including Fusarium oxysporum and Aspergillus niger in bioassays and but also showed a control effect against F. oxysporum infected red pepper, strawberry, and tomato in the in vivo pot test. Genome mining of S. collinus Inha504 revealed the presence of the biosynthetic gene cluster (BGC) in the chromosome encoding a polyene macrolide which is highly homologous to the lucensomycin (LCM), a compound known for effective in crop disease control. Through genetic confirmation and bioassays, the antifungal activity of S. collinus Inha504 was attributed to the presence of LCM BGC in the chromosome. These results could serve as an effective strategy to select novel Streptomyces strains with valuable biological activity through bioassay-based screening and identify biosynthetic gene clusters responsible for the metabolites using genome mining approach.

Information on microbial genome sequences is a powerful resource for accessing natural products with significant activities. We herein report the unveiling of lucensomycin production by Streptomyces achromogenes subsp. streptozoticus NBRC14001 based on the genome sequence of the strain. The genome sequence of strain NBRC14001 revealed the presence of a type I polyketide synthase gene cluster with similarities to a biosynthetic gene cluster for natamycin, which is a polyene macrolide antibiotic that exhibits antifungal activity. Therefore, we investigated whether strain NBRC14001 produces antifungal compound(s) and revealed that an extract from the strain inhibited the growth of Candida albicans. A HPLC analysis of a purified compound exhibiting antifungal activity against C. albicans showed that the compound differed from natamycin. Based on HR-ESI-MS spectrometry and a PubChem database search, the compound was predicted to be lucensomycin, which is a tetraene macrolide antibiotic, and this prediction was supported by the results of a MS/MS analysis. Furthermore, the type I polyketide synthase gene cluster in strain NBRC14001 corresponded well to lucesomycin biosynthetic gene cluster (lcm) in S. cyanogenus, which was very recently reported. Therefore, we concluded that the antifungal compound produced by strain NBRC14001 is lucensomycin.

Actinobacteria are among the most prolific sources of medically and agriculturally important compounds, derived from their biosynthetic gene clusters (BGCs) for specialized (secondary) pathways of metabolism. Genomics witnesses that the majority of actinobacterial BGCs are silent, most likely due to their low or zero transcription. Much effort is put into the search for approaches towards activation of silent BGCs, as this is believed to revitalize the discovery of novel natural products. We hypothesized that the global transcriptional factor AdpA, due to its highly degenerate operator sequence, could be used to upregulate the expression of silent BGCs. Using Streptomyces cyanogenus S136 as a test case, we showed that plasmids expressing either full-length adpA or its DNA-binding domain led to significant changes in the metabolome. These were evident as changes in the accumulation of colored compounds, bioactivity, as well as the emergence of a new pattern of secondary metabolites as revealed by HPLC-ESI-mass spectrometry. We further focused on the most abundant secondary metabolite and identified it as the polyene antibiotic lucensomycin. Finally, we uncovered the entire gene cluster for lucensomycin biosynthesis (lcm), that remained elusive for five decades until now, and outlined an evidence-based scenario for its adpA-mediated activation.

Lucilia sericata, a member of the Calliphoridae family, is one of the most common species in the genus Lucilia. Medical importance of L.sericata stems from its use in maggot debridement therapy (MDT). MDT is the name of L.sericata larvae being sterilized and used in the treatment of non-healing wounds. L.sericata maggots used in the treatment of chronic and non-healing wounds (decubitus ulcer, venous leg ulcer, diabetic foot ulcer, etc.) clean the wounds with the help of secreted proteolytic trypsin and lucimycin -like enzymes. The aim of the study was to determine the molecular characterization of lucimycin gene obtained from L.sericata larvae in MDT by using molecular methods and to contribute to the literature. In this study, continuous production of adult colonies of L.sericata species was carried out in insectarium unit where conditions such as light, humidity and temperature were formed. The life cycle of L.sericata was followed and the production of eggs, larvae, pupae, adult flies and fly colonies of the species were formed. In the third stage larvae obtained from adult flies in the insectarium unit, RNA was isolated and subsequently cDNA synthesis was performed by reverse transcription. Polymerase chain reaction (PCR) analysis of the synthesized cDNAs with the specific primers designed for the lucimycin gene of L.sericata was performed and the obtained amplicons were cloned into pJET1.2/blunt vector and the plasmid was purified. The recombinant plasmids were sequenced with vector-specific primers and target gene region sequences were obtained. After the molecular characterization of the isolate with nucleotide sequences was determined, it was registered to GenBank database with the accession number MF964229. The PCR product of 288 bp was obtained from the cDNA obtained from the larvae of L.sericata produced in the insectarium unit by PCR using lucimycin specific primers. The PCR product imaged on the gel was purified by transformation and subsequent colonies were screened to see whether they contained recombinant plasmids. Three of the colonies were identified as recombinant plasmids containing L.sericata lucimycin gene by PCR screening. From three colonies confirmed by PCR screening, recombinant plasmids containing L.sericata lucimycin gene were purified by miniprep. The recombinant plasmid product was confirmed to contain the L.sericata lucimycin gene by PCR from a total of 20 μl of the recombinant plasmid miniprep product. DNA sequencing analysis was performed to confirm the plasmid after cloning. The 288 bp L.sericata lucimycin sequence was confirmed by DNA sequence analysis. The lucimycin gene isolated was confirmed by specific and pJET1.2 forward and reverse primers using Blastn algorithm as a result of species and/or subspecies using the Blastn algorithm and the related isolate was recorded in GenBank database with the MF964229 accessory number. The DNA sequence of the isolated sample was compared with other isolates found in GenBank by Pubmed/Blast program. KJ413251.1 was found to be 99% similar to the GenBank isolate. The 113th nucleotide was C (cytosine) in the sequence of our isolate, while the existence of G (guanine) in the sequence numbered KJ413251.1 GenBank revealed the difference between the two sequences. In this study the molecular characterization of lucimycin gene derived from L.sericata larvae were determined for the first time in Turkey, it is assumed that this molecule which has an antifungal property, can be used in the studies that will be carried out in the future, especially in microorganisms causing cutaneous infections. The study is important since the isolate is registered as a biological asset of Turkey in GenBank and also being the second study in the world.

Comparative analysis of the effects of chemically transformed polyene antibiotics pimaricin, nystatin, lucensomycin, amphotericin B, and levorin on biological objects in vivo and in vitro revealed the greatest biological activity of original amphotericin B and levorin with its derivatives. The study also examined the effects of alkyl derivatives of amphotericin B and levorin modified in certain parts of the lactone ring on the lipid and biological membranes. It is established that methylated levorin possesses larger biological activity than the original antibiotic. Examination of the effects of alkyl derivatives of levorin and amphotericin B on cell cultures C6 (rat glioma) and HeLa (human cervical carcinoma) in vitro revealed the antitumor action of methylated levorin and original amphotericin B.

We report the identification, cloning, heterologous expression and functional characterization of a novel antifungal peptide named lucimycin from the common green bottle fly Lucilia sericata. The lucimycin cDNA was isolated from a library of genes induced during the innate immune response in L. sericata larvae, which are used as therapeutic maggots. The peptide comprises 77 amino acid residues with a molecular mass of 8.2 kDa and a pI of 6.6. It is predicted to contain a zinc-binding motif and to form a random coil, lacking β-sheets or other secondary structures. Lucimycin was active against fungi from the phyla Ascomycota, Basidiomycota and Zygomycota, in addition to the oomycete Phytophtora parasitica, but it was inactive against bacteria. A mutant version of lucimycin, lacking the four C-terminal amino acid residues, displayed 40-fold lower activity. The activity of lucimycin against a number of highly-destructive plant pathogens could be exploited to produce transgenic crops that are resistant against fungal diseases.

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Two methods, biological and spectroscopic, were used to determine the avidity of the polyene antibiotic Etruscomycin for cholesterol and ergosterol. The biological method consisted of measuring the inhibitory potency of both sterols on the Etruscomycin-induced damage to erythrocytes and fungi. The spectroscopic method consisted of recording of series of differential spectra in a number of solvents of different composition. The results obtained showed that cholesterol protected erythrocytes and candida albicans against the damaging action of Etruscomycin more efficiently than ergosterol did and that Etruscomycin-cholesterol complexes were more resistant to interruption by organic solvents than Etruscomycin-ergosterol complexes. These results and their comparison with the results obtained with other polyene antibiotics indicate that Etruscomycin resembles filipin in that it binds more avidly to cholesterol than to ergosterol. This implies that the length of the hydrophobic chain rather than the presence of the amino sugar determines sterol preference. The spectral method that we used can have general application for the quantitative measurement of complex formation between polyenes and sterols.