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Registration Number

0225U004271, (0124U002533) , R & D reports

Title

Biotechnological and pharmacological potential of new antibiotic Je478 with the antimycobacterial activity

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Платформа для продукції, пілотна продукція та очищення, біологічні та фармакологічні властивості антибіотика Je478.

Head

Fedorenko Viktor O., д.б.н.

Registration Date

27-11-2025

Organization

Ivan Franko National University of Lviv

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Elucidate the genetic control of Je478 biosynthesis and using this knowledge to construct the platform for Je478 production based on biotechnologically improved strains of actinomycetes

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The project focuses on a comprehensive investigation of the new antibiotic Je478, produced by the actinomycete Streptomyces sp. Je1-332. It was established that growth in DNPM medium provides the highest production of Je478. A gene cloning system was developed, and efficient expression of reporter genes was demonstrated. The genome sequence of strain Je1-332 is 8.27 Mbp and contains 21 secondary metabolite gene clusters, including 6 terpene biosynthesis clusters. The Je478 biosynthetic gene cluster was identified using Tn5 transposon mutagenesis, and its functionality was confirmed by heterologous expression in S. albus Del14. The minimal set of genes required for Je478 biosynthesis was determined. Deletion analysis demonstrated that the groA–groC and groF genes are critical for forming the cyclic structure of the compound, and their inactivation leads to the accumulation of a linear precursor—hexaprenylguanidine. It was found that the isoprene units are formed via the non-mevalonate pathway, while the guanidine group originates from arginine, as confirmed by labeled substrate feeding experiments. Based on the obtained data, a model of the Je478 biosynthetic pathway was constructed. The process of heterologous expression in S. albus STS-36 was optimized, providing the highest productivity (1.0–1.5 mg/L) and stable compound synthesis. Fermentation of the producer strain in 10-liter medium yielded more than 10 mg of purified Je478, and several of its physicochemical and biological properties were investigated. Preliminary pharmacological tests confirmed the activity of Je478 against both sensitive and antibiotic-resistant bacterial strains, as well as its cytotoxic effects on cell lines and Danio rerio embryos. Genome analysis of spontaneous B. subtilis DSM10 mutants enabled identification of a potential target of Je478— a TetR-family regulatory protein encoded by the pksA gene, mutations in which are associated with the development of resistance to this antibiotic.

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Kurylenko Olena O.
Vasyl Y. Syrvatka
Ostash Iryna S.
Rebets Yurii V.
Білик Богдан Романович
Oleksandr M. Gromyko
Samborskyy Markiyan
Stepan I. Tistechok

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2025-11-27