RESUMEN
In the search for new antibiotics, it is a common occurrence that already known molecules are "rediscovered" while new promising ones remain unnoticed. A possible solution to this problem may be the so-called "target-oriented" search, using special reporter microorganisms that combine increased antibiotic sensitivity with the ability to identify a molecule's damaging effect. The use of such test organisms makes it possible to discover new promising properties even in known metabolites. In this study, we used a high-throughput screening method based on the pDualrep2 dual reporter system, which combines high sensitivity through the use of modified strains of test organisms and makes it possible to easily and accurately identify the interaction mechanisms of a substance and a bacterial cell at the initial stages of screening. This reporter system is unknown in Russia and is significantly superior to its global analogues. In the system, translation inhibition induces the expression of the fluorescent protein Katushka2s, while DNA damage is induced by TurboRFP. Using pDualrep2, we have isolated and described BV-204, an S. phaeochromogenes strain producing K-1115A, the biologically active substance that we have previously described. In our study, K-1115A for the first time has demonstrated antibiotic activity and an ability to inhibit bacterial translation, which was confirmed in vitro in a cell-free translation system for FLuc mRNA. K-1115A's antibacterial activity was tested and confirmed for S. aureus (MRSA) and B. subtilis, its cytotoxicity measured against that for the HEK293 cell line. Its therapeutic index amounted to 2 and 8, respectively. The obtained results open up prospects for further study of K-1115A; so, this can be regarded as the basis for the production of semi-synthetic derivatives with improved therapeutic properties to be manufactured in dosage forms.
RESUMEN
Representatives of the phylum Actinomycetota are one of the main sources of secondary metabolites, including antibiotics of various classes. Modern studies using high-throughput sequencing techniques enable the detection of dozens of potential antibiotic biosynthetic genome clusters in many actinomycetes; however, under laboratory conditions, production of secondary metabolites amounts to less than 5% of the total coding potential of producer strains. However, many of these antibiotics have already been described. There is a continuous "rediscovery" of known antibiotics, and new molecules become almost invisible against the general background. The established approaches aimed at increasing the production of novel antibiotics include: selection of optimal cultivation conditions by modifying the composition of nutrient media; co-cultivation methods; microfluidics, and the use of various transcription factors to activate silent genes. Unfortunately, these tools are non-universal for various actinomycete strains, stochastic in nature, and therefore do not always lead to success. The use of genetic engineering technologies is much more efficient, because they allow for a directed and controlled change in the production of target metabolites. One example of such technologies is mutagenesis-based genome editing of antibiotic biosynthetic clusters. This targeted approach allows one to alter gene expression, suppressing the production of previously characterized molecules, and thereby promoting the synthesis of other unknown antibiotic variants. In addition, mutagenesis techniques can be successfully applied both to new producer strains and to the genes of known isolates to identify new compounds.
RESUMEN
Potential to produce inducible enzymes (several hydrolases and oxidases) and antibiotics as secondary metabolites was studied in soil micromycete strains from the Arctic (Franz Josef Land and Novaya Zemlya) and Antarctica (the oases Thala Hills, Larsemann Hills, Schirmacher, and Marie Byrd Land). Maximal esterase activity was observed in strains of two typical Antarctic species, Hyphozyma variabilis 218 and Thelebolus ellipsoideus 210 (51 and 29 nmol FDA/((g mycelium h), respectively). Cellulolytic activity was maximal (89 µmol glucose/mg biomass) in Ascochyta pisi 192. Extracellular phenol oxidase (laccase) and peroxidase activities were not detected in the strains examined. Antibacterial activity toward Bacillus subtilis ATCC 6633 was observed in 75% of the Antarctic micromycete strains. Higher-activity strains were isolated from organic-rich moist habitats with a moss or lichen cover. Maximal activities were displayed by Paecilomyces marquandii 166, Penicillium janczewskii 165, Penicillium roseopurpureum 169, and Thelebolus ellipsoideus 210. Antagonistic activity toward Antarctic bacterial strains was shown by 77% of the microfungal strains examined. Maximal inhibition was observed with strains of the typical Antarctic species Antarctomyces psychrotrophicus MT303855 and the eurytopic species Sarocladium kiliense MT303856. Antimycotic activity was observed in 42% of the strains. Both activities were detected in 38% of the Antarctic strains.